CN1310945C - Endogenous and non-endogenous versions of human G protein-coupled receptors - Google Patents

Abstract

The invention disclosed in this patent application document relates to transmembrane receptors; in particular, to human G protein-coupled receptors whose endogenous ligands are unknown (orphan GPCR receptors); and more particularly, to mutant (non-endogenous) forms of human GPCRs used to provide evidence of constitutive activity.

Description

Endogenous and non-endogenous forms of human G protein-coupled receptors

field of invention

The invention disclosed in this patent application document relates to transmembrane receptors; in particular, to receptors coupled to human G proteins, particularly endogenous human GPCRs; with particular emphasis on receptors that have been altered to have or enhance Non-endogenous form of a constitutively active GPCR. Preferably, the altered GPCR is used for the direct identification of candidate compounds as receptor agonists, inverse agonists or partial agonists as potential therapeutic agents.

Background of the invention

Although there are many classes of receptors in the human body, by far the most abundant and therapeutically relevant are the G protein-coupled receptors (GPCRs). It is estimated that there are approximately 100,000 genes within the human genome, of which approximately 2% or 2,000 genes are estimated to encode GPCRs. Receptors, including GPCRs, whose endogenous ligands are known are called "known" receptors, and receptors whose endogenous ligands are not known are called "orphan" receptors. GPCRs represent an important area of drug product development: 60% of prescribed drugs are developed from approximately 20 of the 100 known GPCRs.

GPCRs all share a common motif. All of these receptors have seven sequences of 22 to 24 hydrophobic amino acids that form seven α-helices, each of which spans the membrane (each span is represented by a number, e.g., transmembrane-1 (TM -1), transmembrane-2 (TM-2), etc.). The transmembrane helices are connected by chains of amino acids, and the amino acid chains on the outer side of the cell membrane, the "extracellular" side, are respectively transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, transmembrane-6 and transmembrane -7 (these are referred to as "extracellular" regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively). On the inside of the cell membrane, the "intracellular" side, the transmembrane helices are also connected by chains of amino acids in transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, transmembrane-5 and Between transmembrane-6 (these are referred to as "intracellular" regions 1, 2 and 3 (IC-1, IC-2 and IC-3) respectively). The "carboxy" ("C") terminus of the receptor is the intracellular region and the "amino" ("N") terminus of the receptor is the extracellular region.

Generally, when an endogenous ligand binds to a receptor (often referred to as "activation" of the receptor), the conformation of the intracellular domain changes to allow coupling between the intracellular domain and the intracellular "G-protein". couplet. GPCRs have been reported to be "promiscuous" with respect to G proteins, that is, to interact with more than one G protein. See, Kenakin, T., 43, Life Sciences 1095 (1988). Although other G proteins exist, the currently recognized G proteins are Gq, Gs, Gi, Gz, and Go. Coupling of GPCRs activated by endogenous ligands to G-proteins initiates a signaling cascade process (termed "signaling"). In general, signaling ultimately results in cellular activation or cellular inhibition. Both the IC-3 loop and the carboxyl terminus of the receptor are thought to interact with the G protein.

Under physiological conditions, GPCRs exist on the cell membrane in equilibrium between two different conformations, an "inactive" state and an "activated" state. Receptors in the inactive state are unable to couple with intracellular signaling pathways to produce a biological response. The conformational transition of the receptor to the active state couples it to the transduction pathway (via the G-protein) and produces a biological response.

Receptors can be stabilized in their active state by endogenous ligands or compounds such as drugs. Recent discoveries provide methods that, in addition to endogenous ligands or drugs, can promote and stabilize the conformation of the receptor to the active state, including but not limited to modifications to the amino acid sequence of the receptor. These methods effectively stabilize the active state of the receptor by mimicking the action of endogenous ligands bound to the receptor. Stabilization by such a ligand-independent approach is referred to as "constitutive receptor activation".

Summary of the invention

Disclosed herein are endogenous and non-endogenous forms of human GPCRs and their use.

Brief description of the drawings

Figure 1 shows the production of the second messenger _IP3 by an endogenous form of RUP12 ('RUP12') compared to a control ("CMV").

Figure 2 is a graph of the results of a cell-based second messenger loop AMP assay showing a comparison of constitutive signaling of the endogenous form of RUP13 ("RUP13") and a control vector ("CMV").

Figure 3 is a graphical representation comparing signal measurements of CMV, an endogenous form of RUP13 ("RUP13wt") and a non-endogenous form of constitutively activated RUP13 ("RUP13(A268K)"), using the 8XCRE-Luc reporter plasmid.

Figure 4 is a schematic representation of the results of [ ^35S ]GTPyS analysis showing a comparison of constitutive signaling of the RUP13:Gs fusion protein ("RUP13-Gs") and a control vector ("CMV").

Figure 5 is a graphical representation comparing signal measurements of CMV, the endogenous form of RUP14 ("RUP14wt") and the non-endogenous form of constitutively activated RUP13 ("RUP14(L246K)"), using the 8XCRE-Luc reporter plasmid.

Figure 6 is a graphical representation comparing signal measurements of CMV, the endogenous form of RUP15 ("RUP15wt") and the non-endogenous form of constitutively activated RUP15 ("RUP15(A398K)"), using the 8XCRE-Luc reporter plasmid.

Figure 7 is a graphical representation of the results of a cell-based second messenger loop AMP assay showing the endogenous form of RUP15 ("RUP15wt"), the non-endogenous form of constitutively activated RUP15 ("RUP15(A398K)"), and the control vector ("CMV ”) The result of the comparison of constituent signaling.

Figure 8 is a schematic representation of the results of an [ ^35S ]GTPyS assay showing a comparison of constitutive signaling of the RUP15:Gs fusion protein ("RUP15-Gs") and a control vector ("CMV").

Figure 9 shows the production of the second messenger _IP3 in an endogenous form of RUP17 ("RUP17") compared to a control ("CMV").

Figure 10 shows the production of the second messenger _IP3 in an endogenous form of RUP21 ("RUP21") compared to a control ("CMV").

Figure 11 is a graphical representation comparing signal measurements of CMV, an endogenous form of RUP23 ("RUP23wt") and a non-endogenous form of constitutively activated RUP23 ("RUP23(W275K)"), using the 8XCRE-Luc reporter plasmid.

Figure 12 is a schematic diagram of the preliminary screening results of several candidate compounds against RUP13; the results of "compound A" are provided in well A2, and the results of "compound B" are provided in well G9.

A detailed description

The scientific literature deals with receptors and adopts some terms to describe ligands that have different effects on receptors. For clarity and consistency, the following definitions will be used throughout the present document. Where these definitions conflict with other definitions of these words, the following definitions are chosen:

Agonists mean substances (eg, ligands, candidate compounds) that activate intracellular responses when they bind receptors or promote GTP binding to membranes.

The amino acid abbreviations used herein are listed in Table A below:

Table A Alanine ALA A arginine ARG R Asparagine ASN N aspartic acid ASP D. cysteine CYS C glutamic acid Glu E. Glutamine GLN Q Glycine GLY G Histidine HIS h Isoleucine ILE I Leucine LEU L Lysine Lys K Methionine MET m Phenylalanine PHE f proline PRO P serine SER S threonine THR T Tryptophan TRP W Tyrosine TYR Y Valine VAL V

Partial agonists mean substances (eg, ligands, candidate compounds) that, upon binding to a receptor, activate intracellular responses or promote GTP binding to membranes to a lesser extent than agonists.

Antagonist means a substance (e.g., a ligand, a candidate compound) that competes for binding to a receptor at the same site as an agonist, but does not activate the intracellular response elicited by the active form of the receptor, and can Thus inhibiting intracellular responses promoted by agonists or partial agonists. Antagonists do not impair fundamental intracellular responses in the absence of agonists or partial agonists.

Candidate compound means a molecule (such as but not limited to a chemical compound) that will be subjected to screening techniques. Preferred "candidate compounds" do not include compounds known to the public as inverse agonists, agonists or antagonists of receptors, which have previously been identified by indirect identification methods ("indirect identification compounds ”); more preferably do not include indirectly identified compounds that have previously been determined to have a therapeutic effect in at least one mammal; and, most preferably do not include indirectly identified compounds that have previously been determined to have therapeutic use in humans identified compounds.

Composition means a substance comprising at least one ingredient; a pharmaceutical composition is an example of a composition.

Compound effect means a measure of a compound's ability to inhibit or stimulate receptor function, as opposed to receptor binding affinity. Typical methods for determining the effects of compounds are further disclosed in the Examples section of this patent application.

Codon refers to a group of three nucleotides (or words equivalent to nucleotides), which are usually composed of a nucleoside (adenosine (A), guanosine (G), cytidine (C), Uridine (U) and thymidine (T)) are coupled to a phosphate group, and one codon codes for one amino acid during translation.

A constitutively activated receptor means a receptor susceptible to constitutive receptor activation. Receptors that are constitutively activated can be endogenous or non-endogenous.

Constitutive receptor activation means the stabilization of a receptor in an active state without the use of its endogenous ligand or its chemical equivalent to bind to the receptor.

Contacting means bringing at least two parts together, whether in an in vitro system or an in vivo system.

Directly identifying or being directly identified, in connection with the term "candidate compound", means screening against a constitutively activated receptor, preferably against a constitutively activated orphan receptor, most preferably against a constitutively activated G protein-coupled Candidate compounds for cell surface orphan receptors. Under no circumstances should this term be construed or understood to include or include the terms "indirectly identified" or "indirectly identified".

Endogenous means a substance naturally produced by a mammal. These are by way of illustration only and not limitation. In contrast, the term "non-endogenous" herein means not naturally produced by a mammal (such as, but not limited to, a human) or a virus. In contrast, the term "non-endogenous" herein means a substance that is not naturally produced by a mammal (such as, but not limited to, a human) or a virus. For example, a receptor that is not constitutively activated in its endogenous form, when manipulated so as to be constitutively activated, is most preferably referred to as a "non-endogenous constitutively activated receptor". ”, which are for illustration only and not limitation. Both terms can be used to describe "in vivo" and "in vitro" systems. For example, in the screening process, endogenous or non-endogenous receptors can be used in in vitro screening systems, again by way of illustration and not limitation. By way of further example and not limitation, when the genome of a mammal is manipulated to include non-endogenous constitutively activated receptors, candidate compounds can be screened by an in vivo system.

In the present context, "G protein-coupled receptor fusion protein" and "GPCR fusion protein" refer to a non-endogenous protein comprising an endogenous constitutively activated GPCR, or a protein in combination with at least one G protein, preferably a G protein A non-endogenous constitutively activated GPCR fused to an α subunit (which is the subunit that binds to GTP), wherein the type of G protein is preferably the type of G protein that is coupled to the endogenous orphan GPCR under natural circumstances and is consistent. For example (but not limitation), in the endogenous state, if the G protein "Gsα" is the primary G protein coupled to a GPCR, a GPCR fusion protein based on this specific GPCR is a non-endogenous protein comprising a GPCR fused to Gsα . In some cases, non-essential G proteins can also be fused to GPCRs, as described below. The G protein may be directly fused to the C-terminus of a constitutively active GPCR, or a spacer may be present between the two.

A host cell means a cell into which a plasmid and/or vector can be inserted. In the case of a prokaryotic host cell, the plasmid typically replicates in an autonomous molecular manner when the host cell replicates (in general, the plasmid is isolated after replication to be introduced into the eukaryotic host cell); in the case of a eukaryotic host cell , the plasmid is integrated into the cellular DNA of the host cell, thus, when the eukaryotic cell replicates, the plasmid replicates. For the purposes of the invention disclosed herein, the host cell is preferably a eukaryotic cell, more preferably a mammalian cell, most preferably a cell selected from among 293, 293T and COS-7 cells.

Recognizing or being recognized indirectly means the traditional approach to drug discovery, which involves the identification of endogenous ligands specific for endogenous receptors, screening of candidate compounds against the receptors, and determination of those interfering or competing ligands - A receptor-interactive compound, measuring the efficiency of the compound's effect on at least one second messenger pathway associated with the activated receptor.

Inhibiting, in connection with the term "reaction", means that a reaction is reduced or prevented in the presence of a compound, as opposed to the absence of the compound.

Inverse agonist means a substance (e.g., ligand, candidate compound) that binds to an endogenous receptor or a constitutively activated form of a receptor and suppresses the basal intracellular response elicited by the active form of the receptor to normal Below basal levels, the level of activity is observed in the absence of agonists or partial agonists, or they reduce GTP binding to the membrane. The basal intracellular response is preferably inhibited by at least 30%, more preferably at least 50%, most preferably at least 75% in the presence of an inverse agonist compared to the basal response in the absence of the inverse agonist.

Known receptor means an endogenous receptor for which a specific endogenous ligand has been recognized.

Ligand means an endogenous naturally occurring molecule specific for an endogenous naturally occurring receptor.

Mutations in the nucleotide and/or amino acid sequences of endogenous receptors imply specific modifications of these endogenous sequences so that mutant forms of endogenous non-constitutively activated receptors can result in constitutive activation of the receptors. Subsequent mutants of the human receptor are considered equivalent to the first mutation of the human receptor for equivalents of a given sequence if (a) the level of constitutive activation of the subsequent mutant receptor is substantially different from that indicated by the first mutation of the receptor. as above; and (b) the percentage of sequence identity between the subsequent mutant receptor and the first mutation of the receptor is at least 80%, more preferably at least 90%, most preferably at least 95%. Ideally, given that the most preferred cassettes disclosed herein for constitutive activation include single amino acids and/or codons that vary between endogenous and non-endogenous GPCRs, sequence homology The percentage shall be at least 98%.

Non-orphan receptors refer to naturally occurring endogenous molecules that exhibit specificity for naturally occurring endogenous ligands whose binding to the receptor activates intracellular signaling pathways.

Orphan receptors mean endogenous receptors for which specific endogenous ligands have not been identified or are not yet known.

A pharmaceutical composition means a composition comprising at least one active ingredient by means of which active ingredient can be studied for a specific effect of the composition in mammals such as but not limited to humans. Those of ordinary skill in the art will be able to understand and properly evaluate techniques suitable for determining whether an active ingredient has the desired effect based on the needs of the skilled person.

Plasmid means a combination of vector and cDNA. Typically, plasmids are introduced into host cells for the purpose of cDNA replication and/or protein expression.

Second messenger means the intracellular response resulting from receptor activation. For example, second messengers include inositol triphosphate (IP3), diacylglycerol (DAG), cyclic AMP (cAMP), and cyclic GMP (cGMP). Detection of second messenger responses can determine whether there is receptor activation. In addition, detection of second messenger responses allows direct identification of candidate compounds including, for example, inverse agonists, agonists, partial agonists and antagonists.

Stimulation, in connection with the term "response", means that the response is enhanced when a compound is present than when it is not present.

A vector for cDNA means a circular DNA into which at least one cDNA can be incorporated and which can be introduced into a host cell.

The order of the following sections is for presentational effect and should not be construed as a limitation on the following disclosure or claims.

A. Introduction

Traditional studies of receptors have been based on the presumption (based on history) that endogenous ligands must first be recognized before antagonists and other molecules that can act on the receptors can be discovered. Even when antagonists are discovered first, the search is immediately extended to search for endogenous ligands. This mindset has continued in receptor research even after the discovery of constitutively activated receptors. What has not been recognized heretofore is that it is the active state of the receptor that is most useful for discovering agonists, partial agonists and inverse agonists of the receptor. For those diseases caused by overactivation and underactivation of receptors, the desired therapeutic drugs are compounds that can reduce the active state of receptors or enhance receptor activity, respectively, and do not need to be against endogenous Ligand antagonists. This is because a compound that decreases or enhances the activity of an activated receptor does not necessarily bind at the same site as the endogenous ligand. Thus, any search for therapeutic compounds may begin by screening compounds against a ligand-independent active state, as taught by one method of the present invention.

B. Identification of Human GPCRs

The implementation of the Human Genome Project led to the identification of a large amount of information about nucleic acid sequences located in the human genome. After this effort, in fact, we do not need to know or know whether any particular genome sequence contains open reading frame information for translation of human proteins. To obtain genetic sequence information, several methods for identifying nucleic acid sequences in the human genome are familiar to those of ordinary skill in the art, such as (but not limited to), the large number of human GPCRs disclosed herein were found by reviewing the GenBank ^™ database of. Table B, below, lists several endogenous GPCRs that we have discovered, as well as other GPCRs that are homologous to the disclosed GPCRs.

Form B Published Human Orphan GPCRs Registration Open reading frame (base pair) reference homologous GPCR Homology ratios to indicated GPCRs wxya AL121755 1,152 bp NPY2R 27% hRUP9 AC0113375 1,260bp GAL2R twenty two% hRUP10 AC008745 1,014bp QUR 40% hRUP11 AC013396 1,272bp HM74 36% hRUP12 AP000808 966bp Masl 34% hRUP13 AC011780 1,356bp Fish GPRX-ORYLA 43% hRUP14 AL137118 1,041bp CysLT1R 35% hRUP15 AL016468 1,527bp RE2 30% hRUP16 AL136106 1,068bp GLR101 37% hRUP17 AC023078 969bp Masl 37% hRUP18 AC008547 1,305 bp Oxytocin 31% hRUP19 AC026331 1,041bp HM74 52% hRUP20 AL161458 1,011bp GPR34 25% hRUP21 AC026756 1,014bp P2Y1R 37% hRUP22 AC027026 993bp RUP17Masl 67% 37% hRUP23 AC007104 1,092 bp Rat GPR26 31% hRUP24 AL355388 1,125bp SALPR 44% hRUP25 AC026331 1,092bp HM74 95% hRUP26 AC023040 1,044bp Rabbit 5HT1D 27% hRUP27 AC027643 158,700 MCH 38%

Receptor homology is useful for further understanding of the role of receptors in humans, and later in this patent application document we disclose techniques for mutating these receptors in order to establish the non-endogenous composition of these receptors type activated form.

The techniques disclosed herein have also been applied to other human orphan GPCRs known in the art, as will become more apparent as this patent application document progresses.

C. Receptor Screening

Screening for candidate compounds corresponding to non-endogenous, constitutively activated forms of the human GPCRs disclosed herein allows for the direct identification of candidate compounds that function at this cell surface receptor without the need for the receptor's endogenous ligand. Using routine, often commercially available techniques, the regions where endogenous forms of the human GPCRs disclosed herein are expressed and/or overexpressed in vivo can be determined. It is also possible to use these techniques to determine associated diseases/disorders associated with receptor expression and/or overexpression, which methods are disclosed in the present patent application documents.

The technique for making mutations that demonstrate constitutive activation of the human GPCRs disclosed here is based on the distance from the proline residue, which is estimated to be located within TM6 of the GPCR, the algorithm rules published on April 20, 2000 Commonly assigned co-pending PCT application PCT/US99/23938 published as WO 00/22129, which is incorporated herein by reference along with other patent documents listed herein. The algorithmic rules are not predicted on the basis of conventional sequence alignments, but rather on specific distances from the above-mentioned TM6 proline residue (or possibly also an endogenous constitutive substitute for this proline residue). This activation can be obtained by mutating an amino acid residue 16 amino acid residues from this residue (estimated to be located in the IC3 region of the receptor), preferably to lysine. Mutations of other amino acids at this position can be used for this purpose.

D. Disease/disorder identification and/or selection

As will be described in detail below, the methods of the invention are most preferably used to identify inverse agonists and agonists directed against non-endogenous, constitutively activated GPCRs. Such inverse agonists and agonists are ideal candidates for lead compounds in the search for drugs to treat diseases associated with these receptors. Since inverse agonists against these receptors can be directly identified, it will be possible to develop and search for pharmaceutical compositions directed against diseases and disorders associated with these receptors. For example, examining the presence of these GPCRs in diseased and normal tissue samples is now not only a matter of academic research, but also a research effort to identify the endogenous ligands of specific GPCRs. Tissue examination can be performed on a wide range of healthy and diseased tissues. Such tissue examination provides a preferred first step in linking specific receptors to a disease/disorder.

DNA sequences of preferably human GPCRs are used to make probes for (a) dot blot targeting of tissue mRNA and (b) RT-PCR identification of expression of said receptor in tissue samples. The presence of a receptor in a tissue or diseased tissue, or an increased concentration of a receptor in a diseased tissue as compared to normal tissue, may be used preferably to identify an association of treatments, including but not limited to, with that disease. Localization of the receptors to organ regions is also very good in this way. A putative functional role for a receptor can be deduced based on the known function of the particular tissue in which the receptor is localized.

E. Screening of Candidate Compounds

1. General GPCR Screening Assay Techniques

When a G protein receptor becomes constitutively active, it couples to the G protein (eg, Gq, Gs, Gi, Gz, Go) and stimulates GTP to bind the G protein. Next, the G protein acts as a GTPase to slowly hydrolyze GTP to GDP, aided by the normal inactivation of the receptor, whereas the constitutively activated receptor continues to convert GDP to GTP. The nonhydrolyzable analog of GTP, [ ³⁵ S]GTPyS, can be used to monitor binding to membranes expressing constitutively activated receptors. It has been reported that [ ³⁵ S]GTPγS can be used to monitor G protein coupling to membranes in the presence or absence of ligand. One example of such monitoring is reported by Traynor and Nahorski in 1995, among others well known and available in the art. A preferred application of this assay system is for initial screening of candidate compounds, since this system is generally feasible for all protein-coupled receptors, regardless of which particular G protein interacts with the intracellular domain of the receptor .

2. Specific GPCR Screening Assay Techniques

Once candidate compounds have been identified using "general" G protein-coupled receptor assays (i.e., methods that screen for compounds that are agonists, partial agonists, or inverse agonists), further screening is preferred to confirm the presence of receptors acting at the receptor site. compound. For example, a compound identified using a "general" assay may not bind to the receptor, but may also "uncouple" the G protein only from the intracellular domain.

a. Gs, Gz and Gi

Gs stimulates adenylyl cyclase. On the other hand, Gi (and Gz and Go) inhibit the enzyme. Adenylate cyclase catalyzes the conversion of ATP to cAMP; thus, constitutively active GPCRs coupled to Gs proteins are associated with elevated intracellular cAMP levels. On the other hand, constitutively activated GPCRs coupled to Gi (and Gz or Go) proteins are associated with decreased intracellular cAMP levels. See generally "Indirect Mechanisms of Synaptic Transmission", Chapter 8, From Neuron To Brain ( Third Edition), edited by Nichols, JG et al., Sinauer Associates, Inc. (1992). Thus, methods for detecting cAMP can be used to determine whether a competing compound is an inverse agonist of the receptor (ie such a compound would lower cAMP levels) and the like. Different methods known in the art for measuring cAMP can be utilized; the most preferred method relies on the use of anti-cAMP antibodies in an ELISA-based method. Another type of assay that can be used is a whole cell second messenger reporter gene system assay. A promoter on a gene drives the expression of the protein encoded by a particular gene. Cyclic AMP promotes gene expression through the following steps: it responds to the binding of a DNA-binding protein or transcription factor (CREB) that promotes cAMP, which then binds to the promoter at a specific site called the cAMP response element and drives gene expression . Reporter gene systems can be constructed with a promoter containing multiple cAMP-responsive elements, such as β-galactosidase or luciferase, in front of the reporter gene. Thus, a constitutively activated Gs-linked receptor causes the accumulation of cAMP, which in turn activates the gene and expression of the reporter protein. Reporter proteins such as β-galactosidase or luciferase can be detected using standard biochemical methods (Chen et al., 1995).

b. Go and Gq

Go and Gq are associated with the activation of phospholipase C, which then hydrolyzes the phosphate ester PIP ₂ and releases two intracellular messengers: diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP ₃ ). Increased accumulation of _IP3 associates with Gq- and Go-associated receptors. See generally "Indirect Mechanisms of Synaptic Transmission", Chapter 8, From Neuron To Brain (Third Edition), edited by Nichols, JG et al., Sinauer Associates, Inc .(1992). Methods for measuring _IP3 accumulation can be used to determine whether a candidate compound is an inverse agonist (ie such a compound reduces _IP3 levels), for example, at a Gq- or Go-associated receptor or the like. Gq-linked receptors can also be detected using the AP1 reporter gene assay, since Gq-dependent phospholipase C causes activation of genes containing AP1 elements; thus, activated Gq-linked receptors will lead to increased expression of such genes, whereas inverse agonists An agonist will cause a decrease in, and an agonist will cause an increase in, such expression. Commercially available methods for performing such assays are available.

3. GPCR fusion protein

The use of endogenous constitutively activated orphan GPCRs or non-endogenous constitutively activated orphan GPCRs for screening candidate compounds and the direct identification of inverse agonists, agonists, and partial agonists presents an interesting screening challenge, specifically, Receptors remain active in the absence of endogenous ligand binding. Therefore, in order to distinguish a non-endogenous receptor in the presence or absence of a candidate compound, the purpose of this distinction is to know whether the compound is an inverse agonist, agonist, partial agonist or body has no effect at all, the best way is to enhance this difference, the use of GPCR fusion protein is one such method.

In general, once the non-endogenous orphan GPCR is determined to be constitutively active using the above-mentioned analytical techniques (and others), it is possible to identify the dominant G protein coupled to the endogenous GPCR, and the coupling of the G protein to the GPCR provides signaling pathways that can be estimated. Because selection is best performed using mammalian expression systems, it is desirable to have endogenous G proteins present in this system, specifically, non-endogenous constitutively activated orphan GPCRs in which continuous signal is generated. In this regard, it is preferred that the signal be enhanced so that, for example, in the presence of an inverse agonist for the receptor, it is likely to be more convenient to distinguish between different receptors exposed to the inverse agonist, especially throughout the screening process.

The role of GPCR fusion proteins is to increase the effect of G protein coupling to non-endogenous GPCRs. GPCR fusion proteins are preferred for screening non-endogenous constitutively activated GPCRs because this method enhances the signal that is very useful for such screening techniques, Importantly, this facilitates a large "signal-to-noise" ratio, which is particularly preferred for screening the candidate compounds disclosed herein.

The construction technology for the construct of GPCR fusion protein expression is familiar to those of ordinary skill in the art, and commercially available expression vectors and systems provide various methods that can meet special needs for experimenters. This GPCR fusion protein construct Important criteria are that both the endogenous GPCR sequence and the G protein sequence are in-frame (preferably, the endogenous GPCR sequence is located upstream of the G protein sequence), and the "stop" codon of the GPCR must be removed or replaced, so that Along with the expression of GPCRs, G proteins can also be expressed. The GPCR may be directly attached to the G protein, or there may be spacer residues in between (preferably no more than 12, although those skilled in the art can easily know this number). We like to use spacers (based on convenience), and restriction sites that are not efficiently utilized in expression make up spacers. Prior to making GPCR fusion protein constructs, it is preferred to first identify the G protein coupled to the non-endogenous GPCR, as only few G proteins have been identified, so constructs containing G protein sequences (e.g. universal G protein constructs) are preferred The body can insert the endogenous GPCR sequence into it, so that it can efficiently screen a large number of endogenous GPCRs with different sequences on a large scale.

As noted above, constitutively active GPCRs coupled to Gi, Gz, and Go are expected to inhibit cAMP formation, requiring one to find assays based on these types of GPCRs (e.g., cAMP signal decreases with activation, such that direct recognition (e.g. ) inverse agonists (to further attenuate this signal) are even more interesting, as disclosed in this paper, we have shown that for these types of receptors it is possible to make GPCR fusion proteins that are not based on the endogenous G protein of the endogenous GPCR, trying to Establish viable cyclase-based assays. For example, endogenous Gi-coupled receptors can be fused to Gs proteins—we believe that such fusion constructs, when expressed, "drive" or "push" endogenous GPCRs are coupled to proteins such as Gs instead of "native" Gi, enabling the development of cyclase-based assays. For receptors coupled to Gi, Gz, and Go, when using GPCR fusion proteins and assaying with When based on the adenylyl cyclase activity assay, we prefer to use Gs (or a G protein analog that stimulates adenylyl cyclase formation) to create fusion constructs.

G protein fusion constructs in which the Gq protein is fused to a Gs, Gi, Gz or Go protein are also effective. More preferred is a fusion construct obtained by deleting the first six amino acids of the G protein alpha subunit ("Gαq") of the Gq protein, and replacing the last five amino acids at the C-terminal end of Gαq with the corresponding amino acids of the desired G protein alpha subunit. For example, a fusion construct may be a fusion of Gq (deleted by 6 amino acids) and Gi proteins, resulting in a "Gq/Gi fusion construct". We believe this fusion construct facilitates the coupling of endogenous Gi-coupled receptors to their non-endogenous G protein-Gq, thereby enabling the production of second messengers (e.g., inositol triphosphate or diacylglycerol) rather than cAMP. detection.

4. Co-transfection of a GPCR coupled to a target Gi and a GPCR coupled to a signal enhancer Gs (cAMP-based assay)

Gi-coupled receptors are known to inhibit adenylyl cyclase, and thus reduce cAMP production, which makes it difficult to assess cAMP levels. Detecting a decrease in cAMP production as indicative of constitutive activation of receptors that couple predominantly to Gi upon activation can be achieved by coupling the Gi of the GPCR to a signaling enhancer such as the non-endogenous constitutive activation that predominantly couples to Gs upon activation. Co-transfection of receptors, such as the TSHR-A623I published below, was achieved. Apparently, constitutive activation of Gs-coupled receptors can be judged based on an increase in cAMP production. Constitutive activation of Gi-coupled receptors leads to decreased production of cAMP. Therefore, this method of co-transfection is intended to effectively exploit these "opposite" effects. For example, co-transfection of Gs-coupled non-endogenous constitutively activated receptors ("signal enhancers") and endogenous Gi-coupled receptors ("target receptors") provides essential cAMP signaling (i.e., although Gi-coupled receptors reduce cAMP levels, but this reducing effect is associated with a marked increase in cAMP caused by constitutively activated Gs-coupled signal enhancers). Thus, by co-transfection of a signal enhancer with a constitutively activated target receptor, the activity of Gi targets increases (ie, reduces cAMP levels), and cAMP levels are expected to decrease further (relative to basal levels).

Analysis of candidate compounds using cAMP-based assays will then be possible, subject to two constraints: first, an "opposite" effect can be produced relative to the Gi-coupled target receptor, i.e., the Gi-coupled target An inverse agonist of the receptor will enhance the measured cAMP signal, whereas an agonist of the Gi-coupled target receptor will decrease this signal; second, as will also be shown below, candidate compounds for direct identification using this approach should be individually to ensure that it does not target signal-enhancing receptors (this can be done before or after screening for co-transfected receptors).

F. Medicinal Chemistry

Typically, but not often, direct identification of candidate compounds is used in conjunction with compounds generated by combinatorial chemistry techniques, where thousands of compounds are randomly prepared for this analysis. The results of such screening will generally be compounds with unique central structures; these compounds are then preferably subjected to additional chemical modifications around a preferred central structure to further enhance their medicinal properties. Such techniques are known in the art and need not be described in detail in this patent document.

G. Pharmaceutical composition

Candidate compounds selected for further development can be formulated into pharmaceutical compositions using techniques well known in the art. Suitable pharmaceutically acceptable carriers are available in the art; see, for example, Remington's Pharmaceutical Sciences, 16th Ed., 1980, Mack Publishing Co. (Oslo et al. eds.).

H. Other applications

Although a preferred application of the disclosed non-endogenous human GPCRs is for the direct identification of candidate compounds as inverse agonists, agonists or partial agonists, preferably for use as drugs, these forms of human GPCRs can also be used in research for. For example, in vitro or in vivo systems with GPCRs can be used to elucidate and understand the role of these receptors in normal and diseased human conditions, as well as the role of constitutive activation when it is applied to understanding signaling cascades . The value of these non-endogenous human GPCRs is enhanced by their use as research tools due to their unique characteristics, and the disclosed receptors can be used to understand the role of these receptors in humans even when their endogenous ligands are blocked. before recognition. Other applications of the disclosed receptors will be apparent to those skilled in the art, particularly after reading this specification.

Example

The following examples are provided for the purpose of illustrating, not limiting, the invention. While specific nucleic acid and amino acid sequences are disclosed herein, one of ordinary skill in the art would be able to make minor modifications to these sequences and obtain the same or substantially similar results as reported below. The traditional way to use or understand one sequence frame after another (such as from mouse receptor to human receptor or from human receptor A to human receptor B) is generally based on sequence comparison techniques, by aligning the sequences Try to identify common areas. The mutagenesis method disclosed here does not rely on this approach, but is based on algorithmic rules and positional distances from conserved proline residues located in the TM6 region of the human GPCR. Once this approach is secure, it is believed that one of ordinary skill in the art can make minor modifications to obtain substantially the same results as disclosed herein (eg, constitutive activation). Such modifications are considered to be within the scope of the present disclosure.

Example 1

endogenous human GPCR

Identification of 1 human GPCR

On the basis of browsing the GenBank( ^TM) database information, several published endogenous human GPCRs were identified. While searching the database, the following cDNA clones were also identified and are listed below (Table C).

Form C Published Human Orphan GPCRs Accession Registration Number Full DNA sequence (base pairs) Open reading frame (base pair) Nucleic acid sequence SEQ.ID.NO.: Amino acid sequence SEQ.ID.NO. wxya AL121755 147,566bp 1,152bp 1 2 hRUP9 AC0113375 143,181bp 1,260bp 3 4 hRUP10 AC008745 94,194bp 1,014bp 5 6 hRUP11 AC013396 155,086bp 1,272bp 7 8 hRUP12 AP000808 177,764bp 966bp 9 10 hRUP13 AC011780 167,819 bp 1,356bp 11 12 hRUP14 AL137118 168,297bp 1,041bp 13 14 hRUP15 AL016468 138,828b 1,527bp 15 16 hRUP16 AL136106 208,042 bp 1,068bp 17 18 hRUP17 AC023078 161,735bp 969bp 19 20 hRUP18 AC008547 117,304bp 1,305 bp twenty one twenty two hRUP19 AC026331 145,183bp 1,041bp twenty three twenty four hRUP20 AL161458 163,511bp 1,011bp 25 26 hRUP21 AC026756 156,534bp 1,014bp 27 28 hRUP22 AC027026 151,811bp 993bp 29 30 hRUP23 AC007104 200,000bp 1,092bp 31 32 hRUP24 AL355388 190,538bp 1,125bp 33 34 hRUP25 AC026331 145,183 bp 1,092 bp 35 36 hRUP26 AC023040 178,508 bp 1,044bp 37 38 hRUP27 AC027643 158,700bp 1,020bp 39 40

2. Full-length cloning

a.hRUP8(Seq.Id.Nos.1&2)

Published human RUP8 was identified using EST database information (dbEST). A cDNA clone with accession number AL121755 was found to encode a novel GPCR when searching in dbEST. The following PCR primers were used for RT-PCR cloning using human testis Marathon-Ready cDNA (Clontech) as template:

5'-CTTGCAGACATCACCATGGCAGCC-3' (SEQ.ID.NO.: 41; Sense) and

5'-GTGATGCTCTGAGTACTGGACTGG-3' (SEQ.ID.NO.: 42; antisense)

Use Advantage cDNA polymerase (Clontech; operate according to the manufacturer's instructions) to carry out PCR in 50 μl reaction solution, the program is: 94°C for 30 seconds; 94°C for 10 seconds; 65°C for 20 seconds, 72°C for 1.5 minutes, 72°C for 7 minutes, The second step to the fourth step cycle 35 times.

A 1.2 kb PCR fragment was isolated, cloned into pCRII-TOPO (Invitrogen), and sequenced using the ABI Big Dye Terminator kit (P.E. Biosystem). See SEQ.ID.NO.:1, the possible amino acid sequence of RUP8 is presented in SEQ.ID.NO.:2.

b.hRUP9(Seq.Id.Nos.3&4)

Published human RUP9 was identified based on GenBank information. A cDNA clone with accession number AC011375 was found in the search database from the human genome sequence of chromosome 5. The full-length RUP9 was PCR cloned with the following primers:

5'-GAAGCTGTGAAGAGTGATGC-3' (SEQ.ID.NO.: 43; Sense) and

5'-GTCAGCAATATTGATAAGCAGCAG-3' (SEQ.ID.NO.: 44; antisense),

And human genomic DNA (Promega) was used as a template. Amplification was performed in 100 μl reactions containing 5% DMSO using Taq Plus Precision Polymerase (Stratagene). The program is: 94°C for 1 minute; 94°C for 30 seconds; 56°C for 30 seconds; 72°C for 2 minutes; 72°C for 5 minutes, cycle 35 times from the second step to the fourth step.

A 1.3 kb PCR fragment was isolated from a 1% agarose gel, cloned into pCRII-TOPO vector (Invitrogen), and completely sequenced using the ABI Big Dye Terminator kit (P.E. Biosystem). See SEQ.ID.NO.:3, the possible amino acid sequence of RUP8 is shown in SEQ.ID.NO.:4. The sequence of the RUP9 clone isolated from human genomic DNA was consistent with the sequence obtained from the database.

c.hRUP10(Seq.Id.Nos.5&6)

Published human RUP10 was identified based on GenBank information. A cDNA clone with accession number AC008754 was found in the search database as the human genome sequence from chromosome 19. The full-length RUP10 was PCR cloned with the following primers:

5'-CCATGGGGAACGATTCTGTCAGCTACG-3' (SEQ.ID.NO.: 45; Sense) and

5'-GCTATGCCTGAAGCCAGTCTTGTG-3' (SEQ.ID.NO.: 46; antisense),

And human leukocyte Marathon-Ready cDNA (Clontech) was used as template. PCR was performed in 50 μl reactions with Advantage cDNA polymerase (Clontech). The program is: 94°C for 30 seconds; 94°C for 10 seconds; 62°C for 20 seconds, 72°C for 1.5 minutes, 72°C for 7 minutes, cycle 35 times from the second step to the fourth step. A 1.0 kb PCR fragment was isolated, cloned into pCRII-TOPO vector (Invitrogen), and sequenced using the ABI Big Dye Terminator kit (P.E. Biosystem). The nucleic acid sequence of this novel human receptor RUP10 is set forth in SEQ.ID.NO.:5, and its possible amino acid sequence is shown in SEQ.ID.NO.:6.

d.hRUP11(Seq.Id.Nos.7&8)

Published human RUP11 was identified based on GenBank information. A cDNA clone with accession number AC013396 was found in the search database as the human genome sequence from chromosome 2. The full-length RUP11 clone was PCR cloned with the following primers:

5'-CCAGGATGTTGTGTCACCGTGGTGGC-3' (SEQ.ID.NO.: 47; sense) and

5'-CACAGCGCTGCAGCCCTGCAGCTGGC-3' (SEQ.ID.NO.: 48; antisense),

And human genomic DNA (Clontech) was used as a template. Amplification was performed in 50 μl reactions using TaqPlus Precision DNA polymerase (Stratagene). The program is: 94°C for 3 minutes; 94°C for 20 seconds; 67°C for 20 seconds; 72°C for 1.5 minutes; 72°C for 7 minutes, cycle 35 times from the second step to the fourth step. A 1.3 kb PCR fragment was isolated, cloned into pCRII-TOPO vector (Invitrogen), and sequenced using the ABI Big Dye Terminator kit (P.E. Biosystem). The nucleic acid sequence of this novel human receptor RUP11 is set forth in SEQ.ID.NO.:7, and its possible amino acid sequence is shown in SEQ.ID.NO.:8.

e.hRUP12(Seq.Id.Nos.9&10)

Published human RUP12 was identified based on GenBank information. A cDNA clone with the accession number AP000808 was found in the search database to encode a new GPCR with significant homology to the rat RTA and human mas1 oncogene GPCR. The full-length RUP12 was PCR cloned with the following primers:

5'-CTTCCTCTCGTAGGGATGAACCAGAC-3' (SEQ.ID.NO.: 49; Sense) and

5'-CTCGCACAGGTGGGAAGCACCTGTGG-3' (SEQ.ID.NO.: 50; antisense),

And human genome cDNA (Clontech) was used as template. Amplified using TaqPlus Precision DNA polymerase (Stratagene). The program is: 94°C for 3 minutes; 94°C for 20 seconds; 65°C for 20 seconds; 72°C for 2 minutes; 72°C for 7 minutes, cycle 35 times from the second step to the fourth step. A 1.0 kb PCR fragment was isolated, cloned into the pCRII-TOPO vector (Invitrogen), and completely sequenced with the ABI Big Dye Terminator kit (P.E. Biosystem) (its nucleotide sequence is illustrated in SEQ.ID.NO.: 9 , its possible amino acid sequence is shown in SEQ.ID.NO.: 10).

f.hRUP13(Seq.Id.Nos.11&12)

Published human RUP13 was identified based on GenBank information. A cDNA clone with accession number AC011780 was found in the search database to encode a new GPCR with significant homology to the GPCR fish GPRX-ORYLA. The full-length RUP13 was PCR cloned with the following primers:

5'-GCCTGTGACAGGAGGTACCCTGG-3' (SEQ.ID.NO.: 51; sense) and

5'-CATATCCCTCCGAGTGTCCAGCGGC-3' (SEQ.ID.NO.: 52; antisense),

And human genome cDNA (Clontech) was used as template. Amplified using TaqPlus Precision DNA polymerase (Stratagene). The program is: 94°C for 3 minutes; 94°C for 20 seconds; 65°C for 20 seconds; 72°C for 2 minutes; 72°C for 7 minutes, cycle 35 times from the second step to the fourth step. A PCR fragment of 1.35kb was isolated, cloned into the pCRII-TOPO vector (Invitrogen), and completely sequenced with the ABI Big Dye Terminator kit (P.E.Biosystem) (see SEQ.ID.NO. for its nucleotide sequence: 11, possible For the amino acid sequence, see SEQ.ID.NO.: 12).

g.hRUP14(Seq.Id.Nos.13&14)

Published human RUP14 was identified based on GenBank information. A cDNA clone with accession number AL137118 was found in the search database as the human genome sequence from chromosome 13. The full-length RUP14 was PCR cloned with the following primers:

5'-GCATGGAGAGAAAATTTATGTCCTTGCAACC-3' (SEQ.ID.NO.: 53; sense) and

5'-CAAGAACAGGTCTCATCTAAGAGCTCC-3' (SEQ.ID.NO.: 54; antisense),

And human genomic DNA (Promega) was used as a template. Amplified using TaqPlus Precision polymerase (Stratagene) and 5% DMSO. The program is: 94°C for 3 minutes; 94°C for 20 seconds; 58°C for 2 minutes; 72°C for 10 minutes, cycle 35 times from the second step to the third step.

A 1.1 kb PCR fragment was isolated, cloned into pCRII-TOPO vector (Invitrogen), and sequenced using the ABI Big Dye Terminator kit (P.E. Biosystem). (The nucleotide sequence is shown in SEQ.ID.NO.: 13, and the possible amino acid sequence is shown in SEQ.ID.NO.: 14.) The sequence of the RUP14 clone isolated from human genomic DNA is consistent with the sequence obtained from the database .

h.hRUP15(Seq.Id.Nos.15&16)

Published human RUP15 was identified based on GenBank information. A cDNA clone with accession number AC016468 was found to be human genome sequence in the search database. The full-length RUP15 was PCR cloned with the following primers:

5'-GCTGTTGCCATGACGTCCACCTGCAC-3' (SEQ.ID.NO.: 55; sense) and

5'-GGACAGTTCAAGGTTTGCCTTAGAAC-3' (SEQ.ID.NO.: 56; antisense),

And human genomic DNA (Promega) was used as a template. Amplified using TaqPlus Precision Polymerase (Stratagene). The program is: 94°C for 3 minutes; 94°C for 20 seconds; 65°C for 20 seconds; 72°C for 2 minutes; 72°C for 7 minutes, cycle 35 times from the second step to the fourth step.

A 1.5 kb PCR fragment was isolated, cloned into pCRII-TOPO vector (Invitrogen), and fully sequenced using the ABI Big Dye Terminator kit (P.E. Biosystem). (The nucleotide sequence is shown in SEQ.ID.NO.: 15, and the possible amino acid sequence is shown in SEQ.ID.NO.: 16.) The sequence of the RUP15 clone isolated from human genomic DNA is consistent with the sequence obtained from the database .

i.hRUP16(Seq.Id.Nos.17&18)

Published human RUP16 was identified based on GenBank information. A cDNA clone with the accession number AL136106 was found in the search database as the human genome sequence from chromosome 13. The full-length RUP16 was PCR cloned with the following primers:

5'-CTTTCGATACTGCTCCTATGCTC-3' (SEQ.ID.NO.: 57; sense, 5' end of the start codon) and

5'-GTAGTCCACTGAAAGTCCAGTGATCC-3' (SEQ.ID.NO.: 58; antisense, 3' end of the stop codon),

And human skeletal muscle Marathon-Ready cDNA (Clontech) as a template. PCR was performed in 50 μl reactions using the Advantage cDNA Polymerase Kit (Clontech). The program is: 94°C for 30 seconds; 94°C for 5 seconds; 69°C for 15 seconds; 72°C for 1 minute; 72°C for 5 minutes, cycle 35 times from the second step to the fourth step.

A 1.1 kb PCR fragment was isolated, cloned into pCRII-TOPO vector (Invitrogen), and completely sequenced using the T7 Sequenase kit (Amsham). (See SEQ.ID.NO.: 17 for its nucleotide sequence, and SEQ.ID.NO.: 18 for its possible amino acid sequence). The sequence of the RUP16 clone was consistent with the four non-sequential segments of AL136106, indicating that the cDNA of RUP16 contained 4 exons.

j.hRUP17(Seq.Id.Nos.19&20)

Published human RUP17 was identified based on GenBank information. A cDNA clone with accession number AC023078 was found in the search database as the human genome sequence from chromosome 11. The full-length RUP17 was PCR cloned with the following primers:

5'-TTTCTGAGC ATG GATCCAACCATCTC-3' (SEQ.ID.NO.: 59; sense, including start codon) and

5'-CTGTCTGACAGGGCAGAGGCTCTTC-3' (SEQ.ID.NO.: 60; antisense strand, 3' end of the stop codon),

And human genomic DNA (Promega) was used as a template. PCR (Clontech) was performed in 100 μl reactions with Advantage cDNA polymerase mix and 5% DMSO. The program is: 94°C for 1 minute; 94°C for 15 seconds; 67°C for 20 seconds; 72°C for 1 minute and 30 seconds; 72°C for 5 minutes, cycle 30 times from the second step to the fourth step.

A 970bp PCR fragment was isolated from 1% agarose gel, cloned into pCRII-TOPO vector (Invitrogen), and sequenced using ABI Big Dye Termiantor kit (P.E.Biosystem). (See SEQ.ID.NO.: 19 for its nucleotide sequence, and SEQ.ID.NO.: 20 for its possible amino acid sequence).

k.hRUP18(Seq.Id.Nos.21&22)

Published human RUP18 was identified based on GenBank information. A cDNA clone with accession number AC008547 was found in the search database as the human genome sequence from chromosome 5. The full-length RUP18 was PCR cloned with the following primers:

5'-GGAACTCGTATAGACCCAGCGTCGCTCC-3' (SEQ.ID.NO.: 61; sense, 5' end of the start codon) and

5'-GGAGGTTGCGCCTTAGCGACAGATGACC-3' (SEQ.ID.NO.: 62; antisense, 3' end of the stop codon),

And human genomic DNA (Promega) was used as a template. PCR (Clontech) was performed with TaqPlus Precision DNA Polymerase (Stratagene) and 5% DMSO in 100 [mu]l reactions. The program is: 5 minutes at 95°C; 30 seconds at 95°C; 30 seconds at 65°C; 2 minutes at 72°C; 5 minutes at 72°C, 35 cycles from the second step to the fourth step.

A 1.3 kb PCR fragment was isolated from 1% agarose gel, cloned into pCRII-TOPO vector (Invitrogen), and sequenced using ABI Big Dye Termiantor kit (P.E. Biosystem). (See SEQ.ID.NO.: 21 for its nucleotide sequence, and SEQ.ID.NO.: 22 for its possible amino acid sequence).

l.hRUP19(Seq.Id.Nos.23&24)

Published human RUP19 was identified based on GenBank information. A cDNA clone with accession number AC026331 was found in the search database as the human genome sequence from chromosome 12. The full-length RUP19 was PCR cloned with the following primers:

5'-CTGCACCCGGACACTTGCTCTG-3' (SEQ.ID.NO.: 63; sense, 5' end of the start codon) and

5'- GTCTGCTTGTTCA GTGCCACTCAAC-3' (SEQ.ID.NO.: 64; antisense, containing stop codon),

And human genomic DNA (Promega) was used as a template. Amplification was performed in a 100 μl reaction with TaqPlus Precision DNA polymerase (Stratagene) and 5% DMSO. The program is: 94°C for 1 minute; 94°C for 15 seconds; 70°C for 20 seconds; 72°C for 1 minute and 30 seconds; 72°C for 5 minutes, cycle 35 times from the second step to the fourth step.

A 1.1 kb PCR fragment was isolated from a 1% agarose gel, cloned into pCRII-TOPO vector (Invitrogen), and completely sequenced using the ABI Big Dye Termiantor kit (P.E. Biosystem). Its nucleotide sequence is shown in SEQ.ID.NO.: 23, and its possible amino acid sequence is shown in SEQ.ID.NO.: 24).

m.hRUP20(Seq.Id.Nos.25&26)

Published human RUP20 was identified based on GenBank information. A cDNA clone with accession number AL161458 was found in the search database as the human genome sequence from chromosome 1. The full-length RUP20 was PCRed with the following primers:

5'-TATCTGCAATTCTATTCTAGCTCCTG-3' (SEQ.ID.NO.: 65; sense, start codon 5' end) and

5'-TGTCCCTAATAAAGTCACATGAATGC-3' (SEQ.ID.NO.: 66; antisense, stop codon 3' end),

And human genomic DNA (Promega) was used as a template. Amplification was performed with Advantage cDNA Polymerase Mix (Clonetech) with 5% DMSO. The program is: 94°C for 1 minute; 94°C for 15 seconds; 60°C for 20 seconds; 72°C for 1 minute and 30 seconds; 72°C for 5 minutes, cycle 35 times from the second step to the fourth step.

A 1.0 kb PCR fragment was isolated from 1% agarose gel, cloned into pCRII-TOPO vector (Invitrogen), and sequenced using ABI Big Dye Termiantor kit (P.E. Biosystem). (See SEQ.ID.NO.: 25 for its nucleotide sequence, and SEQ.ID.NO.: 26 for its possible amino acid sequence).

n.hRUP21(Seq.Id.Nos.27&28)

Published human RUP21 was identified based on GenBank information. A cDNA clone with accession number AC026756 was found in the search database as the human genome sequence from chromosome 13. The full-length RUP21 was PCR cloned with the following primers:

5'-GGAGACAACCATGAATGAGCCAC-3' (SEQ.ID.NO.: 67; Sense) and

5'-TATTTCAAGGGTTGTTTGAGTAAC-3' (SEQ.ID.NO.: 68; antisense strand),

And human genomic DNA (Promega) was used as a template. Amplification was performed in 100 μl reactions with TaqPlus Precision Polymerase (Stratagene) and 5% DMSO. The program is: 94°C for 1 minute; 94°C for 15 seconds; 55°C for 20 seconds; 72°C for 1 minute and 30 seconds; 72°C for 5 minutes, cycle 30 times from the second step to the fourth step.

A 1014bp PCR fragment was isolated from 1% agarose gel, cloned into pCRII-TOPO vector (Invitrogen), and sequenced using ABI Big Dye Termiantor kit (P.E.Biosystem). (See SEQ.ID.NO.: 27 for its nucleotide sequence, and SEQ.ID.NO.: 28 for its possible amino acid sequence).

o.hRUP 22(Seq.Id.Nos.29&30)

Published human RUP22 was identified based on GenBank information. A cDNA clone with accession number AC027026 was found in the search database as the human genome sequence from chromosome 11. The full-length RUP22 was PCR cloned with the following primers:

5'-GGCACCAGTGGAGGTTTTCTGAGC ATG -3' (SEQ.ID.NO.: 69; sense, including start codon) and

5'-CTGATGGAAGTAGAGGCTGTCCATCTC-3' (SEQ.ID.NO.: 70; antisense, stop codon 3' end),

And human genomic DNA (Promega) was used as a template. Amplification was performed in a 100 μl reaction with TaqPlus Precision DNA polymerase (Stratagene) and 5% DMSO. The program is: 94°C for 1 minute; 94°C for 15 seconds; 55°C for 20 seconds; 72°C for 1 minute and 30 seconds; 72°C for 5 minutes, cycle 30 times from the second step to the fourth step.

A 970bp PCR fragment was isolated from 1% agarose gel, cloned into pCRII-TOPO vector (Invitrogen), and completely sequenced using ABI Big Dye Termiantor kit (P.E. Biosystem). (See SEQ.ID.NO.29 for its nucleotide sequence, and SEQ.ID.NO.30 for its possible amino acid sequence).

p.hRUP 23(Seq.Id.Nos.31&32)

Published human RUP23 was identified based on GenBank information. A cDNA clone with accession number AC007104 was found in the search database as the human genome sequence from chromosome 4. The full-length RUP23 was PCRed with the following primers:

5'-CCTGGCGAGCCGCTAGCGCC ATG -3'(SEQ.ID.NO.71; sense, ATG is the initiation codon) and

5'-ATGAGCCCTGCCAGGCCC TCA GT-3'(SEQ.ID.NO.72; antisense, TCA is a stop codon),

And human placenta Marathon-Ready cDNA (Clontech) as a template. Amplification was performed in 50 μl reactions with Advantage cDNA polymerase (Clontech). The program is: 95°C for 30 seconds; 95°C for 15 seconds; 66°C for 20 seconds; 72°C for 1 minute and 20 seconds; 72°C for 5 minutes, cycle 35 times from the second step to the fourth step.

A 1.0 kb PCR fragment was isolated from 1% agarose gel, cloned into pCRII-TOPO vector (Invitrogen), and completely sequenced using the ABI Big Dye Terminator kit (P.E. Biosystem). (See SEQ.ID.NO.31 for its nucleotide sequence, and SEQ.ID.NO.32 for its possible amino acid sequence).

q.HRUP24(Seq.Id.Nos.33&34)

Published human RUP25 was identified based on GenBank information. A cDNA clone with accession number AC026331 was found in the search database as the human genome sequence from chromosome 12. The full-length RUP25 was PCR cloned with the following primers:

5'-GCTGGAGCATTCACTAGGCGAG-3' (SEQ.ID.NO.73; sense, start codon 5' end) and

5'-AGATCCTGGTTCTTGGTGACAATG-3' (SEQ.ID.NO.74; antisense, stop codon 3' end),

And human genomic DNA (Promega) was used as a template. PCR was performed in 100 [mu]l reactions with Advantage cDNA polymerase mix (Clontech) and 5% DMSO. The program is: 94°C for 1 minute; 94°C for 15 seconds; 56°C for 20 seconds; 72°C for 1 minute and 30 seconds; 72°C for 5 minutes, cycle 35 times from the second step to the fourth step.

A 1.2 kb PCR fragment was isolated from a 1% agarose gel, cloned into pCRII-TOPO vector (Invitrogen), and completely sequenced using the ABI Big Dye Terminator kit (P.E. Biosystem). (See SEQ.ID.NO.33 for its nucleotide sequence, and SEQ.ID.NO.34 for its possible amino acid sequence).

r.hRUP 25(Seq.Id.Nos.35&36)

Published human RUP25 was identified based on GenBank information. A cDNA clone with accession number AC026331 was found in the search database as the human genome sequence from chromosome 12. The full-length RUP25 was PCR cloned with the following primers:

5'-GCTGGAGCATTCACTAGGCGAG-3' (SEQ.ID.NO.75; sense, start codon 5' end) and

5'-AGATCCTGGTTCTTGGTGACAATG-3' (SEQ.ID.NO.76; antisense, stop codon 3' end),

And human genomic DNA (Promega) was used as a template. Amplify with Advantage cDNA Polymerase Mixture (Clontech) and 5% DMSO. The program is: 94°C for 1 minute; 94°C for 15 seconds; 56°C for 20 seconds; 72°C for 1 minute and 30 seconds; 72°C for 5 minutes. Go to the fourth step and cycle 35 times.

A 1.2 kb PCR fragment was isolated from a 1% agarose gel, cloned into pCRII-TOPO vector (Invitrogen), and completely sequenced using the ABI Big Dye Terminator kit (P.E. Biosystem). (See SEQ.ID.NO.35 for its nucleotide sequence, and SEQ.ID.NO.36 for its possible amino acid sequence).

s.hRUP 26 (Seq.Id.Nos.37&38)

Published human RUP26 was identified based on GenBank information. A cDNA clone with accession number AC023040 was found in the search database as the human genome sequence from chromosome 2. The full-length RUP26 was cloned by RT-PCR with RUP26-specific primers:

5'-AGCCATCCCTGCCAGGAAGC ATG G-3', (SEQ.ID.NO.77; sense, including start codon) and

5'-CCAGACTGTGGACTCAAGAACT CTA GG-3 (SEQ.ID.NO.78; antisense, containing stop codon),

And human pancreas Marathon-Ready cDNA (Clontech) was used as template. PCR (Clontech) was carried out in 100 [mu]l reactions with Advantage cDNA polymerase mix (Clontech) and 5% DMSO. The program is: 94°C for 5 minutes; 95°C for 30 seconds; 65°C for 30 seconds; 72°C for 2 minutes; 72°C for 5 minutes, cycle 35 times from the second step to the fourth step.

A 1.1 kb PCR fragment was isolated from a 1% agarose gel, cloned into pCRII-TOPO vector (Invitrogen), and completely sequenced using the ABI Big Dye Termiantor kit (P.E. Biosystem). (See SEQ.ID.NO.37 for its nucleotide sequence, and SEQ.ID.NO.38 for its possible amino acid sequence).

t.hRUP27(Seq.Id.Nos.39&40)

Published human RUP27 was identified based on GenBank information. A cDNA clone with accession number AC027643 was found in the search database as the human genome sequence from chromosome 12. The full-length RUP27 was PCR cloned with RUP27-specific primers:

5'-AGTCCACGAACA ATG AATCCATTTCATG-3 (SEQ.ID.NO.79; sense, including start codon) and

5'-ATCATGTCTAGACTCATGGTGATCC-3' (SEQ.ID.NO.80; antisense, stop codon 3' end),

And adult brain Marathon-Ready cDNA (Clontech) was used as template. PCR was performed in 50 μl reactions with Advantage cDNA polymerase mix (Clontech) and 5% DMSO. The program is: 94°C for 1 minute; 94°C for 10 seconds; 58°C for 20 seconds; 72°C for 1 minute and 30 seconds; 72°C for 5 minutes, cycle 35 times from the second step to the fourth step.

A 1.1 kb PCR fragment was isolated from a 1% agarose gel, cloned into pCRII-TOPO vector (Invitrogen), and completely sequenced using the ABI Big Dye Termiantor kit (P.E. Biosystem). (See SEQ.ID.NO.35 for its nucleotide sequence, and SEQ.ID.NO.36 for its possible amino acid sequence). The cDNA clone sequence of RUP27 isolated from human brain was determined to be consistent with five non-sequential segments of AC027643, indicating that the cDNA of RUP27 contains 5 exons.

Example 2

Preparation of non-endogenous constitutively activated GPCRs

It is believed that one of ordinary skill in the art is capable of selecting a technique for nucleic acid sequence mutation, and provided below are methods for making several non-endogenous forms of human GPCRs disclosed above. The mutations disclosed below are based on an algorithmic rule approach whereby amino acid position 16 (located in the IC3 region of the GPCR) is replaced by a conserved proline (or its endogenous conservative substitute) residue (located in the TM6 region of the GPCR, close to TM6/ IC3 junction) is mutated, more preferably to an alanine, histidine, arginine or lysine residue, most preferably to a lysine residue.

1. Transformer Sited-Directed ^TM mutagenesis

Production of non-endogenous human GPCRs from human GPCRs can be achieved using the Transformer Sited-Directed ^™ mutagenesis kit following the manufacturer's instructions. Two mutagenesis primers are used, first preferably a lysine mutagenesis oligonucleotide that produces lysine mutations, and a selectable marker oligonucleotide. For convenience, codon mutations incorporated into human GPCRs are indicated in a standard manner (Table D):

Form D Receptor name codon mutation wxya V274K hRUP9 T249K hRUP10 R232K hRUP11 M294K hRUP12 F220K hRUP16 A238K hRUP17 Y215K hRUP18 L294K hRUP19 T219K hRUP20 K248AK248HK248R hRUP21 R240K hRUP22 Y222K hRUP24 A245K hRUP25 I230K hRUP26 V285K hRUP27 T248K

2. QuikChange ^™ Site-Directed ^™ mutagenesis

Production of non-endogenous human GPCRs can be achieved using the QuikChange ^™ Sited-Directed ^™ Mutagenesis Kit (Stratagene, according to the manufacturer's instructions). It is preferred to use an endogenous GPCR as a template and use two mutagenic primers, also preferred is a lysine mutagenic oligonucleotide and a selectable marker oligonucleotide (included in the kit). For convenience, the codon mutations and individual oligonucleotides incorporated into the novel human GPCRs are indicated in a standard manner (Table E):

Form E Receptor name codon mutation 5'-3' direction (sense), (SEQ.ID.NO.) mutant sequence underlined 5'-3' orientation (antisense) (SEQ.ID.NO.) Loop condition, (minutes'seconds") loops 16 times from 2-4 steps hRUP13 A268K GGGGAGGGAAAGCA AA G GTGGTCCTCCTGG (81) CCAGGAGAACCAC CTT TGCTTTTCCCTCCCC (82) 98°2'98°30"56°C30"72°11'40"72°5' hRUP14 L246K CAGGAAGGCA AAG ACCACCATCATCATC (85) GATGATGATGGTGGT C TT TGCCTTCCTG(86) 98°2'98°30"55°C30"72°11'40"72°5' hRUP15 A398K CCAGTGCAAAGCT AAG AAAGTGATCTTC (89) GAAGATCACTTT CTT AGCTTTGCACTGG(90) 98°2'98°30"55°C30"72°11'40"72°5' hRUP23 W275K GCCGCCACCGCGCC AAG AGGAAGATTGGC(93) GCCAATCTTCCT CTT -GCGCGGTGGCGGC(94) 98°2'98°30"56°C30"72°11'40"72°5'

Then, the non-endogenous human GPCR was sequenced, and the obtained and verified nucleic acid and amino acid sequences were included in the "Sequence Listing" attached to this patent application document, and summarized in Table F below.

Form F non-endogenous human GPCR Nucleic Acid Sequence Listing Amino Acid Sequence List hRUP13 SEQ. ID. NO.: 83 SEQ. ID. NO.: 84 hRUP14 SEQ. ID. NO.: 87 SEQ. ID. NO.: 88 hRUP15 SEQ. ID. NO.: 91 SEQ. ID. NO.: 92 hRUP23 SEQ. ID. NO.: 95 SEQ. ID. NO.: 96

Example 3

receptor expression

Although a variety of cells are available in the art for protein expression, mammalian cells are most preferably employed. It is predicted that the basic reason for this is practicality, i.e. the use of, for example, yeast cells expressing GPCRs, it is possible to introduce into the program a non-mammalian cell which may not (actually, in the case of yeast, not) include Coupled receptors, genetic mechanisms, and secretory pathways that have evolved for use in mammalian systems. Thus, results obtained in non-mammalian cells, while potentially useful, are not as favorable as those obtained from mammalian cells. Among mammalian cells, COS-7, 293 and 293T cells are particularly preferred, although the particular mammalian cells used will be determined by the particular needs of the skilled artisan.

a. Transient transfection

On the first day, 6 × ¹⁰⁶ 293 cells were seeded on a 10 cm culture plate. The next day, prepare two test tubes (the ratio is one tube per plate): by mixing 4 μg DNA (e.g. pCMV vector, pCMV vector with receptor cDNA, etc.) in 0.5 ml serum-free DMEM (Gibco BRL) Tube A was prepared in DMEM; tube B was prepared by mixing 24 μl lipofectamine (Gibco BRL) in 0.5 ml serum-free DMEM. Tubes A and B were mixed by inversion (several times), then incubated at room temperature for 30-45 minutes. The composition is referred to as "transfection composition". The explanted 293T cells were washed with 1XPBS, and then 5ml of serum-free DMEM was added. 1 ml of the transfection composition was added to the cells and incubated for 4 hours at 37°C/5% CO ₂ . The transfection composition was then removed by aspiration, followed by the addition of 10 ml of DMEM/10% fetal calf serum. Cells were then incubated at 37°C/5% _CO2 . Cells were harvested after 48 hours and used for analysis.

b. Stable cell line: Gs fusion protein

About 12 × ¹⁰⁶ 293 cells were seeded on a 15 cm tissue culture plate. Grow in DME high glucose medium containing 10% fetal bovine serum and 1% sodium pyruvate, L-glutamine, antibiotics. The 293 cells reached -80% confluency 24 hours after plating and the cells were transfected with 12 μg of DNA. The 12 μg DNA was mixed with 60 μl lipofectamine and 2 ml serum-free DME high glucose medium. Aspirate the medium from the culture plate and rinse the cells once with serum-free medium. A mixture of DNA, lipofectamine and medium was plated along with 10 ml of serum-free medium. After culturing at 37°C for 4-5 hours, the culture medium was aspirated, and 25ml of serum-containing medium was added. 24 hours after transfection, the medium was aspirated again and fresh medium containing serum was added. After 48 hours of transfection, the medium was sucked off, and the medium containing geneticin (G418 drug, final concentration 500 μg/ml) was added. Positively transfected cells containing the G418 resistance gene are selected at this point. Medium was replaced every 4 to 5 hours during selection. During selection, cells grow and produce a stable population of cells, or cells divide for stable clonal selection.

Example 4

Assays to determine the constitutive activity of non-endogenous GPCRs

A variety of methods are available for assessing the constitutive activity of non-endogenous human GPCRs, the following examples are illustrative; those of ordinary skill in the art are well within the ability to determine those techniques that are most beneficial to their needs.

1. Cell membrane binding analysis: [ ³⁵ S]GTPγS analysis

When a G protein-coupled receptor is in its active state, either as a result of ligand binding or as a result of constitutive activation, the receptor couples to a G protein and stimulates the release of GDP and the subsequent binding of GTP to the G protein. The alpha subunit of the G protein-receptor complex acts as a GTPase and slowly hydrolyzes GTP to GDP, at which point the receptor is usually inactivated. Constitutively activated receptors continue to convert GDP to GTP. The non-hydrolyzable GTP analog, [ ³⁵ S]GTPγS, can be used to demonstrate enhanced binding of [ ³⁵ S]GTPγS to membranes expressing constitutively activated receptors. The advantages of constitutive activation assays using [ ³⁵ S]GTPγS binding are that (a) it is universally applicable to all G protein-coupled receptors; Molecules that affect cascades of reactions in cells.

This assay utilizes the ability of G protein-coupled receptors to stimulate [ ^35S ]GTPyS binding to cell membranes expressing the relevant receptors. This assay can therefore be used in a direct recognition approach to screen candidate compounds against known, orphan, and constitutively active G protein-coupled receptors. This assay is universal and can be used for drug discovery against all G protein-coupled receptors.

[ ³⁵ S]GTPγS assay: in 20 mM HEPES, 1 to approximately 20 mM MgCl ₂ (although 20 mM is preferred, this dose can be adjusted for optimization of results), pH 7.4, containing between 0.3 and 1.2 nM [ ³⁵ S]GTPγS (although 1.2 is preferred, this dose can be adjusted for optimal results), 12.5 to 75 μg of membrane protein (e.g., 293 cells expressing Gs fusion protein; this dose can be adjusted for optimal ) and 10 μM GDP (this dose can be tailored for optimal results) in binding buffer for one hour. Wheat germ agglutinin beads (25 [mu]l, Amersham) were then added and the composition was incubated for an additional 30 minutes at room temperature before the tubes were centrifuged at 1500 xg for 5 minutes at room temperature and counted in a scintillation counter.

2. Adenylate cyclase

The Flash Plate ^™ Adenylyl Cyclase Kit (New England Nuclear; Cat# SMP004A), designed for cell-based assays, was modified for application to unprocessed plasma membranes. The wells of the scintillation plate contain a scintillator coating containing specific antibodies that recognize cAMP. cAMP produced in the wells can be quantified by direct competition with the radioactive cAMP tracer for binding to the cAMP antibody. Below is a short description of the procedure for measuring changes in cAMP levels in whole cells expressing the receptor.

Transfected cells were harvested approximately 24 hours after transient transfection. Carefully aspirate the medium and discard. Gently add 10ml of PBS to each cell culture dish and aspirate carefully. Add 1ml Sigma Cell Dissociation Buffer and 3ml PBS per plate. The cells were aspirated from the plate, and the harvested cell suspension was added to a 50 ml conical centrifuge tube, and then centrifuged at 1100 rpm for 5 minutes at room temperature. Carefully resuspend the cell pellet with an appropriate amount of PBS (approximately 3 ml per plate). Cells were counted with a hemocytometer and adjusted to the appropriate cell number with additional PBS (final volume approximately 50 μl per well).

cAMP standards and assay buffer (11 ml assay buffer containing 1 μCi of tracer [ ¹²⁵ I cAMP (50 μl)]) were prepared and maintained according to the manufacturer's instructions. Assay buffer for screening was freshly prepared and contained 50 μl of stimulation buffer, 3 μl of test compound (12 mM final concentration) and 50 μl of cells; assay buffer could be stored on ice for future use. First 50 μl of cAMP standard was added to the appropriate wells followed by 50 μl of PBSA to wells H-11 and H-12. Add 50 μl of stimulation buffer to all wells, add DMSO (or selected candidate compounds) to the appropriate wells using a needle tool that can disperse 3 μl of compound drop, the final concentration of the test compound is 12 mM, the total volume of the experiment for 100 μl. Cells were then added to the wells and incubated at room temperature for 60 minutes before adding 100 [mu]l of detection mix containing traced cAMP. Plates were then incubated for an additional 2 hours before counting using a Wallac MicroBeta liquid scintillation counter. Values for cAMP/well were extrapolated from standard cAMP curves included with each assay plate.

3. Cell-based cAMP analysis of Gi-coupled target GPCRs

TSHR is a Gs-coupled GPCR that causes the accumulation of cAMP when activated. Mutation of amino acid residue 623 (ie, changing an alanine residue to an isoleucine residue) results in constitutive activation of TSHR. Gi-coupled receptors are expected to inhibit adenylate cyclase, and thus reduce cAMP levels, making assessment of cAMP levels difficult. An efficient means of detecting a reduction in cAMP production as indicative of constitutively activated Gi-coupled receptors is by co-transfection, more preferably by non-endogenous, constitutively activated TSHR (TSHR-A623I) (or an endogenous constitutively activated Gs-coupled receptor) as a "signal enhancer", co-transfected with a Gi-coupled target GPCR to establish baseline levels of cAMP. After generating non-endogenous Gi-coupled receptors, non-endogenous target GPCRs were co-transfected with signal enhancers and used as screening material. We will utilize this method to efficiently generate signals when assayed using cAMP; this method is preferably used for the direct identification of candidate compounds against Gi-coupled receptors. Note that for Gi-coupled GPCRs, an inverse agonist of the target GPCR will increase cAMP signal and an agonist will decrease cAMP signal when using this method.

On the first day, 293 and 293 cells were plated at a density of 2 x ¹⁰⁴ cells per well. The next day, prepare two tubes (ratio is one tube per plate): 2 μg of each recipient DNA (e.g. pCMV vector; pCMV vector with mutant THSR (TSHR-A623I); TSHR-A623I) by mixing a total of 4 μg DNA A623 and GPCR, etc.) were prepared in 1.2 ml serum-free DMEM (Irvine Scientific, Irvine, CA); tube A was prepared by mixing 120 μll lipofectamine (Gibco BRL) in 1.2 ml serum-free DMEM. Tubes A and B were mixed by inversion (several times), then incubated at room temperature for 30-45 minutes. The composition is referred to as "transfection composition". The explanted 293 cells were washed with 1XPBS, and then 10ml of serum-free DMEM was added. 2.4 ml of the transfection composition was added to the cells and incubated for 4 hours at 37°C/5% CO ₂ . The transfection composition was then removed by aspiration, followed by the addition of 25 ml of DMEM/10% fetal bovine serum. Cells were then incubated at 37°C/5% _CO2 . Cells were harvested after 24 hours and used for analysis.

The Flash Plate ^(TM) Adenylyl Cyclase Kit (New England Nuclear; Cat. No. SMP004A), designed for cell-based assays, can be modified for use with unprocessed plasma membranes as desired by those skilled in the art. The wells of the scintillation plate contain a scintillator coating containing specific antibodies that recognize cAMP. The cAMP produced in the wells is quantified by direct competition with the radioactive cAMP tracer for binding to the cAMP antibody. Below is a brief description of the procedure for measuring changes in cAMP levels in whole cells expressing the receptor.

Transfected cells were harvested approximately 24 hours after transient transfection. Carefully aspirate the medium and discard. Gently add 10ml PBS to each cell culture dish. Add 1ml Sigma Cell Dissociation Buffer and 3ml PBS per plate. The cells were aspirated from the plate, and the harvested cell suspension was added to a 50 ml conical centrifuge tube, and then centrifuged at 1100 rpm for 5 minutes at room temperature. Carefully resuspend the cell pellet with an appropriate amount of PBS (approximately 3 ml per plate). Count with a hemocytometer and adjust to the appropriate cell number with additional PBS (final volume approximately 50 μl per well).

cAMP standards and assay buffer (11 ml assay buffer containing 1 μCi of tracer [ ¹²⁵ I cAMP (50 μl)]) were prepared and maintained according to the manufacturer's instructions. Assay buffer for screening was freshly prepared and contained 50 μl of stimulation buffer, 3 μl of test compound (12 mM final concentration) and 50 μl of cells; assay buffer could be stored on ice for future use. First 50 μl of cAMP standard was added to the appropriate wells followed by 50 μl of PBSA to wells H-11 and H-12. Add 50 μl of stimulation buffer to all wells, use a needle that can disperse 3 μl of the mixture, add the selected compound (such as TSH) into the appropriate well, the final concentration of the test compound is 12 μM, and the total volume of the experiment is 100 μl. Cells were then added to the wells and incubated at room temperature for 60 minutes before adding 100 [mu]l of detection mix containing tracer cAMP. Plates were then incubated for an additional 2 hours before counting using a Wallac MicroBeta liquid scintillation counter. Values for cAMP/well were extrapolated from standard cAMP curves included with each assay plate.

4. Reporter Gene-Based Assays

a. CRE-Luc reporter gene assay (Gs-related receptor)

293 and 293T cells were planted on a 96-well plate at a density of 2×10 ⁴ cells per well, and transfected with Lipofectamine reagent (BRL) according to the manufacturer’s instructions on the next day, and the DNA/lipid mixture was prepared for each 6-well transfection as follows : 260ng plasmid DNA in 100μl DMEM gently mixed with 2μl lipid in 100μl DMEM (260ng plasmid DNA consisted of, 200ng 8×CRE-Luc reporter plasmid, 50ng pCMV containing endogenous receptors or non-endogenous receptors, Or pCMV alone, and 10 ng of GPRS expression plasmid (GPRS in pcDNA3 (Invitrogen))). The 8×CRE-Luc reporter plasmid was prepared as follows: The promoter of mouse somatostatin (-71/+51) was cloned at the BglV-HindIII site of the pβgal-basic vector (Clontech) to obtain the SRIF-β-gal vector , 8 copies of the cAMP response element were obtained by PCR from the adenovirus template AdpCF126CCRE8 (referring to, 7 Human Gene Therapy (Human GeneTherapy) 1883 (1996)), and it was cloned into the Kpn-BglV site of the SRIF-β-gal vector, To generate an 8×CRE-β-gal reporter vector, replace the β-galactosidase gene in the 8×CRE-β-gal reporter vector with the luciferase gene to generate an 8×CRE-Luc reporter plasmid, the luciferase The gene was taken from the HindIII-BamHI site of the pGL3-basic vector (Promega). After standing at room temperature for 30 minutes, the DNA/lipid mixture was diluted with 400 μl DMEM, 100 μl of the diluted mixture was added to each well, and 100 μl of DMEM containing 10% FCS was added to each well after 4 hours of incubation in the cell culture incubator. On the second day, each well of the transfected cells was replaced with 200 μl of DMEM containing 10% FCS, and after 8 hours, washed once with PBS, and each sample well was changed into 100 μl of DMEM without phenol red. The next day, the luciferase activity was measured with the LucLite ^™ Reporter Gene Assay Kit (Packard) according to the manufacturer's instructions and read on a 1450 MicroBeta ^™ scintillation luminescence counter (Wallac).

b. AP1 reporter gene analysis (Gq-associated receptor)

The method for determining Gq stimulus dependence relies on the known property of Gq-dependent phospholipase C that it induces the activation of genes containing an AP1 element in their promoter. According to the procedure mentioned above for CREB reporter gene assay, use Pathdetect ^TM AP-1 cis-Reporting System (Stratagene, Catalog #219073), in which only the components of calcium phosphate precipitation are changed to 410ng pAP1-Luc, 80ng pCMV-receptor Expression plasmid and 20ng CMV-SEAP.

c. SRF-Luc reporter gene analysis (Gq-related receptor)

One method of detecting Gq stimulation relies on the known property of Gq-dependent phospholipase C to cause activation of genes containing serum response factors in their promoters. Pathdetect ^™ SRF-Luc reporter system (Stratagene) can be used to analyze the activity of Gq coupling, such as analyzing the activity of Gq coupling in COS7 cells. Using the Mammalian Transfection ^TM kit (Stratagene, Catalog #200285), various plasmids in the system and the indicated expression plasmids encoding endogenous or non-endogenous GPCRs were used to transfect cells according to the manufacturer's instructions. Briefly, 410ng SRF-Luc, 80ng pCMV receptor expression plasmid, and 20ng CMV-SEAP (expression plasmid that secretes alkaline phosphatase; measure alkaline phosphatase activity in the culture medium of transfected cells to control for differences in transfection efficiency between samples ), were combined with calcium phosphate precipitation according to the instructions of each manufacturer, and half of the precipitation was equally distributed to 3 wells of a 96-well plate, and the cells were maintained in serum-free medium for 24 hours. Cells were incubated with 1 μM angiotensin for the final 5 hours as required and then lysed using the LucLite ^™ kit (Packard, cat# 6016911) and a "Trilax 1450 Microbeta" liquid scintillation luminescence counter (Wallac), as per the respective vendor. The luciferase activity was analyzed according to the instructions in the protocol, and the data were analyzed with GraphPad Prism ^™ 2.0a software (GraphPad Software Inc.).

d. Analysis of intracellular IP ₃ accumulation (Gq-associated receptors)

On the first day, cells containing the receptor (endogenous and/or non-endogenous) are seeded on 24-well culture plates, typically at 1 x ¹⁰⁵ cells/well (although this number can be optimized). Cells were transfected the next day by first mixing 0.25 μg DNA in 50 μl/well serum-free DMEM and 2 μl lipofectamine in 50 μl/well serum-free DMEM. The solution was mixed gently and incubated at room temperature for 15-30 minutes. The cells were washed with 0.5 ml of PBS, and 400 μl of serum-free medium was mixed with the transfection medium and added to the cells. Cells were then incubated at 37°C/5% CO ₂ for 3-4 hours before the transfection medium was removed and replaced with 1 ml/well regular medium. On the third day, cells were labeled ^with3H -inositol. Briefly, the medium was removed and the cells were washed with 0.5ml PBS followed by the addition of 0.5ml/well myo-inositol/serum-free medium (GIBCO BRL) and 0.25μCi/ ^well3H -inositol at 37°C/5 Cells were incubated for 16-18 hours under % _CO2 . On the fourth day, wash the cells with 0.5ml PBS, add 0.45ml test medium containing myo-inositol/serum-free medium 10μM Baguirin 10mM lithium chloride or 0.4ml test medium and 50μl 10×ketaserin (ket) to A final concentration of 10 μM was obtained. Cells were then incubated at 37°C for 30 minutes. Cells were washed with 0.5 ml PBS and 200 μl/well fresh/ice-cold stop solution (1M KOH, 18 mM sodium borate, 3.8 mM EDTA) was added. The solution was kept on ice for 5-10 minutes or until the cells were lysed, then neutralized with 200 [mu]l fresh/ice-cold neutralizing solution (7.5% HCl). The lysate was then transferred to a 1.5ml centrifuge tube, and 1ml/tube of chloroform/methanol (1:2) was added. The solution was then vortexed for 15 seconds and the upper layer was loaded onto Biorad AG1-X8 ^™ anion exchange resin (100-200 mesh). First, the resin was washed with water at a ratio of 1:1.25 W/V, and 0.9 ml of the upper layer solution was loaded into the column. The column was washed with 10 ml 5 mM inositol and 10 ml 5 mM sodium borate/60 mM sodium formate. Inositol triphosphate was eluted into liquid flash vials containing 10 ml liquid flash cocktail with 2 ml 0.1M formic acid/1M ammonium formate. The column was regenerated by washing with 10 ml 0.1M formic acid/3M ammonium formate and twice with _ddH2O , and the column was stored in water at 4°C.

Typical results are listed in Table G below:

Form G

N/A = not applied

As shown in Example 4(1) above, typical results of GTPyS detection constitutive activation assays were achieved using Gs:fusion protein constructs on human RUP13 and human RUP15. The signals produced by this assay and the differences between them are listed in Table H below:

Form H

Example 5

Preparation of fusion protein

a. GPCR:Gs fusion construct

The design of the constitutively active GPCR-G protein fusion construct was accomplished as follows: the 5' and 3' ends of the rat G protein Gsa (long form; ltoch, H. et al., 83 PNAS 3776 (1986)) were constructed to include HindIII (5'-AAGCTT-3') sequence. After confirmation of the correct sequence (including HindIII flanking sequences), the complete sequence was inserted into pcDNA3.1(-) (Invitrogen, cat. no. V795-20) by subcloning using the HindIII restriction site of this vector. After subcloning into pcDNA3.1(-), confirming the correct orientation of the Gsα sequence, and then confirming this modified pcDNA3.1(-) with the rat Gsα gene on the HindIII sequence, this vector can now be used as a "universal The "Gsα protein carrier. pcDNA3.1(-) contains a series of known restriction sites upstream of the HindIII site, which can advantageously insert the endogenous constitutively active GPCR coding sequence upstream of the Gs protein. The same method can be used to create other "universal" G protein vectors, of course, other vectors that are commercially available or known to those skilled in the art can be used - the important criterion is that the GPCR sequence is upstream of the G protein sequence and In-frame with the G protein sequence.

RUP13 is coupled with Gs, and for the following typical GPCR fusion proteins, fusion with Gsα is realized.

The RUP13-Gsα fusion protein construct was prepared as follows:

Primers were designed as follows:

5'-gatc[TCTAGAAT]GGAGTCCTCACCCATCCCCAG-3' (SEQ.ID.NO.: 97; sense)

5'-gcta[GATATC]CGTGACTCCAGCCGGGGTGAGGCGGC-3' (SEQ.ID.NO.: 98, antisense)

Lowercase nucleotides are included in the restriction site (square brackets) between the G protein and RUP13 as a spacer, the sense and antisense primers include XbaI and EcoRV restriction sites, and the spacer (for the restriction site point contributed) exists between this G protein and RUP15.

PCR was then applied to obtain individual receptor sequences for fusion in the Gsα universal vector disclosed above, using the following procedure for each PCR: 100 ng of RUP15 cDNA was added to separate tubes, each tube containing each primer (sense and antisense ) 2 μl, 3 μl 10 mM dNTPs, 10 μl 10×TaqPlus ^™ precision buffer, 1 μl TaqPlus ^™ precision polymerase (Stratagene: #600211 ), and 80 μl water. Reaction temperatures and cycle times for RUP15 were as follows: 1 min at 94 °C, followed by 30 s at 94 °C; 20 s at 62 °C; 1 min 40 s at 72 °C; 5 min at 72 °C, from steps 2 to Step 4 loops 35 times. The PCR products were run on 1% agarose gel and purified (data not shown). The purified products were digested with EcoRV of XbaI, and the desired inserts were purified and ligated into the respective restriction sites in the Gs universal vector. Positive clones were isolated after transformation and confirmed by restriction enzyme digestion; 293 cells were used to express according to the following procedure, and each positive clone of RUP15-Gs fusion protein was sequenced to confirm the correctness. (See SEQ.ID.NO.: 99 for the nucleic acid sequence, and SEQ.ID.NO.: 100 for the amino acid sequence).

RUP15 is coupled with Gs, and for the following typical GPCR fusion proteins, fusion with Gsα is realized.

The RUP15-Gsα fusion protein construct was prepared as follows:

Primers were designed as follows:

5'-TCTAGAATGACGTCCACCTGCACCAACAGC-3' (SEQ.ID.NO.: 101; sense)

5'-gatatcGCAGGAAAAGTAGCAGAATCGTAGGAAG-3' (SEQ.ID.NO.: 102, antisense)

Lowercase nucleotides are included as spacers in the restriction site between the G protein and RUP15, the sense and antisense primers contain the EcoRV and XbaI restriction sites, respectively, and the spacer (contributes to the restriction site) Exists between this G protein and RUP15.

PCR was then applied to obtain individual receptor sequences for fusion in the Gsα universal vector disclosed above, using the following procedure for each PCR: 100 ng of RUP15 cDNA was added to separate tubes, each tube containing each primer (sense and antisense ) 2 μl, 3 μl 10 mM dNTPs, 10 μl 10×TaqPlus ^™ precision buffer, 1 μl TaqPlus ^™ precision polymerase (Stratagene: #600211 ), and 80 μl water. Reaction temperatures and cycle times for RUP15 were as follows: 1 min at 94 °C, followed by 30 s at 94 °C; 20 s at 62 °C; 1 min 40 s at 72 °C; 5 min at 72 °C, from steps 2 to Step 4 loops 35 times. PCR products were run on a 1% agarose gel and then purified (data not shown). Digest the purified product. The purified product was digested with EcoRV and XbaI, and the desired insert was purified and ligated into the respective restriction sites in the Gs universal vector. Positive clones were isolated after transformation and confirmed by restriction enzyme digestion; 293 cells were used to express according to the following procedure, and each positive clone of RUP15-Gs fusion protein was sequenced to confirm the correctness. (See SEQ.ID.NO.: 103 for the nucleotide sequence, and SEQ.ID.NO.: 104 for the amino acid sequence).

b. Gq (6 amino acid deletion)/Gi fusion construct

The design of the Gq (deletion)/Gi fusion construct is as follows: the N-terminal 6 amino acids of the Gαq subunit (from 2 to 7 amino acids, the sequence is TLESIM (SEQ.ID.NO.: 129)) is deleted, and its C The 5 amino acids at the end (sequence: EYNLV, SEQ.ID.NO.: 130) were replaced by the corresponding amino acids of Gαi protein (sequence: DCGLF, SEQ.ID.NO.: 131). This fusion construct was PCR cloned with the following primers:

5'-gatcaagcttcCATGGCGTGCTGCCTGAGCGAGGAG-3' (SEQ.ID.NO.: 132) and

5'-gatcggatccTTAGAACAGGCCGCAGTCCTTCAGGTTCAGCTGCAGGATGGTG-3' (SEQ.ID.NO.: 133)

And plasmid 63313 of mouse wild-type Gαq with hemagglutinin tag was used as template. Lowercase nucleotides are spacers.

Use TaqPlus Precision DNA Polymerase (Stratagene) for the following cyclic amplification reactions: 95 °C for 2 min; 95 °C for 20 sec; 56 °C for 20 sec; 72 °C for 2 min; 72 °C for 7 min from step 2 to step 4 step cycle 35 times. PCR products were cloned into pCRII-TOPO vector (Invitrogen) and sequenced using ABI Big Dye Terminator kit (P.E. Biosystem). Inserts from TOPO clones containing the fusion construct sequence were shuttled into the HindIII/BamHI sites of the expression vector pcDNA3.1(+) by two-step cloning.

Example 6

Tissue distribution of published human GPCRs: RT-PCR

RT-PCR was used to confirm the expression and the distribution of several new human GPCRs in tissues. The oligonucleotides used were GPCR-specific, using human various tissue cDNAs as templates (MTC, Clontech). Amplification was performed in 40 μl reactions using Taq DNA polymerase (Stratagene), used according to the manufacturer's instructions. 20 μl of the reaction solution was subjected to 1.5% agarose gel electrophoresis to analyze the RT-PCR product. Table J below lists the receptors, cycling reaction conditions and primers used.

Form J Receptor type Loop condition (minutes'seconds") loops 30 times from 2-4 steps 5' primer (SEQ.ID.NO) 3' primer (SEQ.ID.NO) DNA fragment tissue expression hRUP10 94°30”94°10”62°20”72°1'72°7' ^* 2-4 steps cycle 35 times CATGTATGCCAGCGTCCTGCTCC(105) GCTATGCCTGAAGCCAGTCTTGTG(106) 730bp Kidney, white blood cells, liver, placenta, spleen hRUP11 94°2'94°15"67°15"72°45"72°5' GCACCTGCTCCTGAGCACCTTCTCC(107) CACAGCGCTGCAGCCCTGCAGCTGGC(108) 630bp liver, kidneys, pancreas, colon, small intestine, spleen, and prostate hRUP12 94°2'94°15"66°15"72°45"72°5' CCAGTGATGACTCTGTCCAGCCTG(109) CAGACACTTGGCAGGGACGAGGTG(110) 490bp Brain, heart, kidneys, colon, white blood cells, pancreas, prostate, small intestine, spleen, testes, and thymus hRUP13 94°1'94°15"68°20"721'45"72°5' CTTGTGGTCTACTGCAGCATGTTCCG(111) CATAACCCTCCGAGTGTCCAGCGGC(112) 700bp placenta and lungs hRUP14 94°1'94°15"68°20"72°1'45"72°5' ATGGATCCTTATCATGGCTTCCTC(113) CAAGAACAGGTCTCATCTAAGAGCTCC(114) 700bp unknown hRUP16 94°30”94°5”69°15”72°30”72°5' CTCTGATGCCATCTGCTGGATTCCTG(115) GTAGTCCACTGAAAGTCCAGTGATCC(116) 370bp Fetal brain, fetal kidney, and fetal skeletal muscle hRUP18 94°2'94°15"60°20"72°1'72°5' TGGTGGCGATGGCCAACAGCGCTC(117) GTTGCGCCTTAGCGACAGATGACC(118) 330bp pancreas hRUP21 94°1'94°15”56°20”72°40” ^* 2-3 steps cycle 30 times TCAACCTGTATAGCAGCATCCTC(119) AAGGAGTAGCAGAATGGTTAGCC (120) kidneys, lungs and testes hRUP22 94°30”94°15”69°20”72°40” ^* 2-3 steps cycle 30 times GACACCTGTCAGCGGTCGTGTGTG(121) CTGATGGAAGTAGAGGCTGTCCATCTC(122) testes, thymus and spleen hRUP23 94°2'94°15"60°20"72°1' GCGCTGAGCGCAGACCAGTGGCTG(123) CACGGTGACGAAGGGCACGAGCTC(124) 520bp placenta 72°5' hRUP26 94°2'94°15"65°20"72°1'72°5' AGCCATCCCTGCCAGGAAGCATGG(125) CCAGGTAGGTGTGCAGCACAATGGC(126) 470bp pancreas hRUP27 94°30”94°10”55°20”72°1'72°3' ^* 2-4 steps cycle 35 times CTGTTCAACAGGGCGTTGGCAAC(127) ATCATGTCTAGACTCATGGTGATCC(128) 890bp brain

Example 7

Protocol: Direct identification of inverse agonists and agonists

A: Analysis of [ ³⁵ S]GTPγS

Although we have used endogenous constitutively active GPCRs to directly identify, for example, candidate compounds as inverse agonists, in-assay errors can become compounded for reasons that are not fully understood. The GPCR fusion proteins disclosed above are then preferably also used with non-endogenous constitutively activated GPCRs. We have determined that with such proteins, the intra-assay error appears to be essentially stable, thus obtaining a valid signal-to-noise ratio. This facilitates more thorough identification of candidate compounds. Therefore, for direct recognition, it is better to use GPCR fusion proteins, and when used, the following assay protocol is preferably used.

1. Membrane Preparation

Membranes comprising a constitutively active orphan GPCR fusion protein of interest and membranes for direct recognition of candidate compounds as inverse agonists, agonists or partial agonists are preferably prepared as follows:

a.Material

"Membrane Extraction Buffer" consists of 20mM HEPES and 10mM EDTA, pH7.4; "Membrane Washing Buffer" consists of 20mM HEPES and 0.1mM EDTA, pH7.4; "Binding Buffer" consists of 20mM HEPES, 100mM NaCl and 10mM MgCl ₂ composition, pH 7.4.

b. steps

All materials were kept on ice throughout the procedure. First, the medium was aspirated from the confluent monolayer of cells, followed by washing with 10 ml of cold PBS, followed by aspiration, after which 5 ml of membrane extraction buffer was added to the extracted cells, and the cell extract was subsequently transferred to a 50 ml centrifuge. tube (20,000 rpm at 4°C for 17 minutes), after which the supernatant was aspirated and the pellet was resuspended in 30 ml membrane wash buffer, followed by centrifugation at 20,000 rpm for 17 minutes at 4°C, then the supernatant was aspirated and the pellet was resuspended in binding buffer, then homogenized with a Brinkman polytron ^(TM) homogenizer (15-20 seconds vigorous shaking until all material is in suspension), referred to herein as "membrane proteins".

2. Bradford protein analysis

After homogenization, the protein concentration of these membranes was determined by Bradford protein assay. (The protein can be diluted to approximately 1.5 mg/ml, aliquoted and frozen (-80°C) for later use; in the frozen state, the procedure used is as follows: on the day of analysis, thaw the frozen membrane protein at room temperature, then vortex Homogenize with a polytron at about 12×1000rpm for about 5-10 seconds; please note that for multiple preparations, the homogenizer should be thoroughly cleaned between homogenization of different preparations.

a.Material

Binding Buffer (as above); Bradford Staining Reagent, Bradford Protein Standards, used according to manufacturer's instructions (Biorad, cat. no. 500-0006).

b. steps

Prepare two test tubes, one containing the membrane and one as a "blank" control. Each tube was filled with 800μl binding buffer, then 10μl Bradford protein standard (1mg/ml) was added to each test tube, then 10μl membrane protein was added to only one test tube (not added to the blank tube), and then 200μl Bradford The dyeing reagent was added to each test tube, and then each test tube was vortexed. After 5 minutes, the test tube was vortexed again, and the material in it was transferred to the cuvette, and then the cuvette was tested with a CECIL 3041 spectrophotometer Read at 595 wavelength.

3. Direct identification analysis

a.Material

GDP buffer consisted of 37.5 ml of binding buffer and 2 mg of GDP (Sigma, cat.no.G.7127), followed by serial dilutions with binding buffer to obtain 0.2 μM GDP (final concentration of GDP in each well was 0.1 μM GDP); each well contains a candidate compound in a final volume of 200 μl, including 100 μl GDP buffer (final concentration 0.1 μM GDP), 50 μl membrane protein suspended in binding buffer, and 50 μl dissolved in binding buffer [ ³⁵ S]GTPγS (0.6 nM) (2.5 μl of [ ³⁵ S]GTPγS per 10 ml of binding buffer).

b. steps

Candidate compounds are best screened in 96-well plates (which can be frozen at -80°C), and membrane proteins (or membranes with expression vectors other than GPCR fusion proteins, as a control) are briefly homogenized to a suspension state, and then used as described above Protein concentration was determined by Bradford protein assay. Then the membrane protein (and control) was diluted to 0.25 mg/ml (final assay concentration, 12.5 μg/well) with binding buffer, after which, 100 μl of GDP buffer was added to each sample well of Wallac Scintistrip ^™ (Wallac), 5 μl of candidate compound was then transferred to these sample wells using a 5 μl needle (5 μl in a 1:40 ratio in a total assay volume of 200 μl thus a final screening concentration of candidate compound of 10 μM). Also, to avoid contamination, needles should be washed three times after each transfer step: water (1X), ethanol (1X) and water (2X) - excess liquid should be shaken off after each wash, with paper and kimwipe dry. After this, 50 μl of membrane protein was added to each sample well (the control sample well contained a membrane without GPCR fusion protein), pre-incubated at room temperature for 5-10 minutes, after which, 50 μl of [ ³⁵ S]GTPγS in binding buffer ( 0.6nM) was added to each sample well, and then incubated on a shaker at room temperature for 60 minutes (again, in this example, the culture plate was covered with metal foil. The plate was then rotated at 4000RPM at 22°C for 15 minutes to terminate Analyze, then use 8 branch pipes to clean the plate, cover it with a plate cover, and finally read the plate with Wallac 1450 set at the "Prot. #37" file (according to the instructions of each manufacturer).

B. Cyclic AMP Analysis

Another assay that directly identifies candidate compounds is the cyclase-based assay. In addition to direct identification, this method can also be used as a stand-alone method to confirm the results obtained from the above analysis of [ ³⁵ S]GTPγS.

Preferably, using the improved Flash Plate ^TM adenylyl cyclase kit (NewEngland Nuclear; Cat. no. SMP004A), the following protocol directly identifies candidate compounds as inverse agonists of constitutively activated orphan GPCRs and agonist.

Transfected cells were harvested approximately three days after transfection and membranes were prepared by homogenizing suspended cells in a buffer containing 20 mM HEPES (pH 7.4) and 10 mM MgCl ₂ using a Brinkman Polytron ^™ on ice For about 10 seconds, the resulting homogenate was centrifuged at 49,000×g for 15 minutes at 4°C, and then the resulting pellet was resuspended in a buffer containing 20 mM HEPES (pH 7.4) and 0.1 mM EDTA, homogenized for 10 seconds, and then 4°C Centrifuge at 49,000×g for 15 minutes, and store the resulting pellet at -80°C for future use. On the day of direct recognition screening, the membrane pellet was slowly thawed at room temperature and resuspended in a buffer containing 20 mM HEPES (pH 7.4) and ₁₀ mM MgCl to yield a final protein concentration of 0.6 mg/ml (the resuspended membrane was placed on ice on standby).

cAMP standards and assay buffer (2 μCi tracer [ ¹²⁵ I cAMP 100 μl] added to 11 ml assay buffer) were prepared and stored according to the manufacturer's instructions. Prepare fresh assay buffer for screening containing 20 mM HEPES (pH 7.4), 10 mM MgCl ₂ , 20 mM creatine phosphate (Sigma), 0.1 unit/ml creatine phosphokinase (Sigma), 50 μM GTP (Sigma), and 0.2mM ATP (Sigma); the assay buffer can be stored on ice for future use.

Preferably, the above-identified candidate compounds (such as freezing, thawing at room temperature) are added to the sample wells of a 96-well plate (3 μl/well, 12 μM final analysis concentration), mixed with 40 μl membrane protein (30 μg/well) and 50 μl analysis buffer combine together. The mixture was then incubated at room temperature for 30 minutes with gentle shaking.

After the incubation is completed, 100 μl of assay buffer is added to each sample well, and then left for 2 to 4 hours, and then the plate is counted in the Wallac MicroBeta ^TM plate reader with the "Prot.#31" file (operate according to the instructions of each manufacturer) .

A representative screening assay plate (96-well plate) is shown in FIG. 12 . Each bar represents the results for a different compound in each well, and for the RUP13-Gsa fusion protein construct prepared in Example 5(a) above. The typical results in Figure 12 also provide the standard deviation, which is obtained based on the individual plate mean ("m"), together with two inverse agonists randomly selected as "leads" from the initial screening process, including the selected Candidate compounds have a rate of decrease in response that is at least the mean response value minus two standard deviations. In contrast, arbitrary preference for "lead" agonists from the initial screening process includes selection of candidate compounds whose response increase rate is at least the mean response value plus two standard deviations. Based on the selection procedure above, candidate compounds in the following wells could be directly identified as possible inverse agonists (compound A) and agonists (compound B) of RUP13 in wells A2 and G9, respectively. See Figure 12. Note for clarity: These compounds were directly recognized without knowledge of the GPCR's endogenous ligand. Using this analytical technique based on receptor function rather than the binding affinity of the compound, we can identify compounds that decrease the activity of receptor function (compound A) and compounds that increase the activity of receptor function (compound B) . Based on the localization of these receptors in the lung (see, eg, hRUP13 and hRUP21 in Example 6), agents potentially useful in the treatment of lung cancer can be discovered.

Every document mentioned in this patent application document, including co-pending applications and related patent applications, is hereby incorporated by reference in its entirety unless otherwise indicated. Modifications and extensions to the disclosed invention that are within the purview of those skilled in the art are within the scope of the foregoing disclosure and the appended claims.

While many different vectors are available to those of ordinary skill in the art for use with endogenous and non-endogenous GPCRs, the pCMV vector is preferred. In accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, the vector was deposited at the American Type Culture Collection (ATCC) (University Blvd, Manassas, VA20110-2209 USA7) on October 13, 1998 . Its DNA was assayed and confirmed to be viable by ATCC, which assigned the following deposit number ATCC #203351 for pCMV.

<110>Arena Pharmaceuticals, Inc.

  Ruoping CHEN; Huong T.DANG; Kevin P.LOWITZ

<l20> Non-endogenous, constitutively activated human G protein-coupled receptors (Non-Endogenous, Constitutively

  Activated Human G Protein-Coupled Receptors)

<130>AREN0087

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<160>133

<170>PatentIn version 3.0

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<211>1155

<212>DNA

<213> Homo sapiens

<400>1

atggcagccc agaatggaaa caccagtttc acacccaact ttaatccacc ccaagaccat 60

gcctcctccc tctcctttaa cttcagttat ggtgattatg acctccctat ggatgaggat 120

gaggacatga ccaagacccg gaccttcttc gcagccaaga tcgtcattgg cattgcactg 180

gcaggcatca tgctggtctg cggcatcggt aactttgtct ttatcgctgc cctcacccgc 240

tataagaagt tgcgcaacct caccaatctg ctcattgcca acctggccat ctccgacttc 300

ctggtggcca tcatctgctg ccccttcgag atggactact acgtggtacg gcagctctcc 360

tgggagcatg gccacgtgct ctgtgcctcc gtcaactacc tgcgcaccgt ctccctctac 420

gtctccacca atgccttgct ggccattgcc attgacagat atctcgccat cgttcacccc 480

ttgaaaccac ggatgaatta tcaaacggcc tccttcctga tcgccttggt ctggatggtg 540

tccattctca ttgccatccc atcggcttac tttgcaacag aaacggtcct ctttatgtc 600

aagagccagg agaagatctt ctgtggccag atctggcctg tggatcagca gctctactac 660

aagtcctact tcctcttcat ctttggtgtc gagttcgtgg gccctgtggt caccatgacc 720

ctgtgctatg ccaggatctc ccgggagctc tggttcaagg cagtccctgg gttccagacg 780

gagcagattc gcaagcggct gcgctgccgc aggaagacgg tcctggtgct catgtgcatt 840

ctcacggcct atgtgctgtg ctgggcaccc ttctacggtt tcaccatcgt tcgtgacttc 900

ttccccactg tgttcgtgaa ggaaaagcac tacctcactg ccttctacgt ggtcgagtgc 960

atcgccatga gcaacagcat gatcaacacc gtgtgcttcg tgacggtcaa gaacaacacc 1020

atgaagtact tcaagaagat gatgctgctg cactggcgtc cctcccagcg ggggagcaag 1080

tccagtgctg accttgacct cagaaccaac ggggtgccca ccacagaaga ggtggactgt 1140

atcaggctga agtga 1155

<210>2

<211>384

<212>PRT

<213> Homo sapiens

<400>2

Met Ala Ala Gln Asn Gly Asn Thr Ser Phe Thr Pro Asn Phe Asn Pro

1 5 10 15

Pro Gln Asp His Ala Ser Ser Leu Ser Phe Asn Phe Ser Tyr Gly Asp

20 25 30

Tyr Asp Leu Pro Met Asp Glu Asp Glu Asp Met Thr Lys Thr Arg Thr

35 40 45

Phe Phe Ala Ala Lys Ile Val Ile Gly Ile Ala Leu Ala Gly Ile Met

50 55 60

Leu Val Cys Gly Ile Gly Asn Phe Val Phe Ile Ala Ala Leu Thr Arg

65 70 75 80

Tyr Lys Lys Leu Arg Asn Leu Thr Asn Leu Leu Ile Ala Asn Leu Ala

85 90 95

Ile Ser Asp Phe Leu Val Ala Ile Ile Cys Cys Pro Phe Glu Met Asp

100 105 110

Tyr Tyr Val Val Arg Gln Leu Ser Trp Glu His Gly His Val Leu Cys

115 120 125

Ala Ser Val Asn Tyr Leu Arg Thr Val Ser Leu Tyr Val Ser Thr Asn

130 135 140

Ala Leu Leu Ala Ile Ala Ile Asp Arg Tyr Leu Ala Ile Val His Pro

145 150 155 160

Leu Lys Pro Arg Met Asn Tyr Gln Thr Ala Ser Phe Leu Ile Ala Leu

165 170 175

Val Trp Met Val Ser Ile Leu Ile Ala Ile Pro Ser Ala Tyr Phe Ala

180 185 190

Thr Glu Thr Val Leu Phe Ile Val Lys Ser Gln Glu Lys Ile Phe Cys

195 200 205

Gly Gln Ile Trp Pro Val Asp Gln Gln Leu Tyr Tyr Lys Ser Tyr Phe

210 215 220

Leu Phe Ile Phe Gly Val Glu Phe Val Gly Pro Val Val Thr Met Thr

225 230 235 240

Leu Cys Tyr Ala Arg Ile Ser Arg Glu Leu Trp Phe Lys Ala Val Pro

245 250 255

Gly Phe Gln Thr Glu Gln Ile Arg Lys Arg Leu Arg Cys Arg Arg Lys

260 265 270

Thr Val Leu Val Leu Met Cys Ile Leu Thr Ala Tyr Val Leu Cys Trp

275 280 285

Ala Pro Phe Tyr Gly Phe Thr Ile Val Arg Asp Phe Phe Pro Thr Val

290 295 300

Phe Val Lys Glu Lys His Tyr Leu Thr Ala Phe Tyr Val Val Glu Cys

305 310 315 320

Ile Ala Met Ser Asn Ser Met Ile Asn Thr Val Cys Phe Val Thr Val

325 330 335

Lys Asn Asn Thr Met Lys Tyr Phe Lys Lys Met Met Leu Leu His Trp

340 345 350

Arg Pro Ser Gln Arg Gly Ser Lys Ser Ser Ala Asp Leu Asp Leu Arg

355 360 365

Thr Asn Gly Val Pro Thr Thr Glu Glu Val Asp Cys Ile Arg Leu Lys

370 375 380

<210>3

<211>1260

<2l2>DNA

<213> Homo sapiens

<400>3

atgctggcag ctgcctttgc agactctaac tccagcagca tgaatgtgtc ctttgctcac 60

ctccactttg ccggagggta cctgccctct gattcccagg actggagaac catcatcccg 120

gctctcttgg tggctgtctg cctggtgggc ttcgtgggaa acctgtgtgt gattggcatc 180

ctccttcaca atgcttggaa aggaaagcca tccatgatcc actccctgat tctgaatctc 240

agcctggctg atctctccct cctgctgttt tctgcaccta tccgagctac ggcgtactcc 300

aaaagtgttt gggatctagg ctggtttgtc tgcaagtcct ctgactggtt tatccacaca 360

tgcatggcag ccaagagcct gacaatcgtt gtggtggcca aagtatgctt catgtatgca 420

agtgacccag ccaagcaagt gagtatccac aactaccacca tctggtcagt gctggtggcc 480

atctggactg tggctagcct gttacccctg ccggaatggt tctttagcac catcaggcat 540

catgaaggtg tggaaatgtg cctcgtggat gtaccagctg tggctgaaga gtttatgtcg 600

atgtttggta agctctaccc actcctggca tttggccttc cattattttt tgccagcttt 660

tatttctgga gagcttatga ccaatgtaaa aaacgaggaa ctaagactca aaatcttaga 720

aaccagatac gctcaaagca agtcacagtg atgctgctga gcattgccat catctctgct 780

ctcttgtggc tccccgaatg ggtagcttgg ctgtgggtat ggcatctgaa ggctgcaggc 840

ccggccccac cacaaggttt catagccctg tctcaagtct tgatgttttc catctcttca 900

gcaaatcctc tcatttttct tgtgatgtcg gaagagttca gggaaggctt gaaaggtgta 960

tggaaatgga tgataaccaa aaaacctcca actgtctcag agtctcagga aacaccagct 1020

ggcaactcag agggtcttcc tgacaaggtt ccatctccag aatccccagc atccatacca 1080

gaaaaagaga aacccagctc tccctcctct ggcaaaggga aaactgagaa ggcagagatt 1140

cccatccttc ctgacgtaga gcagttttgg catgagaggg acagtccc ttctgtacag 1200

gacaatgacc ctatcccctg ggaacatgaa gatcaagaga caggggaagg tgttaaatag 1260

<210>4

<211>419

<212>PRT

<213> Homo sapiens

<400>4

Met Leu Ala Ala Ala Phe Ala Asp Ser Asn Ser Ser Ser Met Asn Val

1 5 10 15

Ser Phe Ala His Leu His Phe Ala Gly Gly Tyr Leu Pro Ser Asp Ser

20 25 30

Gln Asp Trp Arg Thr Ile Ile Pro Ala Leu Leu Val Ala Val Cys Leu

35 40 45

Val Gly Phe Val Gly Asn Leu Cys Val Ile Gly Ile Leu Leu His Asn

50 55 60

Ala Trp Lys Gly Lys Pro Ser Met Ile His Ser Leu Ile Leu Asn Leu

65 70 75 80

Ser Leu Ala Asp Leu Ser Leu Leu Leu Phe Ser Ala Pro Ile Arg Ala

85 90 95

Thr Ala Tyr Ser Lys Ser Val Trp Asp Leu Gly Trp Phe Val Cys Lys

100 105 110

Ser Ser Asp Trp Phe Ile His Thr Cys Met Ala Ala Lys Ser Leu Thr

115 120 125

Ile Val Val Val Ala Lys Val Cys Phe Met Tyr Ala Ser Asp Pro Ala

130 135 140

Lys Gln Val Ser Ile His Asn Tyr Thr Ile Trp Ser Val Leu Val Ala

l45 150 155 160

Ile Trp Thr Val Ala Ser Leu Leu Pro Leu Pro Glu Trp Phe Phe Ser

165 170 175

Thr Ile Arg His His Glu Gly Val Glu Met Cys Leu Val Asp Val Pro

180 185 190

Ala Val Ala Glu Glu Phe Met Ser Ser Met Phe Gly Lys Leu Tyr Pro Leu

195 200 205

Leu Ala Phe Gly Leu Pro Leu Phe Phe Ala Ser Phe Tyr Phe Trp Arg

210 215 220

Ala Tyr Asp Gln Cys Lys Lys Arg Gly Thr Lys Thr Gln Asn Leu Arg

225 230 235 240

Asn Gln Ile Arg Ser Lys Gln Val Thr Val Met Leu Leu Ser Ile Ala

245 250 255

Ile Ile Ser Ala Leu Leu Trp Leu Pro Glu Trp Val Ala Trp Leu Trp

260 265 270

Val Trp His Leu Lys Ala Ala Gly Pro Ala Pro Pro Gln Gly Phe Ile

275 280 285

Ala Leu Ser Gln Val Leu Met Phe Ser Ile Ser Ser Ala Asn Pro Leu

290 295 300

Ile Phe Leu Val Met Ser Glu Glu Phe Arg Glu Gly Leu Lys Gly Val

305 310 315 320

Trp Lys Trp Met Ile Thr Lys Lys Pro Pro Thr Val Ser Glu Ser Gln

325 330 335

Glu Thr Pro Ala Gly Asn Ser Glu Gly Leu Pro Asp Lys Val Pro Ser

340 345 350

Pro Glu Ser Pro Ala Ser Ile Pro Glu Lys Glu Lys Pro Ser Ser Pro

355 360 365

Ser Ser Gly Lys Gly Lys Thr Glu Lys Ala Glu Ile Pro Ile Leu Pro

370 375 380

Asp Val Glu Gln Phe Trp His Glu Arg Asp Thr Val Pro Ser Val Gln

385 390 395 400

Asp Asn Asp Pro Ile Pro Trp Glu His Glu Asp Gln Glu Thr Gly Glu

405 410 415

Gly Val Lys

<210>5

<211>1014

<212>DNA

<213> Homo sapiens

<400>5

atggggaacg attctgtcag ctacgagtat ggggattaca gcgacctctc ggaccgccct 60

gtggactgcc tggatggcgc ctgcctggcc atcgacccgc tgcgcgtggc cccgctccca 120

ctgtatgccg ccatcttcct ggtgggggtg ccgggcaatg ccatggtggc ctgggtggct 180

gggaaggtgg cccgccggag ggtgggtgcc acctggttgc tccacctggc cgtggcggat 240

ttgctgtgct gtttgtctct gcccatcctg gcagtgccca ttgcccgtgg aggccactgg 300

ccgtatggtg cagtgggctg tcgggcgctg ccctccatca tcctgctgac catgtatgcc 360

agcgtcctgc tcctggcagc tctcagtgcc gacctctgct tcctggctct cgggcctgcc 420

tggtggtcta cggttcagcg ggcgtgcggg gtgcaggtgg cctgtggggc agcctggaca 480

ctggccttgc tgctcaccgt gccctccgcc atctaccgcc ggctgcacca ggagcacttc 540

ccagcccggc tgcagtgtgt ggtggactac ggcggctcct ccagcaccga gaatgcggtg 600

actgccatcc ggtttctttt tggcttcctg gggcccctgg tggccgtggc cagctgccac 660

agtgccctcc tgtgctgggc agcccgacgc tgccggccgc tgggcacagc cattgtggtg 720

gggttttttg tctgctgggc acctaccac ctgctggggc tggtgctcac tgtggcggcc 780

ccgaactccg cactcctggc cagggccctg cgggctgaac ccctcatcgt gggccttgcc 840

ctcgctcaca gctgcctcaa tcccatgctc ttcctgtatt ttgggagggc tcaactccgc 900

cggtcactgc cagctgcctg tcactgggcc ctgagggagt cccagggcca ggacgaaagt 960

gtggacagca agaaatccac cagccatgac ctggtctcgg agatggaggt gtag 1014

<210>6

<211>337

<212>PRT

<213> Homo sapiens

<400>6

Met Gly Asn Asp Ser Val Ser Tyr Glu Tyr Gly Asp Tyr Ser Asp Leu

1 5 10 15

Ser Asp Arg Pro Val Asp Cys Leu Asp Gly Ala Cys Leu Ala Ile Asp

20 25 30

Pro Leu Arg Val Ala Pro Leu Pro Leu Tyr Ala Ala Ile Phe Leu Val

35 40 45

Gly Val Pro Gly Asn Ala Met Val Ala Trp Val Ala Gly Lys Val Ala

50 55 60

Arg Arg Arg Val Gly Ala Thr Trp Leu Leu His Leu Ala Val Ala Asp

65 70 75 80

Leu Leu Cys Cys Leu Ser Leu Pro Ile Leu Ala Val Pro Ile Ala Arg

85 90 95

Gly Gly His Trp Pro Tyr Gly Ala Val Gly Cys Arg Ala Leu Pro Ser

100 105 110

Ile Ile Leu Leu Thr Met Tyr Ala Ser Val Leu Leu Leu Ala Ala Leu

115 120 125

Ser Ala Asp Leu Cys Phe Leu Ala Leu Gly Pro Ala Trp Trp Ser Thr

130 135 140

Val Gln Arg Ala Cys Gly Val Gln Val Ala Cys Gly Ala Ala Trp Thr

145 150 155 160

Leu Ala Leu Leu Leu Thr Val Pro Ser Ala Ile Tyr Arg Arg Leu His

165 170 175

Gln Glu His Phe Pro Ala Arg Leu Gln Cys Val Val Asp Tyr Gly Gly

180 185 190

Ser Ser Ser Thr Glu Asn Ala Val Thr Ala Ile Arg Phe Leu Phe Gly

195 200 205

Phe Leu Gly Pro Leu Val Ala Val Ala Ser Cys His Ser Ala Leu Leu

210 215 220

Cys Trp Ala Ala Arg Arg Cys Arg Pro Leu Gly Thr Ala Ile Val Val

225 230 235 240

Gly Phe Phe Val Cys Trp Ala Pro Tyr His Leu Leu Gly Leu Val Leu

245 250 255

Thr Val Ala Ala Pro Asn Ser Ala Leu Leu Ala Arg Ala Leu Arg Ala

260 265 270

Glu Pro Leu Ile Val Gly Leu Ala Leu Ala His Ser Cys Leu Asn Pro

275 280 285

Met Leu Phe Leu Tyr Phe Gly Arg Ala Gln Leu Arg Arg Ser Leu Pro

290 295 300

Ala Ala Cys His Trp Ala Leu Arg Glu Ser Gln Gly Gln Asp Glu Ser

305 310 315 320

Val Asp Ser Lys Lys Ser Thr Ser His Asp Leu Val Ser Glu Met Glu

325 330 335

Val

<210>7

<211>1272

<212>DNA

<213> Homo sapiens

<400>7

atgttgtgtc accgtggtgg ccagctgata gtgccaatca tcccactttg ccctgagcac 60

tcctgcaggg gtagaagact ccagaacctt ctctcaggcc catggcccaa gcagcccatg 120

gaacttcata acctgagctc tccatctccc tctctctcct cctctgttct ccctccctcc 180

ttctctccct caccctcctc tgctccctct gcctttacca ctgtgggggg gtcctctgga 240

gggccctgcc accccacctc ttcctcgctg gtgtctgcct tcctggcacc aatcctggcc 300

ctggagtttg tcctgggcct ggtggggaac agtttggccc tcttcatctt ctgcatccac 360

acgcggccct ggacctccaa cacggtgttc ctggtcagcc tggtggccgc tgacttcctc 420

ctgatcagca acctgcccct ccgcgtggac tactacctcc tccatgagac ctggcgcttt 480

ggggctgctg cctgcaaagt caacctcttc atgctgtcca ccaaccgcac ggccagcgtt 540

gtcttcctca cagccatcgc actcaaccgc tacctgaagg tggtgcagcc ccaccacgtg 600

ctgagccgtg cttccgtggg ggcagctgcc cgggtggccg ggggactctg ggtgggcatc 660

ctgctcctca acgggcacct gctcctgagc accttctccg gcccctcctg cctcagctac 720

agggtgggca cgaagccctc ggcctcgctc cgctggcacc aggcactgta cctgctggag 780

ttcttcctgc cactggcgct catcctcttt gctattgtga gcattgggct caccatccgg 840

aaccgtggtc tgggcgggca ggcaggcccg cagagggcca tgcgtgtgct ggccatggtg 900

gtggccgtct aacaccatctg cttcttgccc agcatcatct ttggcatggc ttccatggtg 960

gctttctggc tgtccgcctg ccgatccctg gacctctgca cacagctctt ccatggctcc 1020

ctggccttca cctacctcaa cagtgtcctg gaccccgtgc tctactgctt ctctagcccc 1080

aacttcctcc accagagccg ggccttgctg ggcctcacgc ggggccggca gggcccagtg 1140

agcgacgaga gctcctacca accctccagg cagtggcgct accgggaggc ctctaggaag 1200

gcggaggcca tagggaagct gaaagtgcag ggcgaggtct ctctggaaaa ggaaggctcc 1260

tcccagggct ga                                                                  

<210>8

<211>423

<212>PRT

<213> Homo sapiens

<400>8

Met Leu Cys His Arg Gly Gly Gln Leu Ile Val Pro Ile Ile Pro Leu

1 5 10 15

Cys Pro Glu His Ser Cys Arg Gly Arg Arg Leu Gln Asn Leu Leu Ser

20 25 30

Gly Pro Trp Pro Lys Gln Pro Met Glu Leu His Asn Leu Ser Ser Pro

35 40 45

Ser Pro Ser Leu Ser Ser Ser Val Leu Pro Pro Ser Phe Ser Pro Ser

50 55 60

Pro Ser Ser Ala Pro Ser Ala Phe Thr Thr Val Gly Gly Ser Ser Gly

65 70 75 80

Gly Pro Cys His Pro Thr Ser Ser Ser Ser Leu Val Ser Ala Phe Leu Ala

85 90 95

Pro Ile Leu Ala Leu Glu Phe Val Leu Gly Leu Val Gly Asn Ser Leu

100 105 110

Ala Leu Phe Ile Phe Cys Ile His Thr Arg Pro Trp Thr Ser Asn Thr

115 120 125

Val Phe Leu Val Ser Leu Val Ala Ala Asp Phe Leu Leu Ile Ser Asn

130 135 140

Leu Pro Leu Arg Val Asp Tyr Tyr Leu Leu His Glu Thr Trp Arg Phe

145 150 155 160

Gly Ala Ala Ala Cys Lys Val Asn Leu Phe Met Leu Ser Thr Asn Arg

165 170 175

Thr Ala Ser Val Val Phe Leu Thr Ala Ile Ala Leu Asn Arg Tyr Leu

180 185 190

Lys Val Val Gln Pro His His Val Leu Ser Arg Ala Ser Val Gly Ala

195 200 205

Ala Ala Arg Val Ala Gly Gly Leu Trp Val Gly Ile Leu Leu Leu Asn

210 215 220

Gly His Leu Leu Leu Ser Thr Phe Ser Gly Pro Ser Cys Leu Ser Tyr

225 230 235 240

Arg Val Gly Thr Lys Pro Ser Ala Ser Leu Arg Trp His Gln Ala Leu

245 250 255

Tyr Leu Leu Glu Phe Phe Leu Pro Leu Ala Leu Ile Leu Phe Ala Ile

260 265 270

Val Ser Ile Gly Leu Thr Ile Arg Asn Arg Gly Leu Gly Gly Gln Ala

275 280 285

Gly Pro Gln Arg Ala Met Arg Val Leu Ala Met Val Val Ala Val Tyr

290 295 300

Thr Ile Cys Phe Leu Pro Ser Ile Ile Phe Gly Met Ala Ser Met Val

305 310 315 320

Ala Phe Trp Leu Ser Ala Cys Arg Ser Leu Asp Leu Cys Thr Gln Leu

325 330 335

Phe His Gly Ser Leu Ala Phe Thr Tyr Leu Asn Ser Val Leu Asp Pro

340 345 350

Val Leu Tyr Cys Phe Ser Ser Pro Asn Phe Leu His Gln Ser Arg Ala

355 360 365

Leu Leu Gly Leu Thr Arg Gly Arg Gln Gly Pro Val Ser Asp Glu Ser

370 375 380

Ser Tyr Gln Pro Ser Arg Gln Trp Arg Tyr Arg Glu Ala Ser Arg Lys

385 390 395 400

Ala Glu Ala Ile Gly Lys Leu Lys Val Gln Gly Glu Val Ser Leu Glu

405 410 415

Lys Glu Gly Ser Ser Gln Gly

420

<210>9

<211>966

<212>DNA

<213> Homo sapiens

<400>9

atgaaccaga ctttgaatag cagtgggacc gtggagtcag ccctaaacta ttccagaggg 60

agcacagtgc acacggccta cctggtgctg agctccctgg ccatgttcac ctgcctgtgc 120

gggatggcag gcaacagcat ggtgatctgg ctgctgggct ttcgaatgca caggaacccc 180

ttctgcatct atatcctcaa cctggcggca gccgacctcc tcttcctctt cagcatggct 240

tccacgctca gcctggaaac ccagcccctg gtcaatacca ctgacaaggt ccacgagctg 300

atgaagagac tgatgtactt tgcctacaca gtgggcctga gcctgctgac ggccatcagc 360

accccagcgct gtctctctgt cctcttccct atctggttca agtgtcaccg gcccaggcac 420

ctgtcagcct gggtgtgtgg cctgctgtgg acactctgtc tcctgatgaa cgggttgacc 480

tcttccttct gcagcaagtt cttgaaattc aatgaagatc ggtgcttcag ggtggacatg 540

gtccaggccg ccctcatcat gggggtctta accccagtga tgactctgtc cagcctgacc 600

ctctttgtct gggtgcggag gagctcccag cagtggcggc ggcagcccac acggctgttc 660

gtggtggtcc tggcctctgt cctggtgttc ctcatctgtt ccctgcctct gagcatctac 720

tggtttgtgc tctactggtt gagcctgccg cccgagatgc aggtcctgtg cttcagcttg 780

tcacgcctct cctcgtccgt aagcagcagc gccaaccccg tcatctactt cctggtgggc 840

agccggagga gccacaggct gcccaccagg tccctgggga ctgtgctcca acaggcgctt 900

cgcgaggagc ccgagctgga aggtggggag acgcccaccg tgggcaccaa tgagatgggg 960

gcttga 966

<210>10

<211>321

<212>PRT

<213> Homo sapiens

<400>10

Met Asn Gln Thr Leu Asn Ser Ser Gly Thr Val Glu Ser Ala Leu Asn

1 5 10 15

Tyr Ser Arg Gly Ser Thr Val His Thr Ala Tyr Leu Val Leu Ser Ser

20 25 30

Leu Ala Met Phe Thr Cys Leu Cys Gly Met Ala Gly Asn Ser Met Val

35 40 45

Ile Trp Leu Leu Gly Phe Arg Met His Arg Asn Pro Phe Cys Ile Tyr

50 55 60

Ile Leu Asn Leu Ala Ala Ala Asp Leu Leu Phe Leu Phe Ser Met Ala

65 70 75 80

Ser Thr Leu Ser Leu Glu Thr Gln Pro Leu Val Asn Thr Thr Asp Lys

85 90 95

Val His Glu Leu Met Lys Arg Leu Met Tyr Phe Ala Tyr Thr Val Gly

100 105 110

Leu Ser Leu Leu Thr Ala Ile Ser Thr Gln Arg Cys Leu Ser Val Leu

115 120 125

Phe Pro Ile Trp Phe Lys Cys His Arg Pro Arg His Leu Ser Ala Trp

130 135 140

Val Cys Gly Leu Leu Trp Thr Leu Cys Leu Leu Met Asn Gly Leu Thr

145 150 155 160

Ser Ser Phe Cys Ser Lys Phe Leu Lys Phe Asn Glu Asp Arg Cys Phe

165 170 175

Arg Val Asp Met Val Gln Ala Ala Leu Ile Met Gly Val Leu Thr Pro

180 185 190

Val Met Thr Leu Ser Ser Leu Thr Leu Phe Val Trp Val Arg Arg Ser

195 200 205

Ser Gln Gln Trp Arg Arg Gln Pro Thr Arg Leu Phe Val Val Val Leu

210 215 220

Ala Ser Val Leu Val Phe Leu Ile Cys Ser Leu Pro Leu Ser Ile Tyr

225 230 235 240

Trp Phe Val Leu Tyr Trp Leu Ser Leu Pro Pro Glu Met Gln Val Leu

245 250 255

Cys Phe Ser Leu Ser Arg Leu Ser Ser Ser Val Ser Ser Ser Ala Asn

260 265 270

Pro Val Ile Tyr Phe Leu Val Gly Ser Arg Arg Ser His Arg Leu Pro

275 280 285

Thr Arg Ser Leu Gly Thr Val Leu Gln Gln Ala Leu Arg Glu Glu Pro

290 295 300

Glu Leu Glu Gly Gly Glu Thr Pro Thr Val Gly Thr Asn Glu Met Gly

305 310 315 320

Ala

<210>11

<211>1356

<212>DNA

<213> Homo sapiens

<400>11

atggagtcct cacccatccc ccagtcatca gggaactctt ccactttggg gagggtccct 60

caaaccccag gtccctctac tgccagtggg gtcccggagg tggggctacg ggatgttgct 120

tcggaatctg tggccctctt cttcatgctc ctgctggact tgactgctgt ggctggcaat 180

gccgctgtga tggccgtgat cgccaagacg cctgccctcc gaaaatttgt cttcgtcttc 240

cacctctgcc tggtggacct gctggctgcc ctgaccctca tgcccctggc catgctctcc 300

agctctgccc tctttgacca cgccctcttt ggggaggtgg cctgccgcct ctacttgttt 360

ctgagcgtgt gctttgtcag cctggccatc ctctcggtgt cagccatcaa tgtggagcgc 420

tactattacg tagtccaccc catgcgctac gaggtgcgca tgacgctggg gctggtggcc 480

tctgtgctgg tgggtgtgtg ggtgaaggcc ttggccatgg cttctgtgcc agtgttggga 540

agggtctcct gggaggaagg agctcccagt gtccccccag gctgttcact ccagtggagc 600

cacagtgcct actgccagct ttttgtggtg gtctttgctg tcctttactt tctgttgccc 660

ctgctcctca tacttgtggt ctactgcagc atgttccgag tggcccgcgt ggctgccatg 720

cagcacgggc cgctgcccac gtggatggag acaccccggc aacgctccga atctctcagc 780

agccgctcca cgatggtcac cagctcgggg gccccccaga ccaccccaca ccggacgttt 840

gggggaggga aagcagcagt ggttctcctg gctgtggggg gacagttcct gctctgttgg 900

ttgccctact tctctttcca cctctatgtt gccctgagtg ctcagcccat ttcaactggg 960

caggtggaga gtgtggtcac ctggattggc tacttttgct tcacttccaa ccctttcttc 1020

tatggatgtc tcaaccggca gatccggggg gagctcagca agcagtttgt ctgcttcttc 1080

aagccagctc cagaggagga gctgaggctg cctagccggg agggctccat tgaggagaac 1140

ttcctgcagt tccttcaggg gactggctgt ccttctgagt cctgggtttc ccgaccccta 1200

cccagcccca agcaggagcc acctgctgtt gactttcgaa tcccaggcca gatagctgag 1260

gagacctctg agttcctgga gcagcaactc accagcgaca tcatcatgtc agacagctac 1320

ctccgtcctg ccgcctcacc ccggctggag tcatga 1356

<210>12

<211>451

<212>PRT

<213> Homo sapiens

<400>12

Met Glu Ser Ser Pro Ile Pro Gln Ser Ser Gly Asn Ser Ser Thr Leu

1 5 10 15

Gly Arg Val Pro Gln Thr Pro Gly Pro Ser Thr Ala Ser Gly Val Pro

20 25 30

Glu Val Gly Leu Arg Asp Val Ala Ser Glu Ser Val Ala Leu Phe Phe

35 40 45

Met Leu Leu Leu Asp Leu Thr Ala Val Ala Gly Asn Ala Ala Val Met

50 55 60

Ala Val Ile Ala Lys Thr Pro Ala Leu Arg Lys Phe Val Phe Val Phe

65 70 75 80

His Leu Cys Leu Val Asp Leu Leu Ala Ala Leu Thr Leu Met Pro Leu

85 90 95

Ala Met Leu Ser Ser Ser Ala Leu Phe Asp His Ala Leu Phe Gly Glu

100 105 110

Val Ala Cys Arg Leu Tyr Leu Phe Leu Ser Val Cys Phe Val Ser Leu

115 120 125

Ala Ile Leu Ser Val Ser Ala Ile Asn Val Glu Arg Tyr Tyr Tyr Val

130 135 140

Val His Pro Met Arg Tyr Glu Val Arg Met Thr Leu Gly Leu Val Ala

145 150 155 160

Ser Val Leu Val Gly Val Trp Val Lys Ala Leu Ala Met Ala Ser Val

165 170 175

Pro Val Leu Gly Arg Val Ser Trp Glu Glu Gly Ala Pro Ser Val Pro

180 185 190

Pro Gly Cys Ser Leu Gln Trp Ser His Ser Ala Tyr Cys Gln Leu Phe

195 200 205

Val Val Val Phe Ala Val Leu Tyr Phe Leu Leu Pro Leu Leu Leu Ile

210 215 220

Leu Val Val Tyr Cys Ser Met Phe Arg Val Ala Arg Val Ala Ala Met

225 230 235 240

Gln His Gly Pro Leu Pro Thr Trp Met Glu Thr Pro Arg Gln Arg Ser

245 250 255

Glu Ser Leu Ser Ser Arg Ser Thr Met Val Thr Ser Ser Gly Ala Pro

260 265 270

Gln Thr Thr Pro His Arg Thr Phe Gly Gly Gly Lys Ala Ala Val Val

275 280 285

Leu Leu Ala Val Gly Gly Gln Phe Leu Leu Cys Trp Leu Pro Tyr Phe

290 295 300

Ser Phe His Leu Tyr Val Ala Leu Ser Ala Gln Pro Ile Ser Thr Gly

305 310 315 320

Gln Val Glu Ser Val Val Thr Trp Ile Gly Tyr Phe Cys Phe Thr Ser

325 330 335

Asn Pro Phe Phe Tyr Gly Cys Leu Asn Arg Gln Ile Arg Gly Glu Leu

340 345 350

Ser Lys Gln Phe Val Cys Phe Phe Lys Pro Ala Pro Glu Glu Glu Leu

355 360 365

Arg Leu Pro Ser Arg Glu Gly Ser Ile Glu Glu Asn Phe Leu Gln Phe

370 375 380

Leu Gln Gly Thr Gly Cys Pro Ser Glu Ser Trp Val Ser Arg Pro Leu

385 390 395 400

Pro Ser Pro Lys Gln Glu Pro Pro Ala Val Asp Phe Arg Ile Pro Gly

405 410 415

Gln Ile Ala Glu Glu Thr Ser Glu Phe Leu Glu Gln Gln Leu Thr Ser

420 425 430

Asp Ile Ile Met Ser Asp Ser Tyr Leu Arg Pro Ala Ala Ser Pro Arg

435 440 445

Leu Glu Ser

450

<210>13

<211>1041

<212>DNA

<213> Homo sapiens

<400>13

atggagagaa aatttatgtc cttgcaacca tccatctccg tatcagaaat ggaaccaaat 60

ggcaccttca gcaataacaa cagcaggaac tgcacaattg aaaacttcaa gagagaattt 120

ttcccaattg tatatctgat aatatttttc tggggagtct tgggaaatgg gttgtccata 180

tatgttttcc tgcagcctta taagaagtcc acatctgtga acgttttcat gctaaatctg 240

gccatttcag atctcctgtt cataagcacg cttcccttca gggctgacta ttatcttaga 300

ggctccaatt ggatatttgg agacctggcc tgcaggatta tgtcttattc cttgtatgtc 360

aacatgtaca gcagtattta tttcctgacc gtgctgagtg ttgtgcgttt cctggcaatg 420

gttcacccct ttcggcttct gcatgtcacc agcatcagga gtgcctggat cctctgtggg 480

atcatatgga tccttatcat ggcttcctca ataatgctcc tggacagtgg ctctgagcag 540

aacggcagtg tcacatcatg cttagagctg aatctctata aaattgctaa gctgcagacc 600

atgaactata ttgccttggt ggtgggctgc ctgctgccat ttttcacact cagcatctgt 660

tatctgctga tcattcgggt tctgttaaaa gtggaggtcc cagaatcggg gctgcgggtt 720

tctcacagga aggcactgac caccatcatc atcaccttga tcatcttctt cttgtgtttc 780

ctgccctatc acacactgag gaccgtccac ttgacgacat ggaaagtggg tttatgcaaa 840

gacagactgc ataaagcttt ggttatcaca ctggccttgg cagcagccaa tgcctgcttc 900

aatcctctgc tctattactt tgctggggag aattttaagg acagactaaa gtctgcactc 960

agaaaaggcc atccacagaa ggcaaagaca aagtgtgttt tccctgttag tgtgtggttg 1020

agaaaggaaa caagagtata a 1041

<210>14

<211>346

<212>PRT

<213> Homo sapiens

<400>14

Met Glu Arg Lys Phe Met Ser Leu Gln Pro Ser Ile Ser Val Ser Glu

1 5 10 15

Met Glu Pro Asn Gly Thr Phe Ser Asn Asn Asn Ser Arg Asn Cys Thr

20 25 30

Ile Glu Asn Phe Lys Arg Glu Phe Phe Pro Ile Val Tyr Leu Ile Ile

35 40 45

Phe Phe Trp Gly Val Leu Gly Asn Gly Leu Ser Ile Tyr Val Phe Leu

50 55 60

Gln Pro Tyr Lys Lys Ser Thr Ser Val Asn Val Phe Met Leu Asn Leu

65 70 75 80

Ala Ile Ser Asp Leu Leu Phe Ile Ser Thr Leu Pro Phe Arg Ala Asp

85 90 95

Tyr Tyr Leu Arg Gly Ser Asn Trp Ile Phe Gly Asp Leu Ala Cys Arg

100 105 110

Ile Met Ser Tyr Ser Leu Tyr Val Asn Met Tyr Ser Ser Ile Tyr Phe

115 120 125

Leu Thr Val Leu Ser Val Val Arg Phe Leu Ala Met Val His Pro Phe

130 135 140

Arg Leu Leu His Val Thr Ser Ile Arg Ser Ala Trp Ile Leu Cys Gly

145 150 155 160

Ile Ile Trp Ile Leu Ile Met Ala Ser Ser Ile Met Leu Leu Asp Ser

165 170 175

Gly Ser Glu Gln Asn Gly Ser Val Thr Ser Cys Leu Glu Leu Asn Leu

180 185 190

Tyr Lys Ile Ala Lys Leu Gln Thr Met Asn Tyr Ile Ala Leu Val Val

195 200 205

Gly Cys Leu Leu Pro Phe Phe Thr Leu Ser Ile Cys Tyr Leu Leu Ile

210 215 220

Ile Arg Val Leu Leu Lys Val Glu Val Pro Glu Ser Gly Leu Arg Val

225 230 235 240

Ser His Arg Lys Ala Leu Thr Thr Ile Ile Ile Thr Leu Ile Ile Phe

245 250 255

Phe Leu Cys Phe Leu Pro Tyr His Thr Leu Arg Thr Val His Leu Thr

260 265 270

Thr Trp Lys Val Gly Leu Cys Lys Asp Arg Leu His Lys Ala Leu Val

275 280 285

Ile Thr Leu Ala Leu Ala Ala Ala Asn Ala Cys Phe Asn Pro Leu Leu

290 295 300

Tyr Tyr Phe Ala Gly Glu Asn Phe Lys Asp Arg Leu Lys Ser Ala Leu

305 310 315 320

Arg Lys Gly His Pro Gln Lys Ala Lys Thr Lys Cys Val Phe Pro Val

325 330 335

Ser Val Trp Leu Arg Lys Glu Thr Arg Val

340 345

<210>15

<211>1527

<2l2>DNA

<213> Homo sapiens

<400>15

atgacgtcca cctgcaccaa cagcacgcgc gagagtaaca gcagccaacac gtgcatgccc 60

ctctccaaaa tgcccatcag cctggcccac ggcatcatcc gctcaaccgt gctggttatc 120

ttcctcgccg cctctttcgt cggcaacata gtgctggcgc tagtgttgca gcgcaagccg 180

cagctgctgc aggtgaccaa ccgttttatc tttaacctcc tcgtcaccga cctgctgcag 240

atttcgctcg tggccccctg ggtggtggcc acctctgtgc ctctcttctg gcccctcaac 300

agccacttct gcacggccct ggttagcctc accacctgt tcgccttcgc cagcgtcaac 360

accattgtcg tggtgtcagt ggatcgctac ttgtccatca tccaccctct ctcctacccg 420

tccaagatga cccagcgccg cggttacctg ctcctctatg gcacctggat tgtggccatc 480

ctgcagagca ctcctccact ctacggctgg ggccaggctg cctttgatga gcgcaatgct 540

ctctgctcca tgatctgggg ggccagcccc agctacacta ttctcagcgt ggtgtccttc 600

atcgtcattc cactgattgt catgattgcc tgctactccg tggtgttctg tgcagcccgg 660

aggcagcatg ctctgctgta caatgtcaag agacacagct tggaagtgcg agtcaaggac 720

tgtgtggaga atgaggatga agagggagca gagaagaagg aggagttcca ggatgagagt 780

gagtttcgcc gccagcatga aggtgaggtc aaggccaagg agggcagaat ggaagccaag 840

gacggcagcc tgaaggccaa ggaaggaagc acggggacca gtgagagtag tgtagaggcc 900

aggggcagcg aggaggtcag agagagcagc acggtggcca gcgacggcag catggagggt 960

aaggaaggca gcaccaaagt tgaggagaac agcatgaagg cagacaaggg tcgcacagag 1020

gtcaaccagt gcagcattga cttgggtgaa gatgacatgg agtttggtga agacgacatc 1080

aatttcagtg aggatgacgt cgaggcagtg aacatcccgg agagcctccc acccagtcgt 1140

cgtaacagca acagcaaccc tcctctgccc aggtgctacc agtgcaaagc tgctaaagtg 1200

atcttcatca tcattttctc ctatgtgcta tccctggggc cctactgctt tttagcagtc 1260

ctggccgtgt gggtggatgt cgaaacccag gtaccccagt gggtgatcac cataatcatc 1320

tggcttttct tcctgcagtg ctgcatccac ccctatgtct atggctacat gcacaagacc 1380

attaagaagg aaatccagga catgctgaag aagttcttct gcaaggaaaa gcccccgaaa 1440

gaagatagcc accccagacct gcccggaaca gagggtggga ctgaaggcaa gattgtccct 1500

tcctacgatt ctgctacttt tccttga 1527

<210>16

<211>508

<212>PRT

<213> Homo sapiens

<400>16

Met Thr Ser Thr Cys Thr Asn Ser Thr Arg Glu Ser Asn Ser Ser His

1 5 10 15

Thr Cys Met Pro Leu Ser Lys Met Pro Ile Ser Leu Ala His Gly Ile

20 25 30

Ile Arg Ser Thr Val Leu Val Ile Phe Leu Ala Ala Ser Phe Val Gly

35 40 45

Asn Ile Val Leu Ala Leu Val Leu Gln Arg Lys Pro Gln Leu Leu Gln

50 55 60

Val Thr Asn Arg Phe Ile Phe Asn Leu Leu Val Thr Asp Leu Leu Gln

65 70 75 80

Ile Ser Leu Val Ala Pro Trp Val Val Ala Thr Ser Val Pro Leu Phe

85 90 95

Trp Pro Leu Asn Ser His Phe Cys Thr Ala Leu Val Ser Leu Thr His

100 105 110

Leu Phe Ala Phe Ala Ser Val Asn Thr Ile Val Val Val Ser Val Asp

115 120 125

Arg Tyr Leu Ser Ile Ile His Pro Leu Ser Tyr Pro Ser Lys Met Thr

130 135 110

Gln Arg Arg Gly Tyr Leu Leu Leu Tyr Gly Thr Trp Ile Val Ala Ile

145 150 155 160

Leu Gln Ser Thr Pro Pro Leu Tyr Gly Trp Gly Gln Ala Ala Phe Asp

165 170 175

Glu Arg Asn Ala Leu Cys Ser Met Ile Trp Gly Ala Ser Pro Ser Tyr

180 185 190

Thr Ile Leu Ser Val Val Ser Phe Ile Val Ile Pro Leu Ile Val Met

195 200 205

Ile Ala Cys Tyr Ser Val Val Phe Cys Ala Ala Arg Arg Gln His Ala

210 215 220

Leu Leu Tyr Asn Val Lys Arg His Ser Leu Glu Val Arg Val Lys Asp

225 230 235 240

Cys Val Glu Asn Glu Asp Glu Glu Gly Ala Glu Lys Lys Glu Glu Phe

245 250 255

Gln Asp Glu Ser Glu Phe Arg Arg Gln His Glu Gly Glu Val Lys Ala

260 265 270

Lys Glu Gly Arg Met Glu Ala Lys Asp Gly Ser Leu Lys Ala Lys Glu

275 280 285

Gly Ser Thr Gly Thr Ser Glu Ser Ser Val Glu Ala Arg Gly Ser Glu

290 295 300

Glu Val Arg Glu Ser Ser Thr Val Ala Ser Asp Gly Ser Met Glu Gly

305 310 315 320

Lys Glu Gly Ser Thr Lys Val Glu Glu Asn Ser Met Lys Ala Asp Lys

325 330 335

Gly Arg Thr Glu Val Asn Gln Cys Ser Ile Asp Leu Gly Glu Asp Asp

340 345 350

Met Glu Phe Gly Glu Asp Asp Ile Asn Phe Ser Glu Asp Asp Val Glu

355 360 365

Ala Val Asn Ile Pro Glu Ser Leu Pro Pro Ser Arg Arg Asn Ser Asn

370 375 380

Ser Asn Pro Pro Leu Pro Arg Cys Tyr Gln Cys Lys Ala Ala Lys Val

385 390 395 400

Ile Phe Ile Ile Ile Phe Ser Tyr Val Leu Ser Leu Gly Pro Tyr Cys

405 410 415

Phe Leu Ala Val Leu Ala Val Trp Val Asp Val Glu Thr Gln Val Pro

420 425 430

Gln Trp Val Ile Thr Ile Ile Ile Trp Leu Phe Phe Leu Gln Cys Cys

435 440 445

Ile His Pro Tyr Val Tyr Gly Tyr Met His Lys Thr Ile Lys Lys Glu

450 455 460

Ile Gln Asp Met Leu Lys Lys Phe phe Cys Lys Glu Lys Pro Pro Lys

465 470 475 480

Glu Asp Ser His Pro Asp Leu Pro Gly Thr Glu Gly Gly Thr Glu Gly

485 490 495

Lys Ile Val Pro Ser Tyr Asp Ser Ala Thr Phe Pro

500 505

<210>17

<211>1068

<212>DNA

<213> Homo sapiens

<400>17

atgcccttga cggacggcat ttcttcattt gaggacctct tggctaacaa tatcctcaga 60

atatttgtct gggttatagc tttcattacc tgctttggaa atctttttgt cattggcatg 120

agatctttca ttaaagctga aaatacaact cacgctatgt ccatcaaaat cctttgttgc 180

gctgattgcc tgatgggtgt ttacttgttc tttgttggca ttttcgatat aaaataccga 240

gggcagtatc agaagtatgc cttgctgtgg atggagagcg tgcagtgccg cctcatgggg 300

ttcctggcca tgctgtccac cgaagtctct gttctgctac tgacctactt gactttggag 360

aagttcctgg tcattgtctt ccccttcagt aacattcgac ctggaaaacg gcagacctca 420

gtcatcctca tttgcatctg gatggcggga tttttaatag ctgtaattcc attttggaat 480

aaggattatt ttggaaactt ttatgggaaa aatggagtat gtttcccact ttattatgac 540

caaacagaag atattggaag caaagggtat tctcttggaa ttttcctagg tgtgaacttg 600

ctggcttttc tcatcattgt gttttcctat attackatgt tctgttccat tcaaaaaacc 660

gccttgcaga ccacagaagt aaggaattgt tttggaagag aggtggctgt tgcaaatcgt 720

ttctttttta taggtgttctc tgatgccatc tgctggattc ctgtatttgt agttaaaatc 780

ctttccctct tccgggtgga aataccagac acaatgactt cctggatagt gatttttttc 840

cttccagtta acagtgcttt gaatccaatc ctctatactc tcacaaccaa cttttttaag 900

gacaagttga aacagctgct gcacaaacat cagaggaaat caattttcaa aattaaaaaa 960

aaaagtttat ctacatccat tgtgtggata gaggactcct cttccctgaa acttggggtt l020

ttgaacaaaa taacacttgg agacagtata atgaaaccag tttcctag 1068

<210>18

<211>355

<212>PRT

<213> Homo sapiens

<400>18

Met Pro Leu Thr Asp Gly Ile Ser Ser Phe Glu Asp Leu Leu Ala Asn

1 5 10 15

Asn Ile Leu Arg Ile Phe Val Trp Val Ile Ala Phe Ile Thr Cys Phe

20 25 30

Gly Asn Leu Phe Val Ile Gly Met Arg Ser Phe Ile Lys Ala Glu Asn

35 40 45

Thr Thr His Ala Met Ser Ile Lys Ile Leu Cys Cys Ala Asp Cys Leu

50 55 60

Met Gly Val Tyr Leu Phe Phe Val Gly Ile Phe Asp Ile Lys Tyr Arg

65 70 75 80

Gly Gln Tyr Gln Lys Tyr Ala Leu Leu Trp Met Glu Ser Val Gln Cys

85 90 95

Arg Leu Met Gly Phe Leu Ala Met Leu Ser Thr Glu Val Ser Val Leu

100 105 110

Leu Leu Thr Tyr Leu Thr Leu Glu Lys Phe Leu Val Ile Val Phe Pro

115 120 125

Phe Ser Asn Ile Arg Pro Gly Lys Arg Gln Thr Ser Val Ile Leu Ile

130 135 140

Cys Ile Trp Met Ala Gly Phe Leu Ile Ala Val Ile Pro Phe Trp Asn

145 150 155 160

Lys Asp Tyr Phe Gly Asn Phe Tyr Gly Lys Asn Gly Val Cys Phe Pro

165 170 175

Leu Tyr Tyr Asp Gln Thr Glu Asp Ile Gly Ser Lys Gly Tyr Ser Leu

180 185 190

Gly Ile Phe Leu Gly Val Asn Leu Leu Ala Phe Leu Ile Ile Val Phe

195 200 205

Ser Tyr Ile Thr Met Phe Cys Ser Ile Gln Lys Thr Ala Leu Gln Thr

210 215 220

Thr Glu Val Arg Asn Cys Phe Gly Arg Glu Val Ala Val Ala Asn Arg

225 230 235 240

Phe Phe Phe Ile Val Phe Ser Asp Ala Ile Cys Trp Ile Pro Val Phe

245 250 255

Val Val Lys Ile Leu Ser Leu Phe Arg Val Glu Ile Pro Asp Thr Met

260 265 270

Thr Ser Trp Ile Val Ile Phe Phe Leu Pro Val Asn Ser Ala Leu Asn

275 280 285

Pro Ile Leu Tyr Thr Leu Thr Thr Asn Phe Phe Lys Asp Lys Leu Lys

290 295 300

Gln Leu Leu His Lys His Gln Arg Lys Ser Ile Phe Lys Ile Lys Lys

305 310 315 320

Lys Ser Leu Ser Thr Ser Ile Val Trp Ile Glu Asp Ser Ser Ser Leu

325 330 335

Lys Leu Gly Val Leu Asn Lys Ile Thr Leu Gly Asp Ser Ile Met Lys

340 345 350

Pro Val Ser

355

<210>19

<211>969

<212>DNA

<213> Homo sapiens

<400>19

atggatccaa ccatctcaac cttggacaca gaactgacac caatcaacgg aactgaggag 60

actctttgct acaagcagac cttgagcctc acggtgctga cgtgcatcgt ttcccttgtc 120

gggctgacag gaaacgcagt tgtgctctgg ctcctgggct gccgcatgcg caggaacgcc 180

ttctccatct acatcctcaa cttggccgca gcagacttcc tcttcctcag cggccgcctt 240

atatattccc tgttaagctt catcagtatc ccccatacca tctctaaaat cctctatcct 300

gtgatgatgt tttccctactt tgcaggcctg agctttctga gtgccgtgag caccgagcgc 360

tgcctgtccg tcctgtggcc catctggtac cgctgccacc gccccacaca cctgtcagcg 420

gtggtgtgtg tcctgctctg ggccctgtcc ctgctgcgga gcatcctgga gtggatgtta 480

tgtggcttcc tgttcagtgg tgctgattct gcttggtgtc aaacatcaga tttcatcaca 540

gtcgcgtggc tgattttttt atgtgtggtt ctctgtgggt ccagcctggt cctgctgatc 600

aggattctct gtggatcccg gaagataccg ctgaccaggc tgtacgtgac catcctgctc 660

acagtactgg tcttcctcct ctgtggcctg ccctttggca ttcagttttt cctattttta 720

tggatccacg tggacaggga agtcttattt tgtcatgttc atctagtttc tattttcctg 780

tccgctctta acagcagtgc caaccccatc atttacttct tcgtgggctc ctttaggcag 840

cgtcaaaata ggcagaacct gaagctggtt ctccagaggg ctctgcagga cgcgtctgag 900

gtggatgaag gtggagggca gcttcctgag gaaatcctgg agctgtcggg aagcagattg 960

gagcagtga 969

<210>20

<211>322

<212>PRT

<213> Homo sapiens

<400>20

Met Asp Pro Thr Ile Ser Thr Leu Asp Thr Glu Leu Thr Pro Ile Asn

1 5 10 15

Gly Thr Glu Glu Thr Leu Cys Tyr Lys Gln Thr Leu Ser Leu Thr Val

20 25 30

Leu Thr Cys Ile Val Ser Leu Val Gly Leu Thr Gly Asn Ala Val Val

35 40 45

Leu Trp Leu Leu Gly Cys Arg Met Arg Arg Asn Ala Phe Ser Ile Tyr

50 55 60

Ile Leu Asn Leu Ala Ala Ala Asp Phe Leu Phe Leu Ser Gly Arg Leu

65 70 75 80

Ile Tyr Ser Leu Leu Ser Phe Ile Ser Ile Pro His Thr Ile Ser Lys

85 90 95

Ile Leu Tyr Pro Val Met Met Phe Ser Tyr Phe Ala Gly Leu Ser Phe

100 105 110

Leu Ser Ala Val Ser Thr Glu Arg Cys Leu Ser Val Leu Trp Pro Ile

115 120 125

Trp Tyr Arg Cys His Arg Pro Thr His Leu Ser Ala Val Val Cys Val

130 135 140

Leu Leu Trp Ala Leu Ser Leu Leu Arg Ser Ile Leu Glu Trp Met Leu

145 150 155 160

Cys Gly Phe Leu Phe Ser Gly Ala Asp Ser Ala Trp Cys Gln Thr Ser

165 170 175

Asp Phe Ile Thr Val Ala Trp Leu Ile Phe Leu Cys Val Val Leu Cys

180 185 190

Gly Ser Ser Leu Val Leu Leu Ile Arg Ile Leu Cys Gly Ser Arg Lys

195 200 205

Ile Pro Leu Thr Arg Leu Tyr Val Thr Ile Leu Leu Thr Val Leu Val

210 215 220

Phe Leu Leu Cys Gly Leu Pro Phe Gly Ile Gln Phe Phe Leu Phe Leu

225 230 235 240

Trp Ile His Val Asp Arg Glu Val Leu Phe Cys His Val His Leu Val

245 250 255

Ser Ile Phe Leu Ser Ala Leu Asn Ser Ser Ala Asn Pro Ile Ile Tyr

260 265 270

Phe Phe Val Gly Ser Phe Arg Gln Arg Gln Asn Arg Gln Asn Leu Lys

275 280 285

Leu Val Leu Gln Arg Ala Leu Gln Asp Ala Ser Glu Val Asp Glu Gly

290 295 300

Gly Gly Gln Leu Pro Glu Glu Ile Leu Glu Leu Ser Gly Ser Arg Leu

305 310 315 320

Glu Gln

<210>21

<211>1305

<212>DNA

<213> Homo sapiens

<400>21

atggaggatc tctttagccc ctcaattctg ccgccggcgc ccaacatttc cgtgcccatc 60

ttgctgggct gggtctcaa cctgaccttg gggcaaggag cccctgcctc tgggccgccc 120

agccgccgcg tccgcctggt gttcctgggg gtcatcctgg tggtggcggt ggcaggcaac 180

accacagtgc tgtgccgcct gtgcggcggc ggcgggccct gggcgggccc caagcgtcgc 240

aagatggact tcctgctggt gcagctggcc ctggcggacc tgtacgcgtg cgggggcacg 300

gcgctgtcac agctggcctg ggaactgctg ggcgagcccc gcgcggccac gggggacctg 360

gcgtgccgct tcctgcagct gctgcaggca tccgggcggg gcgcctcggc ccacctcgtg 420

gtgctcatcg ccctcgagcg ccggcgcgcg gtgcgtcttc cgcacggccg gccgctgccc 480

gcgcgtgccc tcgccgccct gggctggctg ctggcactgc tgctggcgct gcccccggcc 540

ttcgtggtgc gcggggactc cccctcgccg ctgccgccgc cgccgccgcc aacgtccctg 600

cagccaggcg cgcccccggc cgcccgcgcc tggccggggg agcgtcgctg ccacgggatc 660

ttcgcgcccc tgccgcgctg gcacctgcag gtctacgcgt tctacgaggc cgtcgcgggc 720

ttcgtcgcgc ctgttacggt cctgggcgtc gcttgcggcc acctactctc cgtctggtgg 780

cggcaccggc cgcaggcccc cgcggctgca gcgccctggt cggcgagccc aggtcgagcc 840

cctgcgccca gcgcgctgcc ccgcgccaag gtgcagagcc tgaagatgag cctgctgctg 900

gcgctgctgt tcgtgggctg cgagctgccc tactttgccg cccggctggc ggccgcgtgg 960

tcgtccgggc ccgcggggaga ctgggaggga gagggcctgt cggcggcgct gcgcgtggtg 1020

gcgatggcca acagcgctct caatcccttc gtctacctct tcttccaggc gggcgactgc 1080

cggctccggc gacagctgcg gaagcggctg ggctctctgt gctgcgcgcc gcagggaggc 1140

gcggaggacg aggaggggcc ccggggccac caggcgctct accgccaacg ctggccccac 1200

cctcattatc accatgctcg gcgggaaccg ctggacgagg gcggcttgcg cccaccccct 1260

ccgcgcccca gacccctgcc ttgctcctgc gaaagtgcct tctag 1305

<210>22

<211>434

<212>PRT

<213> Homo sapiens

<400>22

Met Glu Asp Leu Phe Ser Pro Ser Ile Leu Pro Pro Ala Pro Asn Ile

1 5 10 15

Ser Val Pro Ile Leu Leu Gly Trp Gly Leu Asn Leu Thr Leu Gly Gln

20 25 30

Gly Ala Pro Ala Ser Gly Pro Pro Ser Arg Arg Val Arg Leu Val Phe

35 40 45

Leu Gly Val Ile Leu Val Val Ala Val Ala Gly Asn Thr Thr Val Leu

50 55 60

Cys Arg Leu Cys Gly Gly Gly Gly Pro Trp Ala Gly Pro Lys Arg Arg

65 70 75 80

Lys Met Asp Phe Leu Leu Val Gln Leu Ala Leu Ala Asp Leu Tyr Ala

85 90 95

Cys Gly Gly Thr Ala Leu Ser Gln Leu Ala Trp Glu Leu Leu Gly Glu

100 105 110

Pro Arg Ala Ala Thr Gly Asp Leu Ala Cys Arg Phe Leu Gln Leu Leu

115 120 125

Gln Ala Ser Gly Arg Gly Ala Ser Ala His Leu Val Val Leu Ile Ala

130 135 140

Leu Glu Arg Arg Arg Ala Val Arg Leu Pro His Gly Arg Pro Leu Pro

145 150 155 160

Ala Arg Ala Leu Ala Ala Leu Gly Trp Leu Leu Ala Leu Leu Leu Ala

165 170 175

Leu Pro Pro Ala Phe Val Val Arg Gly Asp Ser Pro Ser Pro Leu Pro

180 185 190

Pro Pro Pro Pro Pro Thr Ser Leu Gln Pro Gly Ala Pro Pro Ala Ala

195 200 205

Arg Ala Trp Pro Gly Glu Arg Arg Cys His Gly Ile Phe Ala Pro Leu

210 215 220

Pro Arg Trp His Leu Gln Val Tyr Ala Phe Tyr Glu Ala Val Ala Gly

225 230 235 240

Phe Val Ala Pro Val Thr Val Leu Gly Val Ala Cys Gly His Leu Leu

245 250 255

Ser Val Trp Trp Arg His Arg Pro Gln Ala Pro Ala Ala Ala Ala Pro

260 265 270

Trp Ser Ala Ser Pro Gly Arg Ala Pro Ala Pro Ser Ala Leu Pro Arg

275 280 285

Ala Lys Val Gln Ser Leu Lys Met Ser Leu Leu Leu Ala Leu Leu Phe

290 295 300

Val Gly Cys Glu Leu Pro Tyr Phe Ala Ala Arg Leu Ala Ala Ala Trp

305 310 315 320

Ser Ser Gly Pro Ala Gly Asp Trp Glu Gly Glu Gly Leu Ser Ala Ala

325 330 335

Leu Arg Val Val Ala Met Ala Asn Ser Ala Leu Asn Pro Phe Val Tyr

340 345 350

Leu Phe Phe Gln Ala Gly Asp Cys Arg Leu Arg Arg Gln Leu Arg Lys

355 360 365

Arg Leu Gly Ser Leu Cys Cys Ala Pro Gln Gly Gly Ala Glu Asp Glu

370 375 380

Glu Gly Pro Arg Gly His Gln Ala Leu Tyr Arg Gln Arg Trp Pro His

385 390 395 400

Pro His Tyr His His Ala Arg Arg Glu Pro Leu Asp Glu Gly Gly Leu

405 410 415

Arg Pro Pro Pro Pro Arg Pro Arg Pro Leu Pro Cys Ser Cys Glu Ser

420 425 430

Ala Phe

<210>23

<211>1041

<212>DNA

<213> Homo sapiens

<400>23

atgtacaacg ggtcgtgctg ccgcatcgag ggggacacca tctcccaggt gatgccgccg 60

ctgctcattg tggcctttgt gctgggcgca ctaggcaatg gggtcgccct gtgtggtttc 120

tgcttccaca tgaagacctg gaagcccagc actgtttacc ttttcaattt ggccgtggct 180

gatttcctcc ttatgatctg cctgcctttt cggacagact attacctcag acgtagacac 240

tgggcttttg gggacattcc ctgccgagtg gggctcttca cgttggccat gaacagggcc 300

gggagcatcg tgttccttac ggtggtggct gcggacaggt atttcaaagt ggtccacccc 360

caccacgcgg tgaacactat ctccaccgggg gtggcggctg gcatcgtctg caccctgtgg 420

gccctggtca tcctgggaac agtgtatctt ttgctggaga accatctctg cgtgcaagag 480

acggccgtct cctgtgagag cttcatcatg gagtcggcca atggctggca tgacatcatg 540

ttccagctgg agttctttat gcccctcggc atcatcttat tttgctcctt caagattgtt 600

tggagcctga ggcggaggca gcagctggcc agacaggctc ggatgaagaa ggcgacccgg 660

ttcatcatgg tggtggcaat tgtgttcatc acatgctacc tgcccagcgt gtctgctaga 720

ctctatttcc tctggacggt gccctcgagt gcctgcgatc cctctgtcca tggggccctg 780

cacataaccc tcagcttcac ctacatgaac agcatgctgg atcccctggt gtattatttt 840

tcaagcccct cctttcccaa attctacaac aagctcaaaa tctgcagtct gaaacccaag 900

cagccaggac actcaaaaac acaaaggccg gaagagatgc caatttcgaa cctcggtcgc 960

aggagttgca tcagtgtggc aaatagtttc caaagccagt ctgatgggca atgggatccc 1020

cacattgttg agtggcactg a 1041

<210>24

<211>346

<212>PRT

<213> Homo sapiens

<400>24

Met Tyr Asn Gly Ser Cys Cys Arg Ile Glu Gly Asp Thr Ile Ser Gln

1 5 10 15

Val Met Pro Pro Leu Leu Ile Val Ala Phe Val Leu Gly Ala Leu Gly

20 25 30

Asn Gly Val Ala Leu Cys Gly Phe Cys Phe His Met Lys Thr Trp Lys

35 40 45

Pro Ser Thr Val Tyr Leu Phe Asn Leu Ala Val Ala Asp Phe Leu Leu

50 55 60

Met Ile Cys Leu Pro Phe Arg Thr Asp Tyr Tyr Leu Arg Arg Arg His

65 70 75 80

Trp Ala Phe Gly Asp Ile Pro Cys Arg Val Gly Leu Phe Thr Leu Ala

85 90 95

Met Asn Arg Ala Gly Ser Ile Val Phe Leu Thr Val Val Ala Ala Asp

100 105 110

Arg Tyr Phe Lys Val Val His Pro His His Ala Val Asn Thr Ile Ser

115 120 125

Thr Arg Val Ala Ala Gly Ile Val Cys Thr Leu Trp Ala Leu Val Ile

130 135 140

Leu Gly Thr Val Tyr Leu Leu Leu Glu Asn His Leu Cys Val Gln Glu

145 150 155 160

Thr Ala Val Ser Cys Glu Ser Phe Ile Met Glu Ser Ala Asn Gly Trp

165 170 175

His Asp Ile Met Phe Gln Leu Glu Phe Phe Met Pro Leu Gly Ile Ile

180 185 190

Leu Phe Cys Ser Phe Lys Ile Val Trp Ser Leu Arg Arg Arg Gln Gln

195 200 205

Leu Ala Arg Gln Ala Arg Met Lys Lys Ala Thr Arg Phe Ile Met Val

210 215 220

Val Ala Ile Val Phe Ile Thr Cys Tyr Leu Pro Ser Val Ser Ala Arg

225 230 235 240

Leu Tyr Phe Leu Trp Thr Val Pro Ser Ser Ala Cys Asp Pro Ser Val

245 250 255

His Gly Ala Leu His Ile Thr Leu Ser Phe Thr Tyr Met Asn Ser Met

260 265 270

Leu Asp Pro Leu Val Tyr Tyr Phe Ser Ser Pro Ser Phe Pro Lys Phe

275 280 285

Tyr Asn Lys Leu Lys Ile Cys Ser Leu Lys Pro Lys Gln Pro Gly His

290 295 300

Ser Lys Thr Gln Arg Pro Glu Glu Met Pro Ile Ser Asn Leu Gly Arg

305 310 315 320

Arg Ser Cys Ile Ser Val Ala Asn Ser Phe Gln Ser Gln Ser Asp Gly

325 330 335

Gln Trp Asp Pro His Ile Val Glu Trp His

340 345

<210>25

<211>1011

<212>DNA

<213> Homo sapiens

<400>25

atgaacaaca atacaacatg tattcaacca tctatgatct cttccatggc tttaccaatc 60

atttacatcc tcctttgtat tgttggtgtt tttggaaaca ctctctctca atggatattt 120

ttaacaaaaa taggtaaaaa aacatcaacg cacatctacc tgtcacacct tgtgactgca 180

aacttacttg tgtgcagtgc catgcctttc at9agtatct atttcctgaa aggtttccaa 240

tgggaatatc aatctgctca atgcagagtg gtcaattttc tgggaactct atccatgcat 300

gcaagtatgt ttgtcagtct cttaatttta agttggattg ccataagccg ctatgctacc 360

ttaatgcaaa aggattcctc gcaagagact acttcatgct atgagaaaat attttatggc 420

catttactga aaaaatttcg ccagcccaac tttgctagaa aactatgcat ttacatatgg 480

ggagttgtac tgggcataat cattccagtt accgtatact actcagtcat agaggctaca 540

gaaggagaag agagcctatg ctacaatcgg cagatggaac taggagccat gatctctcag 600

attgcaggtc tcattggaac cacatttatt ggattttcct ttttagtagt actaacatca 660

tactactctt ttgtaagcca tctgagaaaa ataagaacct gtacgtccat tatggagaaa 720

gatttgactt acagttctgt gaaaagacat cttttggtca tccagattct actaatagtt 780

tgcttccttc cttatagtat ttttaaaccc attttttatg ttctacacca aagagataac 840

tgtcagcaat tgaattattt aatagaaaca aaaaacattc tcacctgtct tgcttcggcc 900

agaagtagca cagaccccat tatatttctt ttattagata aaacattcaa gaagacacta 960

tataatctct ttacaaagtc taattcagca catatgcaat catatggttg a 1011

<210>26

<211>336

<212>PRT

<213> Homo sapiens

<400>26

Met Asn Asn Asn Thr Thr Cys Ile Gln Pro Ser Met Ile Ser Ser Ser Met

1 5 10 15

Ala Leu Pro Ile Ile Tyr Ile Leu Leu Cys Ile Val Gly Val Phe Gly

20 25 30

Asn Thr Leu Ser Gln Trp Ile Phe Leu Thr Lys Ile Gly Lys Lys Thr

35 40 45

Ser Thr His Ile Tyr Leu Ser His Leu Val Thr Ala Asn Leu Leu Val

50 55 60

Cys Ser Ala Met Pro Phe Met Ser Ile Tyr Phe Leu Lys Gly Phe Gln

65 70 75 80

Trp Glu Tyr Gln Ser Ala Gln Cys Arg Val Val Asn Phe Leu Gly Thr

85 90 95

Leu Ser Met His Ala Ser Met Phe Val Ser Leu Leu Ile Leu Ser Trp

100 105 110

Ile Ala Ile Ser Arg Tyr Ala Thr Leu Met Gln Lys Asp Ser Ser Gln

115 120 125

Glu Thr Thr Ser Cys Tyr Glu Lys Ile Phe Tyr Gly His Leu Leu Lys

l30 135 140

Lys Phe Arg Gln Pro Asn Phe Ala Arg Lys Leu Cys Ile Tyr Ile Trp

145 150 155 160

Gly Val Val Leu Gly Ile Ile Ile Pro Val Thr Val Tyr Tyr Ser Val

165 170 175

Ile Glu Ala Thr Glu Gly Glu Glu Ser Leu Cys Tyr Asn Arg Gln Met

180 185 190

Glu Leu Gly Ala Met Ile Ser Gln Ile Ala Gly Leu Ile Gly Thr Thr

195 200 205

Phe Ile Gly Phe Ser Phe Leu Val Val Leu Thr Ser Tyr Tyr Ser Phe

210 215 220

Val Ser His Leu Arg Lys Ile Arg Thr Cys Thr Ser Ile Met Glu Lys

225 230 235 240

Asp Leu Thr Tyr Ser Ser Val Lys Arg His Leu Leu Val Ile Gln Ile

245 250 255

Leu Leu Ile Val Cys Phe Leu Pro Tyr Ser Ile Phe Lys Pro Ile Phe

260 265 270

Tyr Val Leu His Gln Arg Asp Asn Cys Gln Gln Leu Asn Tyr Leu Ile

275 280 285

Glu Thr Lys Asn Ile Leu Thr Cys Leu Ala Ser Ala Arg Ser Ser Thr

290 295 300

Asp Pro Ile Ile Phe Leu Leu Leu Asp Lys Thr Phe Lys Lys Thr Leu

305 310 315 320

Tyr Asn Leu Phe Thr Lys Ser Asn Ser Ala His Met Gln Ser Tyr Gly

325 330 335

<210>27

<211>1014

<212>DNA

<213> Homo sapiens

<400>27

atgaatgagc cactagacta tttagcaaat gcttctgatt tccccgatta tgcagctgct 60

tttggaaatt gcactgatga aaacatccca ctcaagatgc actacctccc tgttatttat 120

ggcattatct tcctcgtggg atttccaggc aatgcagtag tgatatccac ttacattttc 180

aaaatgagac cttggaagag cagcaccatc attatgctga acctggcctg cacagatctg 240

ctgtatctga ccagcctccc cttcctgatt cactactatg ccagtggcga aaactggatc 300

tttggagatt tcatgtgtaa gtttatccgc ttcagcttcc atttcaacct gtatagcagc 360

atcctcttcc tcacctgttt cagcatcttc cgctactgtg tgatcattca cccaatgagc 420

tgcttttcca ttcacaaaac tcgatgtgca gttgtagcct gtgctgtggt gtggatcatt 480

tcactggtag ctgtcattcc gatgaccttc ttgatcacat caaccaacag gaccaacaga 540

tcagcctgtc tcgacctcac cagttcggat gaactcaata ctattaagtg gtacaacctg 600

attttgactg caactacttt ctgcctcccc ttggtgatag tgacactttg ctataccacg 660

attatccaca ctctgaccca tggactgcaa actgacagct gccttaagca gaaagcacga 720

aggctaacca ttctgctact ccttgcattt tacgtatgtt ttttaccctt ccatatcttg 780

agggtcattc ggatcgaatc tcgcctgctt tcaatcagtt gttccattga gaatcagatc 840

catgaagctt acatcgtttc tagaccatta gctgctctga acacctttgg taacctgtta 900

ctatatgtgg tggtcagcga caactttcag caggctgtct gctcaacagt gagatgcaaa 960

gtaagcggga accttgagca agcaaagaaa attagttact caaacaaccc cttga 1014

<210>28

<211>337

<212>PRT

<213> Homo sapiens

<400>28

Met Asn Glu Pro Leu Asp Tyr Leu Ala Asn Ala Ser Asp Phe Pro Asp

1 5 10 15

Tyr Ala Ala Ala Phe Gly Asn Cys Thr Asp Glu Asn Ile Pro Leu Lys

20 25 30

Met His Tyr Leu Pro Val Ile Tyr Gly Ile Ile Phe Leu Val Gly Phe

35 40 45

Pro Gly Asn Ala Val Val Ile Ser Thr Tyr Ile Phe Lys Met Arg Pro

50 55 60

Trp Lys Ser Ser Thr Ile Ile Met Leu Asn Leu Ala Cys Thr Asp Leu

65 70 75 80

Leu Tyr Leu Thr Ser Leu Pro Phe Leu Ile His Tyr Tyr Ala Ser Gly

85 90 95

Glu Asn Trp Ile Phe Gly Asp Phe Met Cys Lys Phe Ile Arg Phe Ser

100 105 110

Phe His Phe Asn Leu Tyr Ser Ser Ser Ile Leu Phe Leu Thr Cys Phe Ser

115 120 125

Ile Phe Arg Tyr Cys Val Ile Ile His Pro Met Ser Cys Phe Ser Ile

130 135 140

His Lys Thr Arg Cys Ala Val Val Ala Cys Ala Val Val Trp Ile Ile

145 150 155 160

Ser Leu Val Ala Val Ile Pro Met Thr Phe Leu Ile Thr Ser Thr Asn

165 170 175

Arg Thr Asn Arg Ser Ala Cys Leu Asp Leu Thr Ser Ser Asp Glu Leu

180 185 190

Asn Thr Ile Lys Trp Tyr Asn Leu Ile Leu Thr Ala Thr Thr Phe Cys

195 200 205

Leu Pro Leu Val Ile Val Thr Leu Cys Tyr Thr Thr Ile Ile His Thr

210 215 220

Leu Thr His Gly Leu Gln Thr Asp Ser Cys Leu Lys Gln Lys Ala Arg

225 230 235 240

Arg Leu Thr Ile Leu Leu Leu Leu Ala Phe Tyr Val Cys Phe Leu Pro

245 250 255

Phe His Ile Leu Arg Val Ile Arg Ile Glu Ser Arg Leu Leu Ser Ile

260 265 270

Ser Cys Ser Ile Glu Asn Gln Ile His Glu Ala Tyr Ile Val Ser Arg

275 280 285

Pro Leu Ala Ala Leu Asn Thr Phe Gly Asn Leu Leu Leu Tyr Val Val

290 295 300

Val Ser Asp Asn Phe Gln Gln Ala Val Cys Ser Thr Val Arg Cys Lys

305 310 315 320

Val Ser Gly Asn Leu Glu Gln Ala Lys Lys Ile Ser Tyr Ser Asn Asn

325 330 335

Pro

<210>29

<211>993

<212>DNA

<213> Homo sapiens

<400>29

atggatccaa ccaccccggc ctggggaaca gaaagtacaa cagtgaatgg aaatgaccaa 60

gcccttcttc tgctttgtgg caaggagacc ctgatcccgg tcttcctgat ccttttcatt 120

gccctggtcg ggctggtagg aaacgggttt gtgctctggc tcctgggctt ccgcatgcgc 180

aggaacgcct tctctgtcta cgtcctcagc ctggccgggg ccgacttcct cttcctctgc 240

ttccagatta taaattgcct ggtgtacctc agtaacttct tctgttccat ctccatcaat 300

ttccctagct tcttcaccac tgtgatgacc tgtgcctacc ttgcaggcct gagcatgctg 360

agcaccgtca gcaccgagcg ctgcctgtcc gtcctgtggc ccatctggta tcgctgccgc 420

cgccccagac acctgtcagc ggtcgtgtgt gtcctgctc tgggccctgtc cctactgctg 480

agcatcttgg aagggaagtt ctgtggcttc ttattagtg atggtgactc tggttggtgt 540

cagacatttg atttcatcac tgcagcgtgg ctgatttttt tattcatggt tctctgtggg 600

tccagtctgg ccctgctggt caggatcctc tgtggctcca gggtctgcc actgaccagg 660

ctgtacctga ccatcctgct cacagtgctg gtgttcctcc tctgcggcct gccctttggc 720

attcagtggt tcctaatatt atggatctgg aaggattctg atgtcttatt ttgtcatatt 780

catccagttt cagttgtcct gtcatctctt aacagcagtg ccaaccccat catttacttc 840

ttcgtgggct cttttaggaa gcagtggcgg ctgcagcagc cgatcctcaa gctggctctc 900

cagagggctc tgcaggacat tgctgaggtg gatcacagtg aaggatgctt ccgtcagggc 960

accccggaga tgtcgagaag cagtctggtg tag 993

<210>30

<211>330

<212>PRT

<213> Homo sapiens

<400>30

Met Asp Pro Thr Thr Pro Ala Trp Gly Thr Glu Ser Thr Thr Val Asn

1 5 10 15

Gly Asn Asp Gln Ala Leu Leu Leu Leu Cys Gly Lys Glu Thr Leu Ile

20 25 30

Pro Val Phe Leu Ile Leu Phe Ile Ala Leu Val Gly Leu Val Gly Asn

35 40 45

Gly Phe Val Leu Trp Leu Leu Gly Phe Arg Met Arg Arg Asn Ala Phe

50 55 60

Ser Val Tyr Val Leu Ser Leu Ala Gly Ala Asp Phe Leu Phe Leu Cys

65 70 75 80

Phe Gln Ile Ile Asn Cys Leu Val Tyr Leu Ser Asn Phe Phe Cys Ser

85 90 95

Ile Ser Ile Asn Phe Pro Ser Phe Phe Thr Thr Val Met Thr Cys Ala

100 105 110

Tyr Leu Ala Gly Leu Ser Met Leu Ser Thr Val Ser Thr Glu Arg Cys

115 120 125

Leu Ser Val Leu Trp Pro Ile Trp Tyr Arg Cys Arg Arg Pro Arg His

130 135 140

Leu Ser Ala Val Val Cys Val Leu Leu Trp Ala Leu Ser Leu Leu Leu

145 150 155 160

Ser Ile Leu Glu Gly Lys Phe Cys Gly Phe Leu Phe Ser Asp Gly Asp

165 170 175

Ser Gly Trp Cys Gln Thr Phe Asp Phe Ile Thr Ala Ala Trp Leu Ile

180 185 190

Phe Leu Phe Met Val Leu Cys Gly Ser Ser Leu Ala Leu Leu Val Arg

195 200 205

Ile Leu Cys Gly Ser Arg Gly Leu Pro Leu Thr Arg Leu Tyr Leu Thr

210 215 220

Ile Leu Leu Thr Val Leu Val Phe Leu Leu Cys Gly Leu Pro Phe Gly

225 230 235 240

Ile Gln Trp Phe Leu Ile Leu Trp Ile Trp Lys Asp Ser Asp Val Leu

245 250 255

Phe Cys His Ile His Pro Val Ser Val Val Leu Ser Ser Leu Asn Ser

260 265 270

Ser Ala Asn Pro Ile Ile Tyr Phe Phe Val Gly Ser Phe Arg Lys Gln

275 280 285

Trp Arg Leu Gln Gln Pro Ile Leu Lys Leu Ala Leu Gln Arg Ala Leu

290 295 300

Gln Asp Ile Ala Glu Val Asp His Ser Glu Gly Cys Phe Arg Gln Gly

305 310 315 320

Thr Pro Glu Met Ser Arg Ser Ser Leu Val

325 330

<210>31

<211>1092

<212>DNA

<213> Homo sapiens

<400>31

atgggccccg gcgaggcgct gctggcgggt ctcctggtga tggtactggc cgtggcgctg 60

ctatccaacg cactggtgct gctttgttgc gcctacagcg ctgagctccg cactcgagcc 120

tcaggcgtcc tcctggtgaa tctgtcgctg ggccacctgc tgctggcggc gctggacatg 180

cccttcacgc tgctcggtgt gatgcgcggg cggacaccgt cggcgcccgg cgcatgccaa 240

gtcattggct tcctggacac cttcctggcg tccaacgcgg cgctgagcgt ggcggcgctg 300

agcgcagacc agtggctggc agtgggcttc ccactgcgct acgccggacg cctgcgaccg 360

cgctatgccg gcctgctgct gggctgtgcc tggggacagt cgctggcctt ctcaggcgct 420

gcacttggct gctcgtggct tggctacagc agcgccttcg cgtcctgttc gctgcgcctg 480

ccgcccgagc ctgagcgtcc gcgcttcgca gccttcaccg ccacgctcca tgccgtgggc 540

ttcgtgctgc cgctggcggt gctctgcctc acctcgctcc aggtgcaccg ggtggcacgc 600

agccactgcc agcgcatgga caccgtcacc atgaaggcgc tcgcgctgct cgccgacctg 660

caccccagtg tgcggcagcg ctgcctcatc cagcagaagc ggcgccgcca ccgcgccacc 720

aggaagattg gcattgctat tgcgaccttc ctcatctgct ttgccccgta tgtcatgacc 780

aggctggcgg agctcgtgcc cttcgtcacc gtgaacgccc agtggggcat cctcagcaag 840

tgcctgacct acagcaaggc ggtggccgac ccgttcacgt actctctgct ccgccggccg 900

ttccgccaag tcctggccgg catggtgcac cggctgctga agagaaccccc gcgcccagca 960

tccacccatg acagctctct ggatgtggcc ggcatggtgc accagctgct gaagagaacc 1020

ccgcgcccag cgtccaccca caacggctct gtggacacag agaatgattc ctgcctgcag 1080

cagacacact ga l092

<210>32

<211>363

<212>PRT

<213> Homo sapiens

<400>32

Met Gly Pro Gly Glu Ala Leu Leu Ala Gly Leu Leu Val Met Val Leu

1 5 10 15

Ala Val Ala Leu Leu Ser Asn Ala Leu Val Leu Leu Cys Cys Ala Tyr

20 25 30

Ser Ala Glu Leu Arg Thr Arg Ala Ser Gly Val Leu Leu Val Asn Leu

35 40 45

Ser Leu Gly His Leu Leu Leu Ala Ala Leu Asp Met Pro Phe Thr Leu

50 55 60

Leu Gly Val Met Arg Gly Arg Thr Pro Ser Ala Pro Gly Ala Cys Gln

65 70 75 80

Val Ile Gly Phe Leu Asp Thr Phe Leu Ala Ser Asn Ala Ala Leu Ser

85 90 95

Val Ala Ala Leu Ser Ala Asp Gln Trp Leu Ala Val Gly Phe Pro Leu

100 105 110

Arg Tyr Ala Gly Arg Leu Arg Pro Arg Tyr Ala Gly Leu Leu Leu Gly

115 120 125

Cys Ala Trp Gly Gln Ser Leu Ala Phe Ser Gly Ala Ala Leu Gly Cys

130 l35 140

Ser Trp Leu Gly Tyr Ser Ser Ala Phe Ala Ser Cys Ser Leu Arg Leu

145 150 155 160

Pro Pro Glu Pro Glu Arg Pro Arg Phe Ala Ala Phe Thr Ala Thr Leu

165 170 175

His Ala Val Gly Phe Val Leu Pro Leu Ala Val Leu Cys Leu Thr Ser

180 185 190

Leu Gln Val His Arg Val Ala Arg Ser His Cys Gln Arg Met Asp Thr

195 200 205

Val Thr Met Lys Ala Leu Ala Leu Leu Ala Asp Leu His Pro Ser Val

210 2l5 220

Arg Gln Arg Cys Leu Ile Gln Gln Lys Arg Arg Arg His Arg Ala Thr

225 230 235 240

Arg Lys Ile Gly Ile Ala Ile Ala Thr Phe Leu Ile Cys Phe Ala Pro

245 250 255

Tyr Val Met Thr Arg Leu Ala Glu Leu Val Pro Phe Val Thr Val Asn

260 265 270

Ala Gln Trp Gly Ile Leu Ser Lys Cys Leu Thr Tyr Ser Lys Ala Val

275 280 285

Ala Asp Pro Phe Thr Tyr Ser Leu Leu Arg Arg Pro Phe Arg Gln Val

290 295 300

Leu Ala Gly Met Val His Arg Leu Leu Lys Arg Thr Pro Arg Pro Ala

305 310 315 320

Ser Thr His Asp Ser Ser Leu Asp Val Ala Gly Met Val His Gln Leu

325 330 335

Leu Lys Arg Thr Pro Arg Pro Ala Ser Thr His Asn Gly Ser Val Asp

340 345 350

Thr Glu Asn Asp Ser Cys Leu Gln Gln Thr His

355 360

<210>33

<211>1125

<212>DNA

<213> Homo sapiens

<400>33

atgccccacac tcaatacttc tgcctctcca cccacattct tctgggccaa tgcctccgga 60

ggcagtgtgc tgagtgctga tgatgctccg atgcctgtca aattcctagc cctgaggctc 120

atggttgccc tggcctatgg gcttgtgggg gccattggct tgctgggaaa tttggcggtg 180

ctgtgggtac tgagtaactg tgcccggaga gcccctggcc caccttcaga caccttcgtc 240

ttcaacctgg ctctggcgga cctgggactg gcactcactc tccccttttg ggcagccgag 300

tcggcactgg actttcactg gcccttcgga ggtgccctct gcaagatggt tctgacggcc 360

actgtcctca acgtctatgc cagcatcttc ctcatcacag cgctgagcgt tgctcgctac 420

tgggtggtgg ccatggctgc ggggccaggc accacctct cactcttctg ggcccgaata 480

gccaccctgg cagtgtgggc ggcggctgcc ctggtgacgg tgccccacagc tgtcttcggg 540

gtggagggtg aggtgtgtgg tgtgcgcctt tgcctgctgc gtttccccag caggtactgg 600

ctggggggcct accagctgca gagggtggtg ctggctttca tggtgccctt gggcgtcatc 660

accacccagct acctgctgct gctggccttc ctgcagcggc ggcaacggcg gcggcaggac 720

agcaggtcg tggcccgctc tgtccgcatc ctggtggctt ccttcttcct ctgctggttt 780

cccaaccatg tggtcactct ctggggtgtc ctggtgaagt ttgacctggt gccctggaac 840

agtactttct atactatcca gacgtatgtc ttccctgtca ctacttgctt ggcacacagc 900

aatagctgcc tcaaccctgt gctgtactgt ctcctgaggc gggagccccg gcaggctctg 960

gcaggcacct tcagggatct gcggtcgagg ctgtggcccc agggcggagg ctgggtgcaa 1020

caggtggccc taaagcaggt aggcaggcgg tgggtcgcaa gcaacccccg ggagagccgc 1080

ccttctaccc tgctcaccaa cctggacaga gggacacccg ggtga 1125

<210>34

<211>374

<212>PRT

<213> Homo sapiens

<400>34

Met Pro Thr Leu Asn Thr Ser Ala Ser Pro Pro Thr Phe Phe Trp Ala

1 5 10 15

Asn Ala Ser Gly Gly Ser Val Leu Ser Ala Asp Asp Ala Pro Met Pro

20 25 30

Val Lys Phe Leu Ala Leu Arg Leu Met Val Ala Leu Ala Tyr Gly Leu

35 40 45

Val Gly Ala Ile Gly Leu Leu Gly Asn Leu Ala Val Leu Trp Val Leu

50 55 60

Ser Asn Cys Ala Arg Arg Ala Pro Gly Pro Pro Ser Asp Thr Phe Val

65 70 75 80

Phe Asn Leu Ala Leu Ala Asp Leu Gly Leu Ala Leu Thr Leu Pro Phe

85 90 95

Trp Ala Ala Glu Ser Ala Leu Asp Phe His Trp Pro Phe Gly Gly Ala

100 105 110

Leu Cys Lys Met Val Leu Thr Ala Thr Val Leu Asn Val Tyr Ala Ser

115 120 125

Ile Phe Leu Ile Thr Ala Leu Ser Val Ala Arg Tyr Trp Val Val Ala

130 135 140

Met Ala Ala Gly Pro Gly Thr His Leu Ser Leu Phe Trp Ala Arg Ile

145 150 155 160

Ala Thr Leu Ala Val Trp Ala Ala Ala Ala Leu Val Thr Val Pro Thr

165 170 175

Ala Val Phe Gly Val Glu Gly Glu Val Cys Gly Val Arg Leu Cys Leu

180 185 190

Leu Arg Phe Pro Ser Arg Tyr Trp Leu Gly Ala Tyr Gln Leu Gln Arg

195 200 205

Val Val Leu Ala Phe Met Val Pro Leu Gly Val Ile Thr Thr Ser Tyr

210 215 220

Leu Leu Leu Leu Ala Phe Leu Gln Arg Arg Gln Arg Arg Arg Gln Asp

225 230 235 240

Ser Arg Val Val Ala Arg Ser Val Arg Ile Leu Val Ala Ser Phe Phe

245 250 255

Leu Cys Trp Phe Pro Asn His Val Val Thr Leu Trp Gly Val Leu Val

260 265 270

Lys Phe Asp Leu Val Pro Trp Asn Ser Thr Phe Tyr Thr Ile Gln Thr

275 280 285

Tyr Val Phe Pro Val Thr Thr Cys Leu Ala His Ser Asn Ser Cys Leu

290 295 300

Asn Pro Val Leu Tyr Cys Leu Leu Arg Arg Glu Pro Arg Gln Ala Leu

305 310 315 320

Ala Gly Thr Phe Arg Asp Leu Arg Ser Arg Leu Trp Pro Gln Gly Gly

325 330 335

Gly Trp Val Gln Gln Val Ala Leu Lys Gln Val Gly Arg Arg Trp Val

340 345 350

Ala Ser Asn Pro Arg Glu Ser Arg Pro Ser Thr Leu Leu Thr Asn Leu

355 360 365

Asp Arg Gly Thr Pro Gly

370

<210>35

<211>1092

<212>DNA

<213> Homo sapiens

<400>35

atgaatcggc accatctgca ggatcacttt ctggaaatag acaagaagaa ctgctgtgtg 60

ttccgagatg acttcattgt caaggtgttg ccgccggtgt tggggctgga gtttatcttc 120

gggcttctgg gcaatggcct tgccctgtgg atttctgtt tccacctcaa gtcctggaaa 180

tccagccgga ttttcctgtt caacctggca gtggctgact ttctactgat catctgcctg 240

cccttcctga tggacaacta tgtgaggcgt tgggactgga agtttgggga catcccttgc 300

cggctgatgc tcttcatgtt ggctatgaac cgccagggca gcatcatctt cctcacggtg 360

gtggcggtag acaggtattt ccgggtggtc catccccacc acgccctgaa caagatctcc 420

aatcggacag cagccatcat ctcttgcctt ctgtggggca tcactattgg cctgacagtc 480

cacctcctga agaagaagat gccgatccag aatggcggtg caaatttgtg cagcagcttc 540

agcatctgcc ataccttcca gtggcacgaa gccatgttcc tcctggagtt cttcctgccc 600

ctgggcatca tcctgttctg ctcagccaga attatctgga gcctgcggca gagacaaatg 660

gaccggcatg ccaagatcaa gagagccatc accttcatca tggtggtggc catcgtcttt 720

gtcatctgct tccttccccag cgtggttgtg cggatccgca tcttctggct cctgcacact 780

tcgggcacgc agaattgtga agtgtaccgc tcggtggacc tggcgttctt tatcactctc 840

agcttcacct acatgaacag catgctggac cccgtggtgt actacttctc cagcccatcc 900

tttcccaact tcttctccac tttgatcaac cgctgcctcc agaggaagat gacaggtgag 960

ccagataata accgcagcac gagcgtcgag ctcacagggg accccaacaa aaccagaggc 1020

gctccagagg cgttaatggc caactccggt gagccatgga gcccctctta tctgggccca 1080

acctctcctt aa 1092

<210>36

<211>363

<212>PRT

<213> Homo sapiens

<400>36

Met Asn Arg His His Leu Gln Asp His Phe Leu Glu Ile Asp Lys Lys

1 5 10 15

Asn Cys Cys Val Phe Arg Asp Asp Phe Ile Val Lys Val Leu Pro Pro

20 25 30

Val Leu Gly Leu Glu Phe Ile Phe Gly Leu Leu Gly Asn Gly Leu Ala

35 40 45

Leu Trp Ile Phe Cys Phe His Leu Lys Ser Trp Lys Ser Ser Arg Ile

50 55 60

Phe Leu Phe Asn Leu Ala Val Ala Asp Phe Leu Leu Ile Ile Cys Leu

65 70 75 80

Pro Phe Leu Met Asp Asn Tyr Val Arg Arg Trp Asp Trp Lys Phe Gly

85 90 95

Asp Ile Pro Cys Arg Leu Met Leu Phe Met Leu Ala Met Asn Arg Gln

100 105 110

Gly Ser Ile Ile Phe Leu Thr Val Val Ala Val Asp Arg Tyr Phe Arg

115 120 125

Val Val His Pro His His Ala Leu Asn Lys Ile Ser Asn Arg Thr Ala

130 135 140

Ala Ile Ile Ser Cys Leu Leu Trp Gly Ile Thr Ile Gly Lau Thr Val

l45 150 155 160

His Leu Leu Lys Lys Lys Met Pro Ile Gln Asn Gly Gly Ala Asn Leu

165 170 175

Cys Ser Ser Phe Ser Ile Cys His Thr Phe Gln Trp His Glu Ala Met

180 185 190

Phe Leu Leu Glu Phe Phe Leu Pro Leu Gly Ile Ile Leu Phe Cys Ser

195 200 205

Ala Arg Ile Ile Trp Ser Leu Arg Gln Arg Gln Met Asp Arg His Ala

210 215 220

Lys Ile Lys Arg Ala Ile Thr Phe Ile Met Val Val Ala Ile Val Phe

225 230 235 240

Val Ile Cys Phe Leu Pro Ser Val Val Val Arg Ile Arg Ile Phe Trp

245 250 255

Leu Leu His Thr Ser Gly Thr Gln Asn Cys Glu Val Tyr Arg Ser Val

260 265 270

Asp Leu Ala Phe Phe Ile Thr Leu Ser Phe Thr Tyr Met Asn Ser Met

275 280 285

Leu Asp Pro Val Val Tyr Tyr Phe Ser Ser Pro Ser Phe Pro Asn Phe

290 295 300

Phe Ser Thr Leu Ile Asn Arg Cys Leu Gln Arg Lys Met Thr Gly Glu

305 310 315 320

Pro Asp Asn Asn Arg Ser Thr Ser Val Glu Leu Thr Gly Asp Pro Asn

325 330 335

Lys Thr Arg Gly Ala Pro Glu Ala Leu Met Ala Asn Ser Gly Glu Pro

340 345 350

Trp Ser Pro Ser Tyr Leu Gly Pro Thr Ser Pro

355 360

<210>37

<211>1044

<212>DNA

<213> Homo sapiens

<400>37

atgggggatg agctggcacc ttgccctgtg ggcactacag cttggccggc cctgatccag 60

ctcatcagca agacaccctg catgccccaa gcagccagca acacttcctt gggcctgggg 120

gacctcaggg tgcccagctc catgctgtac tggcttttcc ttccctcaag cctgctggct 180

gcagccacac tggctgtcag ccccctgctg ctggtgacca tcctgcggaa ccaacggctg 240

cgacaggagc cccactacct gctcccggct aacatcctgc tctcagacct ggcctacatt 300

ctcctccaca tgctcatctc ctccagcagc ctgggtggct gggagctggg ccgcatggcc 360

tgtggcattc tcactgatgc tgtcttcgcc gcctgcacca gcaccatcct gtccttcacc 420

gccattgtgc tgcacaccta cctggcagtc atccatccac tgcgctacct ctccttcatg 480

tcccatgggg ctgcctggaa ggcagtggcc ctcatctggc tggtggcctg ctgcttcccc 540

acattcctta tttggctcag caagtggcag gatgcccagc tggaggagca aggagcttca 600

tacatcctac caccaagcat gggcacccag ccgggatgtg gcctcctggt cattgttacc 660

taacacctcca ttctgtgcgt tctgttcctc tgcacagctc tcattgccaa ctgtttctgg 720

aggatctatg cagaggccaa gacttcaggc atctgggggc agggctattc ccgggccagg 780

ggcaccctgc tgatccactc agtgctgatc aattgtacg tgagcacagg ggtggtgttc 840

tccctggaca tggtgctgac caggtaccac cacattgact ctgggactca cacatggctc 900

ctggcagcta acagtgaggt actcatgatg cttccccgtg ccatgctccc atacctgtac 960

ctgctccgct accggcagct gttgggcatg gtccggggcc acctcccatc caggaggcac 1020

caggccatct ttaccatttc ctag 1044

<210>38

<211>347

<212>PRT

<213> Homo sapiens

<400>38

Met Gly Asp Glu Leu Ala Pro Cys Pro Val Gly Thr Thr Ala Trp Pro

1 5 10 15

Ala Leu Ile Gln Leu Ile Ser Lys Thr Pro Cys Met Pro Gln Ala Ala

20 25 30

Ser Asn Thr Ser Leu Gly Leu Gly Asp Leu Arg Val Pro Ser Ser Ser Met

35 40 45

Leu Tyr Trp Leu Phe Leu Pro Ser Ser Leu Leu Ala Ala Ala Thr Leu

50 55 60

Ala Val Ser Pro Leu Leu Leu Val Thr Ile Leu Arg Asn Gln Arg Leu

65 70 75 80

Arg Gln Glu Pro His Tyr Leu Leu Pro Ala Asn Ile Leu Leu Ser Asp

85 90 95

Leu Ala Tyr Ile Leu Leu His Met Leu Ile Ser Ser Ser Ser Leu Gly

100 105 110

Gly Trp Glu Leu Gly Arg Met Ala Cys Gly Ile Leu Thr Asp Ala Val

115 120 125

Phe Ala Ala Cys Thr Ser Thr Ile Leu Ser Phe Thr Ala Ile Val Leu

130 135 140

His Thr Tyr Leu Ala Val Ile His Pro Leu Arg Tyr Leu Ser Phe Met

145 150 155 160

Ser His Gly Ala Ala Trp Lys Ala Val Ala Leu Ile Trp Leu Val Ala

165 170 175

Cys Cys Phe Pro Thr Phe Leu Ile Trp Leu Ser Lys Trp Gln Asp Ala

180 185 190

Gln Leu Glu Glu Gln Gly Ala Ser Tyr Ile Leu Pro Pro Ser Met Gly

195 200 205

Thr Gln Pro Gly Cys Gly Leu Leu Val Ile Val Thr Tyr Thr Ser Ile

210 215 220

Leu Cys Val Leu Phe Leu Cys Thr Ala Leu Ile Ala Asn Cys Phe Trp

225 230 235 240

Arg Ile Tyr Ala Glu Ala Lys Thr Ser Gly Ile Trp Gly Gln Gly Tyr

245 250 255

Ser Arg Ala Arg Gly Thr Leu Leu Ile His Ser Val Leu Ile Thr Leu

260 265 270

Tyr Val Ser Thr Gly Val Val Phe Ser Leu Asp Met Val Leu Thr Arg

275 280 285

Tyr His His Ile Asp Ser Gly Thr His Thr Trp Leu Leu Ala Ala Asn

290 295 300

Ser Glu Val Leu Met Met Leu Pro Arg Ala Met Leu Pro Tyr Leu Tyr

305 310 315 320

Leu Leu Arg Tyr Arg Gln Leu Leu Gly Met Val Arg Gly His Leu Pro

325 330 335

Ser Arg Arg His Gln Ala Ile Phe Thr Ile Ser

340 345

<210>39

<211>1023

<212>DNA

<2l3> Homo sapiens

<400>39

atgaatccat ttcatgcatc ttgttggaac acctctgccg aacttttaaa caaatcctgg 60

aataaagagt ttgcttatca aactgccagt gtggtagata cagtcatcct cccttccatg 120

attgggatta tctgttcaac agggctggtt ggcaacatcc tcattgtatt cactataata 180

agatccagga aaaaaacagt ccctgacatc tatatctgca acctggctgt ggctgatttg 240

gtccacatag ttggaatgcc ttttcttatt caccaatggg cccgagggggg agagtgggtg 300

tttggggggc ctctctgcac catcatcaca tccctggata cttgtaacca atttgcctgt 360

agtgccatca tgactgtaat gagtgtggac aggtactttg ccctcgtcca accatttcga 420

ctgacacgtt ggagaacaag gtacaagacc atccggatca atttgggcct ttgggcagct 480

tcctttatcc tggcattgcc tgtctgggtc tactcgaagg tcatcaaatt taaagacggt 540

gttgagagtt gtgcttttga tttgacatcc cctgacgatg tactctggta tacactttat 600

ttgacgataa caactttttt tttccctcta cccttgattt tggtgtgcta tattttaatt 660

ttatgctata cttgggagat gtatcaacag aataaggatg ccagatgctg caatcccagt 720

gtaccaaaac agagagtgat gaagttgaca aagatggtgc tggtgctggt ggtagtcttt 780

atcctgagtg ctgcccctta tcatgtgata caactggtga acttacagat ggaacagccc 840

acactggcct tctatgtggg ttattacctc tccatctgtc tcagctatgc cagcagcagc 900

attaaccctt ttctctacat cctgctgagt ggaaatttcc agaaacgtct gcctcaaatc 960

caaagaagag cgactgagaa ggaaatcaac aatatgggaa acactctgaa atcacacttt 1020

tag 1023

<210>40

<211>340

<212>PRT

<213> Homo sapiens

<400>40

Met Asn Pro Phe His Ala Ser Cys Trp Asn Thr Ser Ala Glu Leu Leu

1 5 10 15

Asn Lys Ser Trp Asn Lys Glu Phe Ala Tyr Gln Thr Ala Ser Val Val

20 25 30

Asp Thr Val Ile Leu Pro Ser Met Ile Gly Ile Ile Cys Ser Thr Gly

35 40 45

Leu Val Gly Asn Ile Leu Ile Val Phe Thr Ile Ile Arg Ser Arg Lys

50 55 60

Lys Thr Val Pro Asp Ile Tyr Ile Cys Asn Leu Ala Val Ala Asp Leu

65 70 75 80

Val His Ile Val Gly Met Pro Phe Leu Ile His Gln Trp Ala Arg Gly

85 90 95

Gly Glu Trp Val Phe Gly Gly Pro Leu Cys Thr Ile Ile Thr Ser Leu

100 105 110

Asp Thr Cys Asn Gln Phe Ala Cys Ser Ala Ile Met Thr Val Met Ser

115 120 125

Val Asp Arg Tyr Phe Ala Leu Val Gln Pro Phe Arg Leu Thr Arg Trp

130 135 140

Arg Thr Arg Tyr Lys Thr Ile Arg Ile Asn Leu Gly Leu Trp Ala Ala

145 150 155 160

Ser Phe Ile Leu Ala Leu Pro Val Trp Val Tyr Ser Lys Val Ile Lys

165 170 175

Phe Lys Asp Gly Val Glu Ser Cys Ala Phe Asp Leu Thr Ser Pro Asp

180 185 190

Asp Val Leu Trp Tyr Thr Leu Tyr Leu Thr Ile Thr Thr Phe Phe Phe

195 200 205

Pro Leu Pro Leu Ile Leu Val Cys Tyr Ile Leu Ile Leu Cys Tyr Thr

210 215 220

Trp Glu Met Tyr Gln Gln Asn Lys Asp Ala Arg Cys Cys Asn Pro Ser

225 230 235 240

Val Pro Lys Gln Arg Val Met Lys Leu Thr Lys Met Val Leu Val Leu

245 250 255

Val Val Val Phe Ile Leu Ser Ala Ala Pro Tyr His Val Ile Gln Leu

260 265 270

Val Asn Leu Gln Met Glu Gln Pro Thr Leu Ala Phe Tyr Val Gly Tyr

275 280 285

Tyr Leu Ser Ile Cys Leu Ser Tyr Ala Ser Ser Ser Ile Asn Pro Phe

290 295 300

Leu Tyr Ile Leu Leu Ser Gly Asn Phe Gln Lys Arg Leu Pro Gln Ile

305 310 315 320

Gln Arg Arg Ala Thr Glu Lys Glu Ile Asn Asn Met Gly Asn Thr Leu

325 330 335

Lys Ser His Phe

340

<210>41

<211>24

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>41

cttgcagaca tcaccatggc agcc 24

<210>42

<211>24

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>42

gtgatgctct gagtactgga ctgg 24

<210>43

<211>20

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>43

gaagctgtga agagtgatgc 20

<210>44

<211>24

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>44

gtcagcaata ttgataagca gcag 24

<210>45

<211>27

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>45

ccatggggaa cgattctgtc agctacg 27

<210>46

<211>24

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>46

gctatgcctg aagccagtct tgtg 24

<210>47

<211>26

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>47

ccaggatgtt gtgtcaccgt ggtggc 26

<210>48

<211>26

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>48

cacagcgctg cagccctgca gctggc 26

<210>49

<211>26

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>49

cttcctctcg tagggatgaa ccagac 26

<210>50

<211>26

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>50

ctcgcacagg tgggaagcac ctgtgg 26

<210>51

<211>23

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>51

gcctgtgaca ggaggtaccc tgg 23

<210>52

<211>25

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>52

catatccctc cgagtgtcca gcggc 25

<210>53

<211>31

<212>DNA

<2l3> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>53

gcatggagag aaaatttatg tccttgcaac c 31

<210>54

<211>27

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>54

caagaacagg tctcatctaa gagctcc 27

<210>55

<211>26

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>55

gctgttgcca tgacgtccac ctgcac 26

<210>56

<211>26

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>56

ggacagttca aggtttgcct tagaac 26

<210>57

<211>23

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>57

ctttcgatac tgctcctatg ctc 23

<210>58

<211>26

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>58

gtagtccact gaaagtccag tgatcc 26

<210>59

<211>26

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>59

tttctgagca tggatccaac catctc 26

<210>60

<211>25

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>60

ctgtctgaca gggcagaggc tcttc 25

<210>61

<211>28

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>61

ggaactcgta tagacccagc gtcgctcc 28

<210>62

<211>28

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>62

ggaggttgcg ccttagcgac agatgacc 28

<210>63

<211>22

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223>Novel Sequence

<400>63

ctgcacccgg acacttgctc tg 22

<210>64

<211>25

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223>Novel Sequence

<400>64

gtctgcttgt tcagtgccac tcaac 25

<210>65

<211>26

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>65

tatctgcaat tctattctag ctcctg 26

<210>66

<211>26

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>66

tgtccctaat aaagtcacat gaatgc 26

<210>67

<211>23

<212>DNA

<2l3> Artificial sequence

<220>

<221>misfc feature

<223> new sequence

<400>67

ggagacaacc atgaatgagc cac 23

<210>68

<211>24

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>68

tatttcaagg gttgtttgag taac 24

<2l0>69

<211>27

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>69

ggcaccagtg gaggttttct gagcatg 27

<210>70

<211>27

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>70

ctgatggaag tagaggctgt ccatctc 27

<210>71

<211>23

<2l2>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>71

cctggcgagc cgctagcgcc atg 23

<210>72

<211>23

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>72

atgagccctg ccaggccctc agt 23

<210>73

<211>27

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>73

ctgcgatgcc cacactcaat acttctg 27

<210>74

<211>27

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>74

aaggatccta cacttggtgg atctcag 27

<210>75

<211>22

<212>DNA

<213> Artificial sequence

<400>75

gctggagcat tcactaggcg ag 22

<210>76

<211>24

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>76

agatcctggt tcttggtgac aatg 24

<210>77

<211>24

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>77

agccatccct gccaggaagc atgg 24

<210>78

<211>27

<212>DNA

<2l3> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>78

ccagactgtg gactcaagaa ctctagg 27

<210>79

<211>28

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>79

agtccacgaa caatgaatcc atttcatg 28

<210>80

<211>25

<212>DNA

<213>Artificial sequence ence

<220>

<221>misc_feature

<223> new sequence

<400>80

atcatgtcta gactcatggt gatcc 25

<210>81

<211>30

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>81

ggggagggaa agcaaaggtg gtcctcctgg 30

<210>82

<211>30

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>82

ccaggagaac cacctttgct ttccctcccc 30

<210>83

<211>1356

<212>DNA

<213> Homo sapiens

<400>83

atggagtcct cacccatccc ccagtcatca gggaactctt ccactttggg gagggtccct 60

caaaccccag gtccctctac tgccagtggg gtcccggagg tggggctacg ggatgttgct 120

tcggaatctg tggccctctt cttcatgctc ctgctggact tgactgctgt ggctggcaat 180

gccgctgtga tggccgtgat cgccaagacg cctgccctcc gaaaatttgt cttcgtcttc 240

cacctctgcc tggtggacct gctggctgcc ctgaccctca tgcccctggc catgctctcc 300

agctctgccc tctttgacca cgccctcttt ggggaggtgg cctgccgcct ctacttgttt 360

ctgagcgtgt gctttgtcag cctggccatc ctctcggtgt cagccatcaa tgtggagcgc 420

tactattacg tagtccaccc catgcgctac gaggtgcgca tgacgctggg gctggtggcc 480

tctgtgctgg tgggtgtgtg ggtgaaggcc ttggccatgg cttctgtgcc agtgttggga 540

agggtctcct gggaggaagg agctcccagt gtccccccag gctgttcact ccagtggagc 600

cacagtgcct actgccagct ttttgtggtg gtctttgctg tcctttactt tctgttgccc 660

ctgctcctca tacttgtggt ctactgcagc atgttccgag tggcccgcgt ggctgccatg 720

cagcacgggc cgctgcccac gtggatggag acaccccggc aacgctccga atctctcagc 780

agccgctcca cgatggtcac cagctcgggg gccccccaga ccaccccaca ccggacgttt 840

gggggaggga aagcaaaggt ggttctcctg gctgtggggg gacagttcct gctctgttgg 900

ttgccctact tctctttcca cctctatgtt gccctgagtg ctcagcccat ttcaactggg 960

caggtggaga gtgtggtcac ctggattggc tacttttgct tcacttccaa ccctttcttc 1020

tatggatgtc tcaaccggca gatccggggg gagctcagca agcagtttgt ctgcttcttc 1080

aagccagctc cagaggagga gctgaggctg cctagccggg agggctccat tgaggagaac 1140

ttcctgcagt tccttcaggg gactggctgt ccttctgagt cctgggtttc ccgaccccta 1200

cccagcccca agcaggagcc acctgctgtt gactttcgaa tcccaggcca gatagctgag 1260

gagacctctg agttcctgga gcagcaactc accagcgaca tcatcatgtc agacagctac 1320

ctccgtcctg ccgcctcacc ccggctggag tcatga 1356

<210>84

<211>451

<212>PRT

<213> Homo sapiens

<400>84

Met Glu Ser Ser Pro Ile Pro Gln Ser Ser Gly Asn Ser Ser Thr Leu

1 5 10 15

Gly Arg Val Pro Gln Thr Pro Gly Pro Ser Thr Ala Ser Gly Val Pro

20 25 30

Glu Val Gly Leu Arg Asp Val Ala Ser Glu Ser Val Ala Leu Phe Phe

35 40 45

Met Leu Leu Leu Asp Leu Thr Ala Val Ala Gly Asn Ala Ala Val Met

50 55 60

Ala Val Ile Ala Lys Thr Pro Ala Leu Arg Lys Phe Val Phe Val Phe

65 70 75 80

His Leu Cys Leu Val Asp Leu Leu Ala Ala Leu Thr Leu Met Pro Leu

85 90 95

Ala Met Leu Ser Ser Ser Ala Leu Phe Asp His Ala Leu Phe Gly Glu

100 105 110

Val Ala Cys Arg Leu Tyr Leu Phe Leu Ser Val Cys Phe Val Ser Leu

115 120 125

Ala Ile Leu Ser Val Ser Ala Ile Asn Val Glu Arg Tyr Tyr Tyr Val

130 135 140

Val His Pro Met Arg Tyr Glu Val Arg Met Thr Leu Gly Leu Val Ala

145 150 155 160

Ser Val Leu Val Gly Val Trp Val Lys Ala Leu Ala Met Ala Ser Val

165 170 175

Pro Val Leu Gly Arg Val Ser Trp Glu Glu Gly Ala Pro Ser Val Pro

180 185 190

Pro Gly Cys Ser Leu Gln Trp Ser His Ser Ala Tyr Cys Gln Leu Phe

195 200 205

Val Val Val Phe Ala Val Leu Tyr Phe Leu Leu Pro Leu Leu Leu Ile

210 215 220

Leu Val Val Tyr Cys Ser Met Phe Arg Val Ala Arg Val Ala Ala Met

225 230 235 240

Gln His Gly Pro Leu Pro Thr Trp Met Glu Thr Pro Arg Gln Arg Ser

245 250 255

Glu Ser Leu Ser Ser Arg Ser Thr Met Val Thr Ser Ser Gly Ala Pro

260 265 270

Gln Thr Thr Pro His Arg Thr Phe Gly Gly Gly Lys Ala Lys Val Val

275 280 285

Leu Leu Ala Val Gly Gly Gln Phe Leu Leu Cys Trp Leu Pro Tyr Phe

290 295 300

Ser Phe His Leu Tyr Val Ala Leu Ser Ala Gln Pro Ile Ser Thr Gly

305 310 315 320

Gln Val Glu Ser Val Val Thr Trp Ile Gly Tyr Phe Cys Phe Thr Ser

325 330 335

Asn Pro Phe Phe Tyr Gly Cys Leu Asn Arg Gln Ile Arg Gly Glu Leu

340 345 350

Ser Lys Gln Phe Val Cys Phe Phe Lys Pro Ala Pro Glu Glu Glu Leu

355 360 365

Arg Leu Pro Ser Arg Glu Gly Ser Ile Glu Glu Asn Phe Leu Gln Phe

370 375 380

Leu Gln Gly Thr Gly Cys Pro Ser Glu Ser Trp Val Ser Arg Pro Leu

385 390 395 400

Pro Ser Pro Lys Gln Glu Pro Pro Ala Val Asp Phe Arg Ile Pro Gly

405 410 415

Gln Ile Ala Glu Glu Thr Ser Glu Phe Leu Glu Gln Gln Leu Thr Ser

420 425 430

Asp Ile Ile Met Ser Asp Ser Tyr Leu Arg Pro Ala Ala Ser Pro Arg

435 440 445

Leu Glu Ser

450

<210>85

<211>28

<212>DNA

<213> Homo sapiens

<400>85

caggaaggca aagaccacca tcatcatc 28

<210>86

<211>28

<212>DNA

<213> Homo sapiens

<400>86

gatgatgatg gtggtctttg ccttcctg 28

<210>87

<211>1041

<212>DNA

<213> Homo sapiens

<400>87

atggagagaa aatttatgtc cttgcaacca tccatctccg tatcagaaat ggaaccaaat 60

ggcaccttca gcaataacaa cagcaggaac tgcacaattg aaaacttcaa gagagaattt 120

ttcccaattg tatatctgat aatatttttc tggggagtct tgggaaatgg gttgtccata 180

tatgttttcc tgcagcctta taagaagtcc acatctgtga acgttttcat gctaaatctg 240

gccatttcag atctcctgtt cataagcacg cttcccttca gggctgacta ttatcttaga 300

ggctccaatt ggatatttgg agacctggcc tgcaggatta tgtcttattc cttgtatgtc 360

aacatgtaca gcagtattta tttcctgacc gtgctgagtg ttgtgcgttt cctggcaatg 420

gttcacccct ttcggcttct gcatgtcacc agcatcagga gtgcctggat cctctgtggg 480

atcatatgga tccttatcat ggcttcctca ataatgctcc tggacagtgg ctctgagcag 540

aacggcagtg tcacatcatg cttagagctg aatctctata aaattgctaa gctgcagacc 600

atgaactata ttgccttggt ggtgggctgc ctgctgccat ttttcacact cagcatctgt 660

tatctgctga tcattcgggt tctgttaaaa gtggaggtcc cagaatcggg gctgcgggtt 720

tctcacagga aggcaaagac caccatcatc atcaccttga tcatcttctt cttgtgtttc 780

ctgccctatc acacactgag gaccgtccac ttgacgacat ggaaagtggg tttatgcaaa 840

gacagactgc ataaagcttt ggttatcaca ctggccttgg cagcagccaa tgcctgcttc 900

aatcctctgc tctattactt tgctggggag aattttaagg acagactaaa gtctgcactc 960

agaaaaggcc atccacagaa ggcaaagaca aagtgtgttt tccctgttag tgtgtggttg 1020

agaaaggaaa caagagtata a 1041

<210>88

<211>346

<212>PRT

<213> Homo sapiens

<400>88

Met Glu Arg Lys Phe Met Ser Leu Gln Pro Ser Ile Ser Val Ser Glu

1 5 10 15

Met Glu Pro Asn Gly Thr Phe Ser Asn Asn Asn Ser Arg Asn Cys Thr

20 25 30

Ile Glu Asn Phe Lys Arg Glu Phe Phe Pro Ile Val Tyr Leu Ile Ile

35 40 45

Phe Phe Trp Gly Val Leu Gly Asn Gly Leu Ser Ile Tyr Val Phe Leu

50 55 60

Gln Pro Tyr Lys Lys Ser Thr Ser Val Asn Val Phe Met Leu Asn Leu

65 70 75 80

Ala Ile Ser Asp Leu Leu Phe Ile Ser Thr Leu Pro Phe Arg Ala Asp

85 90 95

Tyr Tyr Leu Arg Gly Ser Asn Trp Ile Phe Gly Asp Leu Ala Cys Arg

100 105 110

Ile Met Ser Tyr Ser Leu Tyr Val Asn Met Tyr Ser Ser Ile Tyr Phe

115 120 125

Leu Thr Val Leu Ser Val Val Arg Phe Leu Ala Met Val His Pro Phe

130 135 140

Arg Leu Leu His Val Thr Ser Ile Arg Ser Ala Trp Ile Leu Cys Gly

145 150 155 160

Ile Ile Trp Ile Leu Ile Met Ala Ser Ser Ile Met Leu Leu Asp Ser

165 170 175

Gly Ser Glu Gln Asn Gly Ser Val Thr Ser Cys Leu Glu Leu Asn Leu

180 185 190

Tyr Lys Ile Ala Lys Leu Gln Thr Met Asn Tyr Ile Ala Leu Val Val

195 200 205

Gly Cys Leu Leu Pro Phe Phe Thr Leu Ser Ile Cys Tyr Leu Leu Ile

210 215 220

Ile Arg Val Leu Leu Lys Val Glu Val Pro Glu Ser Gly Leu Arg Val

225 230 235 240

Ser His Arg Lys Ala Lys Thr Thr Ile Ile Ile Thr Leu Ile Ile Phe

245 250 255

Phe Leu Cys Phe Leu Pro Tyr His Thr Leu Arg Thr Val His Leu Thr

260 265 270

Thr Trp Lys Val Gly Leu Cys Lys Asp Arg Leu His Lys Ala Leu Val

275 280 285

Ile Thr Leu Ala Leu Ala Ala Ala Asn Ala Cys Phe Asn Pro Leu Leu

290 295 300

Tyr Tyr Phe Ala Gly Glu Asn Phe Lys Asp Arg Leu Lys Ser Ala Leu

305 310 315 320

Arg Lys Gly His Pro Gln Lys Ala Lys Thr Lys Cys Val Phe Pro Val

325 330 335

Ser Val Trp Leu Arg Lys Glu Thr Arg Val

340 345

<210>89

<211>28

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>89

ccagtgcaaa gctaagaaag tgatcttc 28

<210>90

<211>28

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>90

gaagatcact ttcttagctt tgcactgg 28

<210>91

<211>1527

<212>DNA

<213> Homo sapiens

<400>91

atgacgtcca cctgcaccaa cagcacgcgc gagagtaaca gcagccaacac gtgcatgccc 60

ctctccaaaa tgcccatcag cctggcccac ggcatcatcc gctcaaccgt gctggttatc 120

ttcctcgccg cctctttcgt cggcaacata gtgctggcgc tagtgttgca gcgcaagccg 180

cagctgctgc aggtgaccaa ccgttttatc tttaacctcc tcgtcaccga cctgctgcag 240

atttcgctcg tggccccctg ggtggtggcc acctctgtgc ctctcttctg gcccctcaac 300

agccacttct gcacggccct ggttagcctc accacctgt tcgccttcgc cagcgtcaac 360

accattgtcg tggtgtcagt ggatcgctac ttgtccatca tccaccctct ctcctacccg 420

tccaagatga cccagcgccg cggttacctg ctcctctatg gcacctggat tgtggccatc 480

ctgcagagca ctcctccact ctacggctgg ggccaggctg cctttgatga gcgcaatgct 540

ctctgctcca tgatctgggg ggccagcccc agctacacta ttctcagcgt ggtgtccttc 600

atcgtcattc cactgattgt catgattgcc tgctactccg tggtgttctg tgcagcccgg 660

aggcagcatg ctctgctgta caatgtcaag agacacagct tggaagtgcg agtcaaggac 720

tgtgtggaga atgaggatga agagggagca gagaagaagg aggagttcca ggatgagagt 780

gagtttcgcc gccagcatga aggtgaggtc aaggccaagg agggcagaat ggaagccaag 840

gacggcagcc tgaaggccaa ggaaggaagc acggggacca gtgagagtag tgtagaggcc 900

aggggcagcg aggaggtcag agagagcagc acggtggcca gcgacggcag catggagggt 960

aaggaaggca gcaccaaagt tgaggagaac agcatgaagg cagacaaggg tcgcacagag 1020

gtcaaccagt gcagcattga cttgggtgaa gatgacatgg agtttggtga agacgacatc 1080

aatttcagtg aggatgacgt cgaggcagtg aacatcccgg agagcctccc acccagtcgt 1140

cgtaacagca acagcaaccc tcctctgccc aggtgctacc agtgcaaagc taagaaagtg 1200

atcttcatca tcattttctc ctatgtgcta tccctggggc cctactgctt tttagcagtc 1260

ctggccgtgt gggtggatgt cgaaacccag gtaccccagt gggtgatcac cataatcatc 1320

tggcttttct tcctgcagtg ctgcatccac ccctatgtct atggctacat gcacaagacc 1380

attaagaagg aaatccagga catgctgaag aagttcttct gcaaggaaaa gcccccgaaa 1440

gaagatagcc accccagacct gcccggaaca gagggtggga ctgaaggcaa gattgtccct 1500

tcctacgatt ctgctacttt tccttga 1527

<210>92

<211>508

<212>PRT

<213> Homo sapiens

<400>92

Met Thr Ser Thr Cys Thr Asn Ser Thr Arg Glu Ser Asn Ser Ser His

1 5 10 15

Thr Cys Met Pro Leu Ser Lys Met Pro Ile Ser Leu Ala His Gly Ile

20 25 30

Ile Arg Ser Thr Val Leu Val Ile Phe Leu Ala Ala Ser Phe Val Gly

35 40 45

Asn Ile Val Leu Ala Leu Val Leu Gln Arg Lys Pro Gln Leu Leu Gln

50 55 60

Val Thr Asn Arg Phe Ile Phe Asn Leu Leu Val Thr Asp Leu Leu Gln

65 70 75 80

Ile Ser Leu Val Ala Pro Trp Val Val Ala Thr Ser Val Pro Leu Phe

85 90 95

Trp Pro Leu Asn Ser His Phe Cys Thr Ala Leu Val Ser Leu Thr His

100 105 110

Leu Phe Ala Phe Ala Ser Val Asn Thr Ile Val Val Val Ser Val Asp

115 120 125

Arg Tyr Leu Ser Ile Ile His Pro Leu Ser Tyr Pro Ser Lys Met Thr

130 135 140

Gln Arg Arg Gly Tyr Leu Leu Leu Tyr Gly Thr Trp Ile Val Ala Ile

145 150 155 160

Leu Gln Ser Thr Pro Pro Leu Tyr Gly Trp Gly Gln Ala Ala Phe Asp

165 170 175

Glu Arg Asn Ala Leu Cys Ser Met Ile Trp Gly Ala Ser Pro Ser Tyr

180 185 190

Thr Ile Leu Ser Val Val Ser Phe Ile Val Ile Pro Leu Ile Val Met

195 200 205

Ile Ala Cys Tyr Ser Val Val Phe Cys Ala Ala Arg Arg Gln His Ala

210 215 220

Leu Leu Tyr Asn Val Lys Arg His Ser Leu Glu Val Arg Val Lys Asp

225 230 235 240

Cys Val Glu Asn Glu Asp Glu Glu Gly Ala Glu Lys Lys Glu Glu Phe

245 250 255

Gln Asp Glu Ser Glu Phe Arg Arg Gln His Glu Gly Glu Val Lys Ala

260 265 270

Lys Glu Gly Arg Met Glu Ala Lys Asp Gly Ser Leu Lys Ala Lys Glu

275 280 285

Gly Ser Thr Gly Thr Ser Glu Ser Ser Val Glu Ala Arg Gly Ser Glu

290 295 300

Glu Val Arg Glu Ser Ser Thr Val Ala Ser Asp Gly Ser Met Glu Gly

305 310 315 320

Lys Glu Gly Ser Thr Lys Val Glu Glu Asn Ser Met Lys Ala Asp Lys

325 330 335

Gly Arg Thr Glu Val Asn Gln Cys Ser Ile Asp Leu Gly Glu Asp Asp

340 345 350

Met Glu Phe Gly Glu Asp Asp Ile Asn Phe Ser Glu Asp Asp Val Glu

355 360 365

Ala Val Asn Ile Pro Glu Ser Leu Pro Pro Ser Arg Arg Asn Ser Asn

370 375 380

Ser Asn Pro Pro Leu Pro Arg Cys Tyr Gln Cys Lys Ala Lys Lys Val

385 390 395 400

Ile Phe Ile Ile Ile Phe Ser Tyr Val Leu Ser Leu Gly Pro Tyr Cys

405 410 415

Phe Leu Ala Val Leu Ala Val Trp Val Asp Val Glu Thr Gln Val Pro

420 425 430

Gln Trp Val Ile Thr Ile Ile Ile Trp Leu Phe Phe Leu Gln Cys Cys

435 440 445

Ile His Pro Tyr Val Tyr Gly Tyr Met His Lys Thr Ile Lys Lys Glu

450 455 460

Ile Gln Asp Met Leu Lys Lys Phe Phe Cys Lys Glu Lys Pro Pro Lys

465 470 475 480

Glu Asp Ser His Pro Asp Leu Pro Gly Thr Glu Gly Gly Thr Glu Gly

485 490 495

Lys Ile Val Pro Ser Tyr Asp Ser Ala Thr Phe Pro

500 505

<210>93

<211>29

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>93

gccgccaccg cgccaagagg aagattggc 29

<210>94

<211>29

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>94

gccaatcttc ctcttggcgc ggtggcggc 29

<210>95

<211>1092

<212> DNA

<213> Homo sapiens

<400>95

atgggccccg gcgaggcgct gctggcgggt ctcctggtga tggtactg9c cgtggcgctg 60

ctatccaacg cactggtgct gctttgttgc gcctacagcg ctgagctccg cactcgagcc 120

tcaggcgtcc tcctggtgaa tctgtcgctg ggccacctgc tgctggcggc gctggacatg l80

cccttcacgc tgctcggtgt gatgcgcggg cggacaccgt cggcgcccgg cgcatgccaa 240

gtcattggct tcctggacac cttcctggcg tccaacgcgg cgctgagcgt ggcggcgctg 300

agcgcagacc agtggctggc agtgggcttc ccactgcgct acgccggacg cctgcgaccg 360

cgctatgccg gcctgctgct gggctgtgcc tggggacagt cgctggcctt ctcaggcgct 420

gcacttggct gctcgtggct tggctacagc agcgccttcg cgtcctgttc gctgcgcctg 480

ccgcccgagc ctgagcgtcc gcgcttcgca gccttcaccg ccacgctcca tgccgtgggc 540

ttcgtgctgc cgctggcggt gctctgcctc acctcgctcc aggtgcaccg ggtggcacgc 600

agccactgcc agcgcatgga caccgtcacc atgaaggcgc tcgcgctgct cgccgacctg 660

caccccagtg tgcggcagcg ctgcctcatc cagcagaagc ggcgccgcca ccgcgccacc 720

aggaagattg gcattgctat tgcgaccttc ctcatctgct ttgccccgta tgtcatgacc 780

aggctggcgg agctcgtgcc cttcgtcacc gtgaacgccc agaagggcat cctcagcaag 840

tgcctgacct acagcaaggc ggtggccgac ccgttcacgt actctctgct ccgccggccg 900

ttccgccaag tcctggccgg catggtgcac cggctgctga agagaaccccc gcgcccagca 960

tccacccatg acagctctct ggatgtggcc ggcatggtgc accagctgct gaagagaacc 1020

ccgcgcccag cgtccaccca caacggctct gtggacacag agaatgattc ctgcctgcag 1080

cagacacact ga 1092

<210>96

<211>363

<212>PRT

<213> Homo sapiens

<400>96

Met Gly Pro Gly Glu Ala Leu Leu Ala Gly Leu Leu Val Met Val Leu

1 5 10 15

Ala Val Ala Leu Leu Ser Asn Ala Leu Val Leu Leu Cys Cys Ala Tyr

20 25 30

Ser Ala Glu Leu Arg Thr Arg Ala Ser Gly Val Leu Leu Val Asn Leu

35 40 45

Ser Leu Gly His Leu Leu Leu Ala Ala Leu Asp Met Pro Phe Thr Leu

50 55 60

Leu Gly Val Met Arg Gly Arg Thr Pro Ser Ala Pro Gly Ala Cys Gln

65 70 75 80

Val Ile Gly Phe Leu Asp Thr Phe Leu Ala Ser Asn Ala Ala Leu Ser

85 90 95

Val Ala Ala Leu Ser Ala Asp Gln Trp Leu Ala Val Gly Phe Pro Leu

100 105 110

Arg Tyr Ala Gly Arg Leu Arg Pro Arg Tyr Ala Gly Leu Leu Leu Gly

115 120 125

Cys Ala Trp Gly Gln Ser Leu Ala Phe Ser Gly Ala Ala Leu Gly Cys

130 135 140

Ser Trp Leu Gly Tyr Ser Ser Ala Phe Ala Ser Cys Ser Leu Arg Leu

145 150 155 160

Pro Pro Glu Pro Glu Arg Pro Arg Phe Ala Ala Phe Thr Ala Thr Leu

165 170 175

His Ala Val Gly Phe Val Leu Pro Leu Ala Val Leu Cys Leu Thr Ser

180 185 190

Leu Gln Val His Arg Val Ala Arg Ser His Cys Gln Arg Met Asp Thr

195 200 205

Val Thr Met Lys Ala Leu Ala Leu Leu Ala Asp Leu His Pro Ser Val

210 215 220

Arg Gln Arg Cys Leu Ile Gln Gln Lys Arg Arg Arg His Arg Ala Thr

225 230 235 240

Arg Lys Ile Gly Ile Ala Ile Ala Thr Phe Leu Ile Cys Phe Ala Pro

245 250 255

Tyr Val Met Thr Arg Leu Ala Glu Leu Val Pro Phe Val Thr Val Asn

260 265 270

Ala Gln Lys Gly Ile Leu Ser Lys Cys Leu Thr Tyr Ser Lys Ala Val

275 280 285

Ala Asp Pro Phe Thr Tyr Ser Leu Leu Arg Arg Pro Phe Arg Gln Val

290 295 300

Leu Ala Gly Met Val His Arg Leu Leu Lys Arg Thr Pro Arg Pro Ala

305 310 315 320

Ser Thr His Asp Ser Ser Leu Asp Val Ala Gly Met Val His Gln Leu

325 330 335

Leu Lys Arg Thr Pro Arg Pro Ala Ser Thr His Asn Gly Ser Val Asp

340 345 350

Thr Glu Asn Asp Ser Cys Leu Gln Gln Thr His

355 360

<210>97

<211>34

<212>DNA

<213> Artificial sequence

<220>

<221>misc feature

<223> new sequence

<400>97

gatctctaga atggagtcct cacccatccc ccag 34

<210>98

<211>36

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>98

gatcgatatc cgtgactcca gccggggtga ggcggc 36

<210>99

<211>2610

<212>DNA

<213> Homo sapiens and rats

<400>99

atggagtcct cacccatccc ccagtcatca gggaactctt ccactttggg gagggtccct 60

caaaccccag gtccctctac tgccagtggg gtcccggagg tggggctacg ggatgttgct 120

tcggaatctg tggccctctt cttcatgctc ctgctggact tgactgctgt ggctggcaat 180

gccgctgtga tggccgtgat cgccaagacg cctgccctcc gaaaatttgt cttcgtcttc 240

cacctctgcc tggtggacct gctggctgcc ctgaccctca tgcccctggc catgctctcc 300

agctctgccc tctttgacca cgccctcttt ggggaggtgg cctgccgcct ctacttgttt 360

ctgagcgtgt gctttgtcag cctggccatc ctctcggtgt cagccatcaa tgtggagcgc 420

tactattacg tagtccaccc catgcgctac gaggtgcgca tgacgctggg gctggtggcc 480

tctgtgctgg tgggtgtgtg ggtgaaggcc ttggccatgg cttctgtgcc agtgttggga 540

agggtctcct gggaggaagg agctcccagt gtccccccag gctgttcact ccagtggagc 600

cacagtgcct actgccagct ttttgtggtg gtctttgctg tcctttactt tctgttgccc 660

ctgctcctca tacttgtggt ctactgcagc atgttccgag tggcccgcgt ggctgccatg 720

cagcacgggc cgctgcccac gtggatggag acaccccggc aacgctccga atctctcagc 780

agccgctcca cgatggtcac cagctcgggg gccccccaga ccaccccaca ccggacgttt 840

gggggaggga aagcagcagt ggttctcctg gctgtggggg gacagttcct gctctgttgg 900

ttgccctact tctctttcca cctctatgtt gccctgagtg ctcagcccat ttcaactggg 960

caggtggaga gtgtggtcac ctggattggc tacttttgct tcacttccaa ccctttcttc 1020

tatggatgtc tcaaccggca gatccggggg gagctcagca agcagtttgt ctgcttcttc 1080

aagccagctc cagaggagga gctgaggctg cctagccggg agggctccat tgaggagaac 1140

ttcctgcagt tccttcaggg gactggctgt ccttctgagt cctgggtttc ccgaccccta 1200

cccagcccca agcaggagcc acctgctgtt gactttcgaa tcccaggcca gatagctgag 1260

gagacctctg agttcctgga gcagcaactc accagcgaca tcatcatgtc agacagctac 1320

ctccgtcctg ccgcctcacc ccggctggag tcagcgatat ctgcagaatt ccaccacact 1380

ggactagtgg atccgagctc ggtaccaagc ttgggctgca ggtcgatggg ctgcctcggc 1440

aacagtaaga ccgaggacca gcgcaacgag gagaaggcgc agcgcgaggc caacaaaaag 1500

atcgagaagc agctgcagaa ggacaagcag gtctaccggg ccacgcaccg cctgctgctg 1560

ctgggtgctg gagagtctgg caaaagcacc attgtgaagc agatgaggat cctacatgtt 1620

aatgggttta acggagaggg cggcgaagag gacccgcagg ctgcaaggag caacagcgat 1680

ggtgagaagg ccaccaaagt gcaggacatc aaaaacaacc tgaaggaggc cattgaaacc 1740

attgtggccg ccatgagcaa cctggtgccc cccgtggagc tggccaaccc tgagaaccag 1800

ttcagagtgg actacattct gagcgtgatg aacgtgccaa actttgactt cccacctgaa 1860

ttctatgagc atgccaaggc tctgtggggag gatgaggggag ttcgtgcctg ctacgagcgc 1920

tccaacgagt accagctgat cgactgtgcc cagtacttcc tggacaagat tgatgtgatc 1980

aagcaggccg actacgtgcc aagtgaccag gacctgcttc gctgccgcgt cctgacctct 2040

ggaatctttg agaccaagtt ccaggtggac aaagtcaact tccacatgtt cgatgtgggc 2100

ggccagcgcg atgaacgccg caagtggatc cagtgcttca atgatgtgac tgccatcatc 2160

ttcgtggtgg ccagcagcag ctacaacatg gtcatccggg aggacaacca gaccaaccgt 2220

ctgcaggagg ctctgaacct cttcaagagc atctggaaca acagatggct gcgtaccatc 2280

tctgtgatcc tcttcctcaa caagcaagat ctgcttgctg agaaggtcct cgctgggaaa 2340

tcgaagattg aggactactt tccagagttc gctcgctaca ccactcctga ggatgcgact 2400

cccgagcccg gagaggaccc acgcgtgacc cgggccaagt acttcatccg ggatgagttt 2460

ctgagaatca gcactgctag tggagatgga cgtcactact gctaccctca ctttacctgc 2520

gccgtggaca ctgagaacat ccgccgtgtc ttcaacgact gccgtgacat catccagcgc 2580

atgcatcttc gccaatacga gctgctctaa 2610

<210>100

<211>869

<212>PRT

<213> Homo sapiens and rats

<400>100

Met Glu Ser Ser Pro Ile Pro Gln Ser Ser Gly Asn Ser Ser Thr Leu

1 5 10 15

Gly Arg Val Pro Gln Thr Pro Gly Pro Ser Thr Ala Ser Gly Val Pro

20 25 30

Glu Val Gly Leu Arg Asp Val Ala Ser Glu Ser Val Ala Leu Phe Phe

35 40 45

Met Leu Leu Leu Asp Leu Thr Ala Val Ala Gly Asn Ala Ala Val Met

50 55 60

Ala Val Ile Ala Lys Thr Pro Ala Leu Arg Lys Phe Val Phe Val Phe

65 70 75 80

His Leu Cys Leu Val Asp Leu Leu Ala Ala Leu Thr Leu Met Pro Leu

85 90 95

Ala Met Leu Ser Ser Ser Ala Leu Phe Asp His Ala Leu Phe Gly Glu

100 105 110

Val Ala Cys Arg Leu Tyr Leu Phe Leu Ser Val Cys Phe Val Ser Leu

115 120 125

Ala Ile Leu Ser Val Ser Ala Ile Asn Val Glu Arg Tyr Tyr Tyr Val

130 135 140

Val His Pro Met Arg Tyr Glu Val Arg Met Thr Leu Gly Leu Val Ala

145 150 155 160

Ser Val Leu Val Gly Val Trp Val Lys Ala Leu Ala Met Ala Ser Val

165 170 175

Pro Val Leu Gly Arg Val Ser Trp Glu Glu Gly Ala Pro Ser Val Pro

180 185 190

Pro Gly Cys Ser Leu Gln Trp Ser His Ser Ala Tyr Cys Gln Leu Phe

195 200 205

Val Val Val Phe Ala Val Leu Tyr Phe Leu Leu Pro Leu Leu Leu Ile

210 215 220

Leu Val Val Tyr Cys Ser Met Phe Arg Val Ala Arg Val Ala Ala Met

225 230 235 240

Gln His Gly Pro Leu Pro Thr Trp Met Glu Thr Pro Arg Gln Arg Ser

245 250 255

Glu Ser Leu Ser Ser Arg Ser Thr Met Val Thr Ser Ser Gly Ala Pro

260 265 270

Gln Thr Thr Pro His Arg Thr Phe Gly Gly Gly Lys Ala Ala Val Val

275 280 285

Leu Leu Ala Val Gly Gly Gln Phe Leu Leu Cys Trp Leu Pro Tyr Phe

290 295 300

Ser Phe His Leu Tyr Val Ala Leu Ser Ala Gln Pro Ile Ser Thr Gly

305 310 315 320

Gln Val Glu Ser Val Val Thr Trp Ile Gly Tyr Phe Cys Phe Thr Ser

325 330 335

Asn Pro Phe Phe Tyr Gly Cys Leu Asn Arg Gln Ile Arg Gly Glu Leu

340 345 350

Ser Lys Gln Phe Val Cys Phe Phe Lys Pro Ala Pro Glu Glu Glu Leu

355 360 365

Arg Leu Pro Ser Arg Glu Gly Ser Ile Glu Glu Asn Phe Leu Gln Phe

370 375 380

Leu Gln Gly Thr Gly Cys Pro Ser Glu Ser Trp Val Ser Arg Pro Leu

385 390 395 400

Pro Ser Pro Lys Gln Glu Pro Pro Ala Val Asp Phe Arg Ile Pro Gly

405 410 415

Gln Ile Ala Glu Glu Thr Ser Glu Phe Leu Glu Gln Gln Leu Thr Ser

420 425 430

Asp Ile Ile Met Ser Asp Ser Tyr Leu Arg Pro Ala Ala Ser Pro Arg

435 440 445

Leu Glu Ser Ala Ile Ser Ala Glu Phe His His Thr Gly Leu Val Asp

450 455 460

Pro Ser Ser Val Pro Ser Leu Gly Cys Arg Ser Met Gly Cys Leu Gly

465 470 475 480

Asn Ser Lys Thr Glu Asp Gln Arg Asn Glu Glu Lys Ala Gln Arg Glu

485 490 495

Ala Asn Lys Lys Ile Glu Lys Gln Leu Gln Lys Asp Lys Gln Val Tyr

500 505 510

Arg Ala Thr His Arg Leu Leu Leu Leu Gly Ala Gly Glu Ser Gly Lys

515 520 525

Ser Thr Ile Val Lys Gln Met Arg Ile Leu His Val Asn Gly Phe Asn

530 535 540

Gly Glu Gly Gly Glu Glu Asp Pro Gln Ala Ala Arg Ser Asn Ser Asp

545 550 555 560

Gly Glu Lys Ala Thr Lys Val Gln Asp Ile Lys Asn Asn Leu Lys Glu

565 570 575

Ala Ile Glu Thr Ile Val Ala Ala Met Ser Asn Leu Val Pro Pro Val

580 585 590

Glu Leu Ala Asn Pro Glu Asn Gln Phe Arg Val Asp Tyr Ile Leu Ser

595 600 605

Val Met Asn Val Pro Asn Phe Asp Phe Pro Pro Glu Phe Tyr Glu His

610 615 620

Ala Lys Ala Leu Trp Glu Asp Glu Gly Val Arg Ala Cys Tyr Glu Arg

625 630 635 640

Ser Asn Glu Tyr Gln Leu Ile Asp Cys Ala Gln Tyr Phe Leu Asp Lys

645 650 655

Ile Asp Val Ile Lys Gln Ala Asp Tyr Val Pro Ser Asp Gln Asp Leu

660 665 670

Leu Arg Cys Arg Val Leu Thr Ser Gly Ile Phe Glu Thr Lys Phe Gln

675 680 685

Val Asp Lys Val Asn Phe His Met Phe Asp Val Gly Gly Gln Arg Asp

690 695 700

Glu Arg Arg Lys Trp Ile Gln Cys Phe Asn Asp Val Thr Ala Ile Ile

705 710 715 720

Phe Val Val Ala Ser Ser Ser Tyr Asn Met Val Ile Arg Glu Asp Asn

725 730 735

Gln Thr Asn Arg Leu Gln Glu Ala Leu Asn Leu Phe Lys Ser Ile Trp

740 745 750

Asn Asn Arg Trp Leu Arg Thr Ile Ser Val Ile Leu Phe Leu Asn Lys

755 760 765

Gln Asp Leu Leu Ala Glu Lys Val Leu Ala Gly Lys Ser Lys Ile Glu

770 775 780

Asp Tyr Phe Pro Glu Phe Ala Arg Tyr Thr Thr Pro Glu Asp Ala Thr

785 790 795 800

Pro Glu Pro Gly Glu Asp Pro Arg Val Thr Arg Ala Lys Tyr Phe Ile

805 810 815

Arg Asp Glu Phe Leu Arg Ile Ser Thr Ala Ser Gly Asp Gly Arg His

820 825 830

Tyr Cys Tyr Pro His Phe Thr Cys Ala Val Asp Thr Glu Asn Ile Arg

835 840 845

Arg Val Phe Asn Asp Cys Arg Asp Ile Ile Gln Arg Met His Leu Arg

850 855 860

Gln Tyr Glu Leu Leu

865

<210>101

<211>30

<2l2>DNA

<213> Artificial sequence

<220>

<221>misc feature

<223> new sequence

<400>101

tctagaatga cgtccacctg caccaacagc 30

<210>102

<211>34

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>102

gatatcgcag gaaaagtagc agaatcgtag gaag 34

<210>103

<211>2781

<212>DNA

<213> Homo sapiens and rats

<400>103

atgacgtcca cctgcaccaa cagcacgcgc gagagtaaca gcagccaacac gtgcatgccc 60

ctctccaaaa tgcccatcag cctggcccac ggcatcatcc gctcaaccgt gctggttatc 120

ttcctcgccg cctctttcgt cggcaacata gtgctggcgc tagtgttgca gcgcaagccg 180

cagctgctgc aggtgaccaa ccgttttatc tttaacctcc tcgtcaccga cctgctgcag 240

atttcgctcg tggccccctg ggtggtggcc acctctgtgc ctctcttctg gcccctcaac 300

agccacttct gcacggccct ggttagcctc accacctgt tcgccttcgc cagcgtcaac 360

accattgtcg tggtgtcagt ggatcgctac ttgtccatca tccaccctct ctcctacccg 420

tccaagatga cccagcgccg cggttacctg ctcctctatg gcacctggat tgtggccatc 480

ctgcagagca ctcctccact ctacggctgg ggccaggctg cctttgatga gcgcaatgct 540

ctctgctcca tgatctgggg ggccagcccc agctacacta ttctcagcgt ggtgtccttc 600

atcgtcattc cactgattgt catgattgcc tgctactccg tggtgttctg tgcagcccgg 660

aggcagcatg ctctgctgta caatgtcaag agacacagct tggaagtgcg agtcaaggac 720

tgtgtggaga atgaggatga agagggagca gagaagaagg aggagttcca ggatgagagt 780

gagtttcgcc gccagcatga aggtgaggtc aaggccaagg agggcagaat ggaagccaag 840

gacggcagcc tgaaggccaa ggaaggaagc acggggacca gtgagagtag tgtagaggcc 900

aggggcagcg aggaggtcag agagagcagc acggtggcca gcgacggcag catggagggt 960

aaggaaggca gcaccaaagt tgaggagaac agcatgaagg cagacaaggg tcgcacagag 1020

gtcaaccagt gcagcattga cttgggtgaa gatgacatgg agtttggtga agacgacatc 1080

aatttcagtg aggatgacgt cgaggcagtg aacatcccgg agagcctccc acccagtcgt 1140

cgtaacagca acagcaaccc tcctctgccc aggtgctacc agtgcaaagc tgctaaagtg 1200

atcttcatca tcattttctc ctatgtgcta tccctggggc cctactgctt tttagcagtc 1260

ctggccgtgt gggtggatgt cgaaacccag gtaccccagt gggtgatcac cataatcatc 1320

tggcttttct tcctgcagtg ctgcatccac ccctatgtct atggctacat gcacaagacc 1380

attaagaagg aaatccagga catgctgaag aagttcttct gcaaggaaaa gcccccgaaa 1440

gaagatagcc accccagacct gcccggaaca gagggtggga ctgaaggcaa gattgtccct 1500

tcctacgatt ctgctacttt tcctgcgata tctgcagaat tccaccacac tggactagtg 1560

gatccgagct cggtaccaag cttgggctgc aggtcgatgg gctgcctcgg caacagtaag 1620

accgaggacc agcgcaacga ggagaaggcg cagcgcgagg ccaacaaaaa gatcgagaag 1680

cagctgcaga aggacaagca ggtctaccgg gccacgcacc gcctgctgct gctgggtgct 1740

ggagagtctg gcaaaagcac cattgtgaag cagatgagga tcctacatgt taatgggttt 1800

aacggagagg gcggcgaaga ggacccgcag gctgcaagga gcaacagcga tggtgagaag 1860

gccaccaaag tgcaggacat caaaaacaac ctgaaggagg ccattgaaac cattgtggcc 1920

gccatgagca acctggtgcc ccccgtggag ctggccaacc ctgagaacca gttcagagtg 1980

gactacattc tgagcgtgat gaacgtgcca aactttgact tcccacctga attctatgag 2040

catgccaagg ctctgtggga ggatgaggga gttcgtgcct gctacgagcg ctccaacgag 2100

taccagctga tcgactgtgc ccagtacttc ctggacaaga ttgatgtgat caagcaggcc 2160

gactacgtgc caagtgacca ggacctgctt cgctgccgcg tcctgacctc tggaatcttt 2220

gagaccaagt tccaggtgga caaagtcaac ttccacatgt tcgatgtggg cggccagcgc 2280

gatgaacgcc gcaagtggat ccagtgcttc aatgatgtga ctgccatcat cttcgtggtg 2340

gccagcagca gctacaacat ggtcatccgg gaggacaacc agaccaaccg tctgcaggag 2400

gctctgaacc tcttcaagag catctggaac aacagatggc tgcgtaccat ctctgtgatc 2460

ctcttcctca acaagcaaga tctgcttgct gagaaggtcc tcgctgggaa atcgaagatt 2520

gaggactact ttccagagtt cgctcgctac accactcctg aggatgcgac tcccgagccc 2580

ggagaggacc cacgcgtgac ccgggccaag tacttcatcc gggatgagtt tctgagaatc 2640

agcactgcta gtggagatgg acgtcactac tgctaccctc actttacctg cgccgtggac 2700

actgagaaca tccgccgtgt cttcaacgac tgccgtgaca tcatccagcg catgcatctt 2760

cgccaatacg agctgctcta a 2781

<210>104

<211>926

<212>PRT

<213> Homo sapiens and rats

<400>104

Met Thr Ser Thr Cys Thr Asn Ser Thr Arg Glu Ser Asn Ser Ser His

1 5 10 15

Thr Cys Met Pro Leu Ser Lys Met Pro Ile Ser Leu Ala His Gly Ile

20 25 30

Ile Arg Ser Thr Val Leu Val Ile Phe Leu Ala Ala Ser Phe Val Gly

35 40 45

Asn Ile Val Leu Ala Leu Val Leu Gln Arg Lys Pro Gln Leu Leu Gln

50 55 60

Val Thr Asn Arg Phe Ile Phe Asn Leu Leu Val Thr Asp Leu Leu Gln

65 70 75 80

Ile Ser Leu Val Ala Pro Trp Val Val Ala Thr Ser Val Pro Leu Phe

85 90 95

Trp Pro Leu Asn Ser His Phe Cys Thr Ala Leu Val Ser Leu Thr His

100 105 110

Leu Phe Ala Phe Ala Ser Val Asn Thr Ile Val Val Val Ser Val Asp

115 120 125

Arg Tyr Leu Ser Ile Ile His Pro Leu Ser Tyr Pro Ser Lys Met Thr

130 135 140

Gln Arg Arg Gly Tyr Leu Leu Leu Tyr Gly Thr Trp Ile Val Ala Ile

145 150 155 160

Leu Gln Ser Thr Pro Pro Leu Tyr Gly Trp Gly Gln Ala Ala Phe Asp

165 170 175

Glu Arg Asn Ala Leu Cys Ser Met Ile Trp Gly Ala Ser Pro Ser Tyr

180 185 190

Thr Ile Leu Ser Val Val Ser Phe Ile Val Ile Pro Leu Ile Val Met

195 200 205

Ile Ala Cys Tyr Ser Val Val Phe Cys Ala Ala Arg Arg Gln His Ala

210 215 220

Leu Leu Tyr Asn Val Lys Arg His Ser Leu Glu Val Arg Val Lys Asp

225 230 235 240

Cys Val Glu Asn Glu Asp Glu Glu Gly Ala Glu Lys Lys Glu Glu Phe

245 250 255

Gln Asp Glu Ser Glu Phe Arg Arg Gln His Glu Gly Glu Val Lys Ala

260 265 270

Lys Glu Gly Arg Met Glu Ala Lys Asp Gly Ser Leu Lys Ala Lys Glu

275 280 285

Gly Ser Thr Gly Thr Ser Glu Ser Ser Val Glu Ala Arg Gly Ser Glu

290 295 300

Glu Val Arg Glu Ser Ser Thr Val Ala Ser Asp Gly Ser Met Glu Gly

305 310 315 320

Lys Glu Gly Ser Thr Lys Val Glu Glu Asn Ser Met Lys Ala Asp Lys

325 330 335

Gly Arg Thr Glu Val Asn Gln Cys Ser Ile Asp Leu Gly Glu Asp Asp

340 345 350

Met Glu Phe Gly Glu Asp Asp Ile Asn Phe Ser Glu Asp Asp Val Glu

355 360 365

Ala Val Asn Ile Pro Glu Ser Leu Pro Pro Ser Arg Arg Asn Ser Asn

370 375 380

Ser Asn Pro Pro Leu Pro Arg Cys Tyr Gln Cys Lys Ala Ala Lys Val

385 390 395 400

Ile Phe Ile Ile Ile Phe Ser Tyr Val Leu Ser Leu Gly Pro Tyr Cys

405 410 415

Phe Leu Ala Val Leu Ala Val Trp Val Asp Val Glu Thr Gln Val Pro

420 425 430

Gln Trp Val Ile Thr Ile Ile Ile Trp Leu Phe Phe Leu Gln Cys Cys

435 440 445

Ile His Pro Tyr Val Tyr Gly Tyr Met His Lys Thr Ile Lys Lys Glu

450 455 460

Ile Gln Asp Met Leu Lys Lys Phe Phe Cys Lys Glu Lys Pro Pro Lys

465 470 475 480

Glu Asp Ser His Pro Asp Leu Pro Gly Thr Glu Gly Gly Thr Glu Gly

485 490 495

Lys Ile Val Pro Ser Tyr Asp Ser Ala Thr Phe Pro Ala Ile Ser Ala

500 505 510

Glu Phe His His Thr Gly Leu Val Asp Pro Ser Ser Val Pro Ser Leu

515 520 525

Gly Cys Arg Ser Met Gly Cys Leu Gly Asn Ser Lys Thr Glu Asp Gln

530 535 540

Arg Asn Glu Glu Lys Ala Gln Arg Glu Ala Asn Lys Lys Ile Glu Lys

545 550 555 560

Gln Leu Gln Lys Asp Lys Gln Val Tyr Arg Ala Thr His Arg Leu Leu

565 570 575

Leu Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr Ile Val Lys Gln Met

580 585 590

Arg Ile Leu His Val Asn Gly Phe Asn Gly Glu Gly Gly Glu Glu Asp

595 600 605

Pro Gln Ala Ala Arg Ser Asn Ser Asp Gly Glu Lys Ala Thr Lys Val

610 615 620

Gln Asp Ile Lys Asn Asn Leu Lys Glu Ala Ile Glu Thr Ile Val Ala

625 630 635 640

Ala Met Ser Asn Leu Val Pro Pro Val Glu Leu Ala Asn Pro Glu Asn

645 650 655

Gln Phe Arg Val Asp Tyr Ile Leu Ser Val Met Asn Val Pro Asn Phe

660 665 670

Asp Phe Pro Pro Glu Phe Tyr Glu His Ala Lys Ala Leu Trp Glu Asp

675 680 685

Glu Gly Val Arg Ala Cys Tyr Glu Arg Ser Asn Glu Tyr Gln Leu Ile

690 695 700

Asp Cys Ala Gln Tyr Phe Leu Asp Lys Ile Asp Val Ile Lys Gln Ala

705 710 715 720

Asp Tyr Val Pro Ser Asp Gln Asp Leu Leu Arg Cys Arg Val Leu Thr

725 730 735

Ser Gly Ile Phe Glu Thr Lys Phe Gln Val Asp Lys Val Asn Phe His

740 745 750

Met Phe Asp Val Gly Gly Gln Arg Asp Glu Arg Arg Lys Trp Ile Gln

755 760 765

Cys Phe Asn Asp Val Thr Ala Ile Ile Phe Val Val Ala Ser Ser Ser

770 775 780

Tyr Asn Met Val Ile Arg Glu Asp Asn Gln Thr Asn Arg Leu Gln Glu

785 790 795 800

Ala Leu Asn Leu Phe Lys Ser Ile Trp Asn Asn Arg Trp Leu Arg Thr

805 810 815

Ile Ser Val Ile Leu Phe Leu Asn Lys Gln Asp Leu Leu Ala Glu Lys

820 825 830

Val Leu Ala Gly Lys Ser Lys Ile Glu Asp Tyr Phe Pro Glu Phe Ala

835 840 845

Arg Tyr Thr Thr Pro Glu Asp Ala Thr Pro Glu Pro Gly Glu Asp Pro

850 855 860

Arg Val Thr Arg Ala Lys Tyr Phe Ile Arg Asp Glu Phe Leu Arg Ile

865 870 875 880

Ser Thr Ala Ser Gly Asp Gly Arg His Tyr Cys Tyr Pro His Phe Thr

885 890 895

Cys Ala Val Asp Thr Glu Asn Ile Arg Arg Val Phe Asn Asp Cys Arg

900 905 910

Asp Ile Ile Gln Arg Met His Leu Arg Gln Tyr Glu Leu Leu

915 920 925

<210>105

<211>23

<212>DNA

<213> Artificial sequence

<220>

<221>misc feature

<223> new sequence

<400>105

catgtatgcc agcgtcctgc tcc 23

<210>106

<211>24

<212>DNA

<2l3> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>106

gctatgcctg aagccagtct tgtg 24

<210>107

<211>25

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>107

gcacctgctc ctgagcacct tctcc 25

<210>108

<211>26

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>108

cacagcgctg cagccctgca gctggc 26

<210>109

<211>24

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>109

ccagtgatga ctctgtccag cctg 24

<210>110

<211>24

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>110

cagacacttg gcagggacga ggtg 24

<210>111

<211>26

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>111

cttgtggtct actgcagcat gttccg 26

<210>112

<211>25

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>112

catatccctc cgagtgtcca gcggc 25

<210>113

<211>24

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>113

atggatcctt atcatggctt cctc 24

<210>114

<211>27

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>114

caagaacagg tctcatctaa gagctcc 27

<210>115

<211>26

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>115

ctctgatgcc atctgctgga ttcctg 26

<210>116

<211>26

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>116

gtagtccact gaaagtccag tgatcc 26

<210>117

<211>24

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>117

tggtggcgat ggccaacagc gctc 24

<210>118

<211>24

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>118

gttgcgcctt agcgacagat gacc 24

<210>119

<211>23

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>119

tcaacctgta tagcagcatc ctc 23

<210>120

<211>23

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>120

aaggagtagc agaatggtta gcc 23

<210>121

<211>24

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>121

gacacctgtc agcggtcgtg tgtg 24

<210>122

<211>27

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>122

ctgatggaag tagaggctgt ccatctc 27

<210>123

<211>24

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>123

gcgctgagcg cagaccagtg gctg 24

<210>124

<211>24

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>124

cacggtgacg aagggcacga gctc 24

<210>125

<211>24

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>125

agccatccct gccaggaagc atgg 24

<210>126

<211>25

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>126

ccaggtaggt gtgcagcaca atggc 25

<210>127

<211>25

<212> DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>127

ctgttcaaca gggctggttg gcaac 25

<210>128

<211>25

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>128

atcatgtcta gactcatggt gatcc 25

<210>129

<211>6

<212>PRT

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>129

Thr Leu Glu Ser Ile Met

1 5

<210>130

<211>5

<212>PRT

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>130

Glu Tyr Asn Leu Val

1 5

<210>131

<211>5

<212>PRT

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>131

Asp Cys Gly Leu Phe

1 5

<210>132

<211>36

<212>PRT

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>132

Gly Ala Thr Cys Ala Ala Gly Cys Thr Thr Cys Cys Ala Thr Gly Gly

1 5 10 15

Cys Gly Thr Gly Cys Thr Gly Cys Cys Thr Gly Ala Gly Cys Gly Ala

20 25 30

Gly Gly Ala Gly

35

<210>133

<2l1>53

<212>PRT

<213> Artificial sequence

<220>

<221>misc_feature

<223> new sequence

<400>133

Gly Ala Thr Cys Gly Gly Ala Thr Cys Cys Thr Thr Ala Gly Ala Ala

1 5 10 15

Cys Ala Gly Gly Cys Cys Gly Cys Ala Gly Thr Cys Cys Thr Thr Cys

20 25 30

Ala Gly Gly Thr Thr Cys Ala Gly Cys Thr Gly Cys Ala Gly Gly Ala

35 40 45

Thr Gly Gly Thr Gly

50

Claims (10)

1. An isolated G protein-coupled receptor whose amino acid sequence is SEQ ID NO.22. 2. An isolated polynucleotide whose nucleotide sequence is SEQ ID NO.21. 3. An isolated polynucleotide, consisting of the nucleotide sequence of a G protein-coupled receptor whose amino acid sequence is SEQ ID NO.22. 4. A vector comprising the polynucleotide of claim 2 or 3, wherein the vector is an expression vector. 5. A host cell comprising the vector of claim 4. 6. A method for identifying one or more candidate compounds as an agonist of a G protein-coupled receptor whose amino acid sequence is SEQ ID NO.22, a partial agonist or an inverse agonist, comprising the following steps: (a) contacting said one or more compounds with a host cell or with a cell membrane of a host cell expressing said receptor; and (b) measuring the ability of said one or more compounds to inhibit or stimulate the functionality of said receptor. 7. the method for claim 6, wherein host cell comprises expression vector, and described expression vector comprises the polynucleotide that is made up of a nucleotide sequence, and described nucleotide sequence coding aminoacid sequence is the G albumen of SEQ ID NO.22 Coupled receptors. 8. The method of claim 6, further comprising medicating said agonist, partial agonist or inverse agonist. 9. The method of any one of claims 6-8, wherein the host cell is a mammalian cell. 10. The method of any one of claims 6-8, wherein the host cell is a yeast cell.

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