GB2374596A

GB2374596A - An isolated histamine like receptor

Info

Publication number: GB2374596A
Application number: GB0030294A
Authority: GB
Inventors: Steven Michael Foord; Diane Michele Ignar
Original assignee: Glaxo Group Ltd
Current assignee: Glaxo Group Ltd
Priority date: 2000-12-12
Filing date: 2000-12-12
Publication date: 2002-10-23
Also published as: GB0030294D0

Abstract

An isolated histamine-like receptor polypeptide comprising the amino acid sequence of SEQ ID No 2 or a variant, or a fragment, of this which is capable of binding histamine or a structurally related molecule. Alternatively the polypeptide may have at least 80% identity to SEQ ID No 2. Methods of screening compounds, in vivo and in vitro, to identify those capable of binding the receptor or modulating its expression are described, as are those substances identified and methods of treatment using them. Antibodies which bind the receptor specifically are also described. The polynucleotide encoding the putative G protein coupled receptor (GPCR) protein is also described which may be used to express the receptor from a vector in a host eukaryotic cell. Methods of gene therapy involving expressing the polynucleotide are described and its use as a probe to determine expression of the receptor gene.

Description

PROTEIN Field of the Invention The present invention relates to histamine receptor polypeptides.

Background of the Invention G-protein coupled receptors (GPCRs) are a super-family of membrane receptors that mediate a wide variety of biological functions. Upon binding of extracellular ligands, GPCRs interact with a specific subset of heterotrimeric G proteins that can, in their activated forms, inhibit or activate various effector enzymes and/or ion channels. All GPCRs are predicted to share a common molecular architecture consisting of seven transmembrane helices linked by alternating intracellular and extracellular loops. The extracellular receptor surface has been shown to be involved in ligand binding whereas the intracellular portions are involved in G protein recognition and activation.

Histamine is a mediator of inflammation and allergy which is released by mast cells. It is also released by parietal cells of the gastric mucosa and is distributed widely in the both neuronal and non-neuronal cell in the brain and spinal cord.

Histamine has been implicated in CNS functions such as arousal, sexual function, and analgesia.

Summary of the Invention

A novel histamine-like receptor, referred to herein as HIPHUM0000099A, is now provided. HIPHUM0000099A is primarily expressed in cerebellum, lung, testes, myometrium, ovary, thyroid, rectum, and urinary bladder. The polypeptide is also expressed in thymus, tonsil, spleen, adenoid, prostate, omentum, jejunum, T cells, dendritic cells, osteoblasts and endothelial cells. The expression of the polypeptide is downreguled in HSV, HBV and HIV-infected cells. The novel histamine-like receptor is a screening target for the identification and development of novel pharmaceutical agents, including modulators of histamine receptor activity. These agents may be used in the treatment and/or prophylaxis of disorders such as neurodegenerative disorders, for example cerebellar and spinocerebellar disorders,

ataxia, spinocerebellar ataxia and atrophy, progressive supranuclear palsy, Huntington's disease, amyotrophic lateral sclerosis, Alzheimer's disease, Bell's palsy, cerebral palsy, Parkinson's disease, tardive dyskinesia, tremor, CNS disorders for example pain, psychogenic erectile dysfunction, seizures, migraine, attention deficit, depression, anxiety, psychosis, mania, schizophrenia, diseases related to immune function, autoimmune function, viruses, inflammation or immunodeficiency, for example asthma, COPD, Crohn's disease, Inflammatory bowel syndrome, ulcerative colitis, gastroenteritis, inflammatory pain, allergy, rhinitis, dermatitis, anaphylaxis and septic shock, viral diseases, for example HBV, HSV and HIV, general inflammation, rheumatoid arthritis, osteoarthritis, osteoporosis, osteopetrosis, male and female reproductive fertility (ovary and testes expression), pre-term labor, dysmenorrhea, induction of labor, polycystic ovary disease, ovarian cysts, urinary incontinence, for example urge, stress, or mixed incontinence, hypothyroidism, hyperthyroidism, control of metabolic rate, obesity and/or insulindependent diabetes.

Accordingly, the present invention provides an isolated histamine-like receptor polypeptide comprising (i) the amino acid sequence of SEQ ID NO: 2; (ii) a variant thereof which is capable of binding histamine or a structurally related molecule; or (iii) a fragment of (i) or (ii) which is capable of binding histamine or a structurally related molecule.

According to another aspect of the invention there is provided a polynucleotide encoding a polypeptide of the invention which polynucleotide includes a sequence comprising: (a) the nucleic acid sequence of SEQ ID NO: 1 and/or a sequence complementary thereto; (b) a sequence which hybridises under stringent conditions to a sequence as defined in (a); (c) a sequence that is degenerate as a result of the genetic code to a sequence as defined in (a) or (b); or (d) a sequence having at least 60% identity to a sequence as defined in

(a), (b) or (c).

The invention also provides : an expression vector which comprises a polynucleotide of the invention and which is capable of expressing a polypeptide of the invention ; a host cell comprising an expression vector of the invention ; a method of producing a polypeptide of the invention which method comprises maintaining a host cell of the invention under conditions suitable for obtaining expression of the polypeptide and isolating the said polypeptide ; an antibody specific for a polypeptide of the invention ; a method for identification of a substance that modulates histamine-like receptor activity and/or expression, which method comprises contacting a polypeptide, polynucleotide, expression vector or host cell of the invention with a test substance and determining the effect of the test substance on the activity and/or expression of the said polypeptide or the polypeptide encoded by the said polynucleotide, thereby to determine whether the test substance modulates histamine-like receptor activity and/or expression ; a compound which or modulates histamine receptor activity and which is identifiable by the method referred to above ; a method of treating a subject having a disorder that is responsive to histamine-like receptor stimulation or modulation, which method comprises administering to said subject an effective amount of substance of the invention ; and use of a substance that stimulates or modulates histamine receptor activity in the manufacture of a medicament for the treatment or prophylaxis of a disorder that is responsive to stimulation or modulation of histamine-like receptor activity.

Preferably the disorder is selected from neurodegenerative disorders, for example cerebellar and spinocerebellar disorders, ataxia, spinocerebellar ataxia and atrophy, progressive supranuclear palsy, Huntington's disease, amyotrophic lateral sclerosis, Alzheimer's disease, Bell's palsy, cerebral palsy, Parkinson's disease, tardive dyskinesia, tremor, CNS disorders, for example pain, psychogenic erectile dysfunction, seizures, migraine, attention deficit, depression, anxiety, psychosis,

mania, schizophrenia, diseases related to immune function, autoimmune function, viruses, inflammation or immunodeficiency, for example asthma, COPD, Crohn's disease, Inflammatory bowel syndrome, ulcerative colitis, gastroenteritis, inflammatory pain, allergy, rhinitis, dermatitis, anaphylaxis and septic shock, viral diseases, for example HBV, HSV and HIV, general inflammation, rheumatoid arthritis, osteoarthritis, osteoporosis, osteopetrosis, male and female reproductive fertility (ovary and testes expression), pre-term labor, dysmenorrhea, induction of labor, polycystic ovary disease, ovarian cysts, urinary incontinence, for example urge, stress, or mixed incontinence, hypothyroidism, hyperthyroidism, control of metabolic rate, obesity and insulin-dependent diabetes.

Brief Description of the Sequences SEQ ID NO: 1 shows the nucleotide and amino acid sequences of human protein HIPHUM0000099A.

SEQ ID NO: 2 is the amino acid sequence alone of HIPHUM0000099A.

Brief Description of the Figures Figure 1 shows the relative expression of HIPHUM 0000099 in human tissues.

Figure 2 shows the relative expression of HIPHUM 0000099 in a variety of normal and diseased human tissues.

Detailed Description of the Invention Throughout the present specification and the accompanying claims the words "comprise"and"include"and variations such as"comprises","comprising", "includes"and"including"are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

The present invention relates to a human histamine-like receptor, referred to herein as HIPHUM0000099A, and variants thereof. Sequence information for HIPHUM0000099A is provided in SEQ ID NO: 1 (nucleotide and amino acid) and in SEQ ID NO: 2. A polypeptide of the invention thus consists essentially of the amino

acid sequence of SEQ ID NO: 2 or of a variant of that sequence, or of a fragment of either thereof.

Polypeptides of the invention may be in a substantially isolated form. It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as substantially isolated. A polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 50%, e. g. more than 80%, 90%, 95% or 99%, by weight of the polypeptide in the preparation is a polypeptide of the invention.

Routine methods, can be employed to purify and/or synthesise the proteins according to the invention. Such methods are well understood by persons skilled in the art, and include techniques such as those disclosed in Sambrook et al, Molecular Cloning: a Laboratory Manual, 2nd Edition, CSH Laboratory Press, 1989, the disclosure of which is included herein in its entirety by way of reference.

The term"variant"refers to a polypeptide which has a same essential character or basic biological functionality as HIPHUM0000099A. The essential character of HIPHUM0000099A can be defined as follows: HIPHUM0000099A is a histamine-like receptor. Preferably a variant polypeptide is one which binds to the same ligand as HIPHUM0000099A. Preferably the polypeptide is capable of binding histamine or a structurally related molecule. A polypeptide having a same essential character as HIPHUM0000099A may be identified by monitoring for a function the histamine-like receptor selected from immunomodulatory or neuromodulatory activity, effects on control of co-ordinated movement and/or CNS function. A variant receptor may be identified by looking for ligand binding. Possible ligands include histamine and structurally related molecules. A full length variant polypeptide is preferably one which includes a seven transmembrane region.

Preferably, a full length variant polypeptide may couple to G-protein to mediate intracellular responses.

In another aspect of the invention, a variant is one which does not show the

same activity as HIPHUM0000099A but is one which inhibits a basic function of HIPHUM0000099A. For example, a variant polypeptide is one which inhibits immunomodulatory and/or neuromodulatory activity of HIPHUM0000099A, for

example by binding to HIPHUM0000099A ligand to prevent activity mediated by ligand binding to HIPHUM0000099A.

Typically, polypeptides with more than about 65% identity preferably at least 80% or at least 90% and particularly preferably at least 95% at least 97% or at least 99% identity, with the amino acid sequences of SEQ ID NO: 2, are considered as variants of the proteins. Such variants may include allelic variants and the deletion, modification or addition of single amino acids or groups of amino acids within the protein sequence, as long as the peptide maintains a basic biological functionality of the HIPHUM0000099A receptor.

Amino acid substitutions may be made, for example from 1, 2 or 3 to 10,20 or 30 substitutions. The modified polypeptide generally retains activity as a histamine-like receptor. Conservative substitutions may be made, for example according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.

ALIPHATIC Non-polar GAP ILV Polar-uncharged C S T M NQ Polar-charged D E KR AROMATIC H F W Y

Shorter polypeptide sequences are within the scope of the invention. For example, a peptide of at least 20 amino acids or up to 50,60, 70,80, 100, 150 or 200 amino acids in length is considered to fall within the scope of the invention as long as it demonstrates a basic biological functionality of HIPHUM0000099A. In particular, but not exclusively, this aspect of the invention encompasses the situation when the

protein is a fragment of the complete protein sequence and may represent a ligandbinding region (N-terminal extracellular domain) or an effector binding region (Cterminal intracellular domain). Such fragments can be used to construct chimeric receptors preferably with another 7-transmembrane receptor, more preferably with another member of the family of histamine receptors. Such fragments of

HIPHUM0000099A or a variant thereof can also be used to raise antiHIPHUM0000099A antibodies. In this embodiment the fragment may comprise an epitope of the HIPHUM0000099A polypeptide and may otherwise not demonstrate the ligand binding or other properties of HIPHUM0000099A.

Polypeptides of the invention may be chemically modified, e. g. posttranslationally modified. For example, they may be glycosylated or comprise modified amino acid residues. They may also be modified by the addition of histidine residues to assist their purification or by the addition of a signal sequence to promote insertion into the cell membrane. Such modified polypeptides fall within the scope of the term"polypeptide"of the invention.

The invention also includes nucleotide sequences that encode for HIPHUM0000099A or variant thereof as well as nucleotide sequences which are complementary thereto. The nucleotide sequence may be RNA or DNA including genomic DNA, synthetic DNA or cDNA. Preferably the nucleotide sequence is a DNA sequence and most preferably, a cDNA sequence. Nucleotide sequence information is provided in SEQ ID NO: 1. Such nucleotides can be isolated from human cells or synthesised according to methods well known in the art, as described by way of example in Sambrook et al, 1989.

Typically a polynucleotide of the invention comprises a contiguous sequence of nucleotides which is capable of hybridizing under selective conditions to the coding sequence or the complement of the coding sequence of SEQ ID NO: 1.

A polynucleotide of the invention can hydridize to the coding sequence or the complement of the coding sequence of SEQ ID NO: 1 at a level significantly above background. Background hybridization may occur, for example, because of other cDNAs present in a cDNA library. The signal level generated by the interaction between a polynucleotide of the invention and the coding sequence or complement of the coding sequence of SEQ ID NO: 1 is typically at least 10 fold, preferably at least

100 fold, as intense as interactions between other polynucleotides and the coding sequence of SEQ ID NO: 1. The intensity of interaction may be measured, for example, by radiolabelling the probe, e. g. with 32p. Selective hybridisation may typically be achieved using conditions of medium to high stringency. However, such hybridisation may be carried out under any suitable conditions known in the art (see Sambrook et al, 1989. For example, if high stringency is required suitable conditions

include from 0. 1 to 0. 2 x SSC at 60 C up to 65 C. If lower stringency is required suitable conditions include 2 x SSC at 60 C.

The coding sequence of SEQ ID NO: 1 may be modified by nucleotide substitutions, for example from 1,2 or 3 to 10,25, 50 or 100 substitutions. The polynucleotide of SEQ ID NO: 1 may alternatively or additionally be modified by one or more insertions and/or deletions and/or by an extension at either or both ends.

A polynucleotide may include one or more introns, for example may comprise genomic DNA. Additional sequences such as signal sequences which may assist in insertion of the polypeptide in a cell membrane may also be included. The modified polynucleotide generally encodes a polypeptide which has a HIPHUM0000099A receptor activity. Alternatively, a polynucleotide encodes a ligand-binding portion of a polypeptide or a polypeptide which inhibits HIPHUM0000099A activity.

Degenerate substitutions may be made and/or substitutions may be made which would result in a conservative amino acid substitution when the modified sequence is translated, for example as shown in the Table above.

A nucleotide sequence which is capable of selectively hybridizing to the complement of the DNA coding sequence of SEQ ID NO: 1 will generally have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the coding sequence of SEQ ID NO: 1 over a region of at least 20, preferably at least 30, for instance at least 40, at least 60, more preferably at least 100 contiguous nucleotides or most preferably over the full length of SEQ ID NO : 1.

For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (typically

on their default settings), for example as described in Altschul (1993) J. Mol. Evol.

36 : 290-300 ; Altschul et al (1990) J. Mol. Biol. 215 : 403-10.

Software for performing BLAST analyses is publicly available through the National Centre for Biotechnology Information (http ://www. ncbi. nlm. nih. gov/).

This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, 1990). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.

The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad.

Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity

between two sequences ; see e. g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci.

USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N) ), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0. 01, and most preferably less than about 0. 001.

Any combination of the above mentioned degrees of sequence identity and minimum sizes may be used to define polynucleotides of the invention, with the more stringent combinations (i. e. higher sequence identity over longer lengths) being

preferred. Thus, for example a polynucleotide which has at least 90% sequence identity over 25, preferably over 30 nucleotides forms one aspect of the invention, as does a polynucleotide which has at least 95% sequence identity over 40 nucleotides.

The nucleotides according to the invention have utility in production of the proteins according to the invention, which may take place in vitro, in vivo or ex vivo.

The nucleotides may be involved in recombinant protein synthesis or indeed as therapeutic agents in their own right, utilised in gene therapy techniques. Nucleotides complementary to those encoding HIPHUM0000099A, or antisense sequences, may also be used in gene therapy.

Polynucleotides of the invention may be used as a primer, e. g. a PCR primer, a primer for an alternative amplification reaction, a probe e. g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors.

Such primers, probes and other fragments will preferably be at least 10, preferably at least 15 or at least 20, for example at least 25, at least 30 or at least 40 nucleotides in length. They will typically be up to 40,50, 60,70, 100 or 150 nucleotides in length. Probes and fragments can be longer than 150 nucleotides in length, for example up to 200,300, 400,500, 600,700 nucleotides in length, or even up to a few nucleotides, such as five or ten nucleotides, short of the coding sequence of SEQ ID NO : 1.

The present invention also includes expression vectors that comprise nucleotide sequences encoding the proteins or variants thereof of the invention. Such expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for protein expression. Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard we refer to Sambrook et al. 1989.

Polynucleotides according to the invention may also be inserted into the vectors described above in an antisense orientation in order to provide for the production of antisense RNA. Antisense RNA or other antisense polynucleotides

may also be produced by synthetic means. Such antisense polynucleotides may be used as test compounds in the assays of the invention or may be useful in a method of treatment of the human or animal body by therapy.

Preferably, a polynucleotide of the invention or for use in the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i. e. the vector is an expression vector. The term"operably linked"refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.

A regulatory sequence, such as a promoter,"operably linked"to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.

The vectors may be for example, plasmid, virus or phage vectors provided with a origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistence gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in vitro, for example for the production of DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell. The vectors may also be adapted to be used in vivo, for example in a method of gene therapy.

Promoters and other expression regulation signals may be selected to be compatible with the host cell for which expression is designed. For example, yeast promoters include S. cerevisiae GAL4 and ADH promoters, S. pombe nmtl and adh promoter. Mammalian promoters include the metallothionein promoter which can be induced in response to heavy metals such as cadmium. Viral promoters such as the SV40 large T antigen promoter or adenovirus promoters may also be used. All these promoters are readily available in the art.

Mammalian promoters, such as-actin promoters, may be used. Tissuespecific promoters are especially preferred. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE promoter, adenovirus, HSV promoters (such as the HSV IE promoters), or HPV promoters, particularly the HPV upstream regulatory region

(URR). Viral promoters are readily available in the art.

The vector may further include sequences flanking the polynucleotide giving rise to polynucleotides which comprise sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences. This will allow the introduction of the polynucleotides of the invention into the genome of eukaryotic cells or viruses by homologous recombination. In particular, a plasmid vector comprising the expression cassette flanked by viral sequences can be used to prepare a viral vector suitable for delivering the polynucleotides of the invention to a mammalian cell. Other examples of suitable viral vectors include herpes simplex viral vectors and retroviruses, including lentiviruses, adenoviruses, adeno-associated viruses and HPV viruses. Gene transfer techniques using these viruses are known to those skilled in the art. Retrovirus vectors for example may be used to stably integrate the polynucleotide giving rise to the polynucleotide into the host genome. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression.

The invention also includes cells that have been modified to express the HIPHUM0000099A polypeptide or a variant thereof. Such cells include transient, or preferably stable higher eukaryotic cell lines, such as mammalian cells or insect cells, using for example a baculovirus expression system, lower eukaryotic cells, such as yeast or prokaryotic cells such as bacterial cells. Particular examples of cells which may be modified by insertion of vectors encoding for a polypeptide according to the invention include mammalian HEK293T, CHO, HeLa and COS cells. Preferably the cell line selected will be one which is not only stable, but also allows for mature glycosylation and cell surface expression of a polypeptide. Expression may be achieved in transformed oocytes. A polypeptide of the invention may be expressed in cells of a transgenic non-human animal, preferably a mouse. A transgenic nonhuman animal expressing a polypeptide of the invention is included within the scope of the invention. A polypeptide of the invention may also be expressed in Xenopus laevis oocytes or melanophores, in particular for use in an assay of the invention.

According to another aspect, the present invention also relates to antibodies, specific for a polypeptide of the invention. Such antibodies are for example useful in purification, isolation or screening methods involving immunoprecipitation

techniques or, indeed, as therapeutic agents in their own right.

Antibodies may be raised against specific epitopes of the polypeptides according to the invention. Such antibodies may be used to block ligand binding to the receptor. An antibody, or other compound,"specifically binds"to a protein when it binds with preferential or high affinity to the protein for which it is specific but does substantially bind not bind or binds with only low affinity to other proteins. A variety of protocols for competitive binding or immunoradiometric assays to determine the specific binding capability of an antibody are well known in the art (see for example Maddox et al, J. Exp. Med. 158,1211-1226, 1993). Such immunoassays typically involve the formation of complexes between the specific protein and its antibody and the measurement of complex formation.

Antibodies of the invention may be antibodies to human polypeptides or fragments thereof. For the purposes of this invention, the term"antibody", unless specified to the contrary, includes fragments which bind a polypeptide of the invention. Such fragments include Fv, F (ab') and F (ab') 2 fragments, as well as single chain antibodies. Furthermore, the antibodies and fragment thereof may be chimeric antibodies, CDR-grafted antibodies or humanised antibodies.

Antibodies may be used in a method for detecting polypeptides of the invention in a biological sample, which method comprises: I providing an antibody of the invention; II incubating a biological sample with said antibody under conditions which allow for the formation of an antibody-antigen complex; and III determining whether antibody-antigen complex comprising said antibody is formed.

A sample may be for example a tissue extract, blood, serum and saliva.

Antibodies of the invention may be bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions, etc.

Antibodies may be linked to a revealing label and thus may be suitable for use in methods of in vivo HIPHUM0000099A imaging.

Antibodies of the invention can be produced by any suitable method. Means for preparing and characterising antibodies are well known in the art, see for example Harlow and Lane (1988)"Antibodies : A Laboratory Manual", Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, NY. For example, an antibody may be produced by raising antibody in a host animal against the whole polypeptide or a fragment thereof, for example an antigenic epitope thereof, herein after the "immunogen".

A method for producing a polyclonal antibody comprises immunising a suitable host animal, for example an experimental animal, with the immunogen and isolating immunoglobulins from the animal's serum. The animal may therefore be inoculated with the immunogen, blood subsequently removed from the animal and the IgG fraction purified.

A method for producing a monoclonal antibody comprises immortalising cells which produce the desired antibody. Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein (1975) Nature 256, 495-497).

An immortalized cell producing the desired antibody may be selected by a conventional procedure. The hybridomas may be grown in culture or injected intraperitoneally for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host. Human antibody may be prepared by in vitro immunisation of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.

For the production of both monoclonal and polyclonal antibodies, the experimental animal is suitably a goat, rabbit, rat or mouse. If desired, the immunogen may be administered as a conjugate in which the immunogen is coupled, for example via a side chain of one of the amino acid residues, to a suitable carrier.

The carrier molecule is typically a physiologically acceptable carrier. The antibody obtained may be isolated and, if desired, purified.

An important aspect of the present invention is the use of polypeptides according to the invention in screening methods. The screening methods may be used to identify substances that bind to histamine receptors and in particular which bind to HIPHUM0000099A such as a ligand for the receptor. Screening methods may also be used to identify agonists or antagonists which may modulate histamine

receptor activity, inhibitors or activators of HIPHUM0000099A activity, and/or agents which up-regulate or down-regulate HIPHUM0000099A expression.

Any suitable format may be used for the assay. In general terms such screening methods may involve contacting a polypeptide of the invention with a test substance and monitoring for binding of the test substance to the polypeptide or measuring receptor activity. A polypeptide of the invention may be incubated with a test substance. Modulation of histamine-like activity may be determined. In a preferred aspect, the assay is a cell-based assay. Preferably the assay may be carried out in a single well of a microtitre plate. Assay formats which allow high throughput screening are preferred.

Modulator activity can be determined by contacting cells expressing a polypeptide of the invention with a substance under investigation and by monitoring an effect mediated by the polypeptide. The cells expressing the polypeptide may be in vitro or in vivo. The polypeptide of the invention may be naturally or recombinantly expressed. Preferably, the assay is carried out in vitro using cells expressing recombinant polypeptide. Preferably, control experiments are carried out on cells which do not express the polypeptide of the invention to establish whether the observed responses are the result of activation of the polypeptide.

The binding of a test substance to a polypeptide of the invention can be determined directly. For example, a radiolabelled test substance can be incubated with the polypeptide of the invention and binding of the test substance to the polypeptide can be monitored. Typically, the radiolabelled test substance can be incubated with cell membranes containing the polypeptide until equilibrium is reached. The membranes can then be separated from a non-bound test substance and dissolved in scintillation fluid to allow the radioactive content to be determined by scintillation counting. Non-specific binding of the test substance may also be determined by repeating the experiment in the presence of a saturating concentration of a non-radioactive ligand.

Assays may be carried out using cells expressing HIPHUM0000099A, and incubating such cells with the test substance optionally in the presence of HIPHUM0000099A ligand. Alternatively an antibody may be used to complex HIPHUM0000099A and thus mediate HIPHUM0000099A activity. Test substances may then be added to assess the effect on such activity. Cells expressing HIPHUM0000099A constitutively may be provided for use in assays for

HIPHUM0000099A function. Such constitutively expressed HIPHUM0000099A may demonstrate HIPHUM0000099A activity in the absence of ligand binding.

Additional test substances may be introduced in any assay to look for inhibitors of ligand binding or inhibitors ofHIPHUM0000099A-mediated activity.

In preferred aspects, a host cell is provided expressing the polypeptide and containing a G-protein coupled pathway responsive reporter construct. The host cell is treated with a substance under test for a defined time. The expression of the reporter gene, such as SP alkaline phosphatase or luciferase is assayed. The assay enables determination of whether the compound modulates the induction of the Gprotein coupled pathway by HIPHUM0000099A in target cells.

Assays may also be carried out to identify modulators of receptor-shedding.

A polypeptide of the invention can be cleaved from the cell surface. Shedding the receptor would act to down regulate receptor signalling. Thus, cell-based assays may be used to screen for compounds which promote or inhibit receptor-shedding.

Assays may also be carried out to identify substances which modify HIPHUM0000099A receptor expression, for example substances which up-or downregulate expression. Such assays may be carried out for example by using antibodies for HIPHUM0000099A to monitor levels of HIPHUM0000099A expression. Other assays which can be used to monitor the effect of a test substance on

HIPHUM0000099A expression include using a reporter gene construct driven by the HIPHUM0000099A regulatory sequences as the promoter sequence and monitoring for expression of the reporter polypeptide. Further possible assays could utilise membrane fractions from overexpression of HIPHUM0000099A polypeptide either in X laevis oocytes or cell lines such as HEK293, CHO, COS7 and HeLa cells and assessment of displacement of a radiolabelled ligand.

Additional control experiments may be carried out. Assays may also be carried out using known ligands of other histamine receptors to identify ligands which are specific for polypeptides of the invention. Preferably, the assays of the invention are carried out under conditions which would result in G-protein coupled pathway mediated activity in the absence of the test substance, to identify inhibitors or activators of histamine-like receptor mediated activity, or agents which inhibit ligand-induced histamine-like receptor activity. An assay of the invention may be

carried out using a known histamine agonist or histamine antagonist to provide a comparison with a compound under test.

Typically, receptor activity can be monitored indirectly for example by measuring a G-protein coupled readout. G-protein coupled readout can typically be monitored using an electrophysiological method to determine the activity of G-

protein regulated Ca2+ or K+ channels or by using a fluorescent dye to measure changed in intracellular Ca2+ levels. The receptor could be coupled to Gs, Gq, Gi and/or Go. Thus cAMP or GTPyS levels or activity, calcium mobilization, inositol triphosphate generation and protein kinase C activation may be monitored.

Following histamine-like receptor stimulation, cyclic AMP accumulation can be measured for example in forskolin stimulated CHO cells transformed with the HIPHUM0000099A receptor either directly, or indirectly by monitoring the expression of cotransfected reporter gene, the expression of which will be controlled by cyclic AMP response elements.

Xenopus dermal melanophores aggregate or disperse pigment in response to the activation or inhibition of G-protein coupled receptors. This feature can be exploited as an assay for receptor activation or inhibition if a specific G-protein coupled receptor is exogenously expressed.

HIPHUM0000099A receptor is likely to couple to G-protein with consequent hydrolysis of GTP. Accumulation of a labelled GTP stable analogue can be measured utilising membrane fractions from overexpression of HIPHUM0000099A receptor either in X laevis oocytes or cell lines such as HEK293, CHO, COS7, HeLa on exposure to agonist ligand.

G-protein coupled receptors have been shown to activate MAPK signalling pathways. Cell lines overexpressing the histamine-like receptor of the invention with MAPK reporter genes may be utilised as assays for receptor activation or inhibition.

The histamine-like receptor of the invention may be heterologously expressed in modified yeast strains containing multiple reporter genes, such as FUS 1- HIS3 and FUSl-lacZ, each linked to an endogenous MAPK cascade-based signal transduction pathway. This pathway is normally linked to pheromone receptors, but can be coupled to foreign receptors by replacement of the yeast G-protein with yeast/mammalian G protein chimeras. Strains may also contain two further gene

deletions, i. e. deletions of SST2 and FART, to potentiate the assay. Ligand activation of the heterologous receptor can be monitored using the reporter genes, for example either as cell growth in the absence of histidine or with a substrate of beta-galactosidase (lacZ).

Suitable test substances which can be tested in the above assays include combinatorial libraries, defined chemical entities and compounds, peptide and peptide mimetics, oligonucleotides and natural product libraries, such as display (e. g. phage display libraries) and antibody products.

Typically, organic molecules will be screened, preferably small organic molecules which have a molecular weight of from 50 to 2500 daltons. Candidate products can be biomolecules including, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

Test substances may be used in an initial screen of, for example, 10 substances per reaction, and the substances of these batches which show inhibition or

activation tested individually. Test substances may be used at a concentration of from InM to lOOOuM, preferably from ljj. M to lOOu. M, more preferably from 1 IlM to 10uM. Preferably, the activity of a test substance is compared to the activity shown by a known activator or inhibitor. A test substance which acts as an inhibitor may produce a 50% inhibition of activity of the receptor. Alternatively a test substance which acts as an activator may produce 50% of the maximal activity produced using a known activator.

Another aspect of the present invention is the use of polynucleotides encoding the HIPHUM0000099A polypeptides of the invention to identify mutations in HIPHUM0000099A genes which may be implicated in human disorders.

Identification of such mutations may be used to assist in diagnosis or susceptibility to such disorders and in assessing the physiology of such disorders. Polynucleotides may also be used in hybridisation studies to monitor for up-or down-regulation of

HIPHUM0000099A expression. Polynucleotides such as SEQ ID NO : 1 or fragments thereof may be used to identify allelic variants, genomic DNA and species variants.

The present invention provides a method for detecting variation in the expressed products encoded by HIPHUM0000099A genes. This may comprise determining the level of an HIPHUM0000099A expressed in cells or determining specific alterations in the expressed product. Sequences of interest for diagnostic purposes include, but are not limited to, the conserved portions as identified by sequence similarity and conservation of intron/exon structure. The diagnosis may be performed in conjunction with kindred studies to determine whether a mutation of interest co-segregates with disease phenotype in a family.

Diagnostic procedures may be performed on polynucleotides isolated from an individual or alternatively, may be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Appropriate procedures are described in, for example, Nuovo, G. J., 1992, "PCR In Situ Hybridization : Protocols And Applications", Raven Press, NY). Such analysis techniques include, DNA or RNA blotting analyses, single stranded conformational polymorphism analyses, in situ hybridization assays, and polymerase chain reaction analyses. Such analyses may reveal both quantitative aspects of the expression pattern of a HIPHUM0000099A, and qualitative aspects of HIPHUM0000099A expression and/or composition.

Alternative diagnostic methods for the detection of HIPHUM0000099A nucleic acid molecules may involve their amplification, e. g. by PCR (the experimental embodiment set forth in U. S. Patent No. 4, 683, 202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA 88 : 189-193), self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87 : 1874- 1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci.

15 USA 86 : 1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology 6 : 1197) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art.

These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

Particularly suitable diagnostic methods are chip-based DNA technologies such as those described by Hacia et al., 1996, Nature Genetics 14 : 441-447 and Shoemaker et al., 1996, Nature Genetics 14 : 450-456. Briefly, these techniques involve quantitative methods for analyzing large numbers of nucleic acid sequence targets rapidly and accurately. By tagging with oligonucleotides or using fixed probe arrays, one can employ chip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization.

Following detection, the results seen in a given patient may be compared with a statistically significant reference group of normal patients and patients that have HIPHUM0000099A related pathologies. In this way, it is possible to correlate the amount or kind of HIPHUM0000099A encoded product detected with various clinical states or predisposition to clinical states.

Another aspect of the present invention is the use of the substances that have been identified by screening techniques referred to above in the treatment of disease states, which are responsive to regulation of histamine receptor activity. The treatment may be therapeutic or prophylactic. The condition of a patient suffering from such a disease state can thus be improved.

In particular, such substances may be used in the treatment of neurodegenerative disorders, for example cerebellar and spinocerebellar disorders, ataxia, spinocerebellar ataxia and atrophy, progressive supranuclear palsy, Huntington's disease, amyotrophic lateral sclerosis, Alzheimer's disease, Bell's palsy, cerebral palsy, Parkinson's disease, tardive dyskinesia, tremor, CNS disorders, for example pain, psychogenic erectile dysfunction, seizures, migraine, attention deficit, depression, anxiety, psychosis, mania, schizophrenia, diseases related to immune function, autoimmune function, viruses, inflammation or immunodeficiency, for example asthma, COPD, Crohn's disease, Inflammatory bowel syndrome, ulcerative colitis, gastroenteritis, inflammatory pain, allergy, rhinitis, dermatitis, anaphylaxis and septic shock, viral diseases, for example HBV, HSV and HIV, general inflammation, rheumatoid arthritis, osteoarthritis, osteoporosis, osteopetrosis, male and female reproductive fertility (ovary and testes expression), pre-term labor, dysmenorrhea, induction of labor, polycystic ovary disease, ovarian cysts, urinary incontinence, for example urge, stress, or mixed incontinence, hypothyroidism,

hyperthyroidism, control of metabolic rate, obesity and/or insulin-dependent diabetes.

Substances identified according to the screening methods outlined above may be formulated with standard pharmaceutically acceptable carriers and/or excipients as is routine in the pharmaceutical art. For example, a suitable substance may be dissolved in physiological saline or water for injections. The exact nature of a formulation ill depend upon several factors including the particular substance to be administered and the desired route of administration. Suitable types of formulation are fully described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Eastern Pennsylvania, 17th Ed. 1985, the disclosure of which is included herein of its entirety by way of reference.

The substances may be administered by enteral or parenteral routes such as via oral, buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraperitoneal, topical or other appropriate administration routes.

A therapeutically effective amount of a modulator is administered to a patient. The dose of a modulator may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 50 mg per kg of body weight, according to the activity of the specific modulator, the age, weight and conditions of the subject to be treated, the type and seventy of the degeneration and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g.

Nucleic acid encoding HIPHUM0000099A or a variant thereof which inhibits HIPHUM0000099A activity may be administered to the mammal. Nucleic acid, such as RNA or DNA, and preferably, DNA, is provided in the form of a vector, such as the polynucleotides described above, which may be expressed in the cells of the mammal.

Nucleic acid encoding the polypeptide may be administered by any available technique. For example, the nucleic acid may be introduced by needle injection, preferably intradermally, subcutaneously or intramuscularly. Alternatively, the

nucleic acid may be delivered directly across the skin using a nucleic acid delivery device such as particle-mediated gene delivery. The nucleic acid may be administered topically to the skin, or to mucosal surfaces for example by intranasal, oral, intravaginal or intrarectal administration.

Uptake of nucleic acid constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents.

Examples of these agents includes cationic agents, for example, calcium phosphate and DEAE-Dextran and lipofectants, for example, lipofectam and transfectam. The dosage of the nucleic acid to be administered can be altered. Typically the nucleic acid is administered in the range of Ipg to lmg, preferably to Ipg to 1 zig nucleic acid for particle mediated gene delivery and 10/-tg to Img for other routes.

The following Examples illustrate the invention.

Example 1 : Characterisation of the sequence A histamine-like receptor, designated as HIPHUM0000099A has been identified. The nucleotide and amino acid sequences of the receptor have been determined. These are set out below in SEQ ID NOs : 1 and 2. Suitable primers and probes were designed and used to analyse tissue expression. Expression patterns for HIPHUM0000099A in a variety of normal human tissues are shown in Figure 1.

Figure 2 shows expression of HIPHUM0000099A in a variety of normal and diseased tissues. HIPHUM0000099A was found to be primarily expressed in cerebellum, lung, testes, myometrium, ovary, thyroid, rectum, and urinary bladder.

The polypeptide is also expressed in thymus, tonsil, spleen, adenoid, prostate, omentum, jejunum, T cells, dendritic cells, osteoblasts and endothelial cells. The expression of the polypeptide is downreguled in HSV, HBV and HIV-infected cells.

The chromosomal localization was also mapped. Human HIPHUM0000099A has been mapped to 14q24 which is associated with insulindependent diabetes, spinocerebellar ataxia and atrophy.

Example 2 : Screening for substances which exhibit protein modulating activity ty Mammalian cells, such as HEK293, CHO and COS7 cells, over-expressing a polypeptide of the invention are generated for screening purposes. 96 and 384 well plate, high throughput screens (HTS) are employed using fluorescence based calcium

indicator molecules, including but not limited to dyes such as Fura-2, Fura-Red, Fluo 3 and Fluo 4 (Molecular Probes). Secondary screening involves the same technology. Tertiary screens involve the study of modulators in rat, mouse and guinea-pig models of disease relevant to the target.

A brief screening assay protocol is as follows :- Mammalian cells stably over-expressing a polypeptide of the invention are cultured in black wall, clear bottom, tissue culture-coated, 96 or 384 well plates with a volume of 100111 cell culture medium in each well 3 days before use in a FLIPR (Fluorescence Imaging Plate Reader-Molecular Devices). Cells are incubated with 4uM FLUO-3AM at 30 C in 5% CO2 for 90 mins and then washed once in Tyrodes buffer containing 3mM probenecid. Basal fluorescence is determined prior to addition of test substances. The polypeptide is activated upon the addition of a known agonist. Activation results in an increase in intracellular calcium which can be measured directly in the FLIPR. For antagonist studies, substances are preincubated with the cells for 4 minutes following dye loading and washing and fluorescence measured for 4 minutes. Agonists are then added and cell fluorescence measured for a further 1 minute.

Assays may also be carried out as follows: Gs-coupled receptors are expressed and assayed in mammalian cells which express the 6xCRE-luciferase reporter gene such as CHO cells. Gq-coupled and Gicoupled receptors are expressed and assayed in mammalian cells which express the Gal4/Elk-l chimeric protein and 5xUAS-luciferase reporter gene. Cells are propagated in either in suspension or adherent cultures.

For adherent culture, cells are propagated in T225 flasks in DMEM/F12 containing 5% fetal bovine serum and 1 mM glutamine. Forty-eight hours prior to assay, cells are harvested with 2 ml of 0.05% trypsin, washed with complete medium and plated at a concentration of 4,000 cells/well in complete medium. Sixteen hours prior to the assay, the medium is removed from the cells and replaced with 90 u. l/well of serum-free DMEM/F12. At the time of the assay, test substances are added to the wells at a final concentration of 10 11M and the plates are incubated for four hours at 37 C in a cell culture incubator. The medium is aspirated by vacuum followed by

the addition of 50 ut of a 1: 1 mixture of LucLiteTM and dPBS/1 mM CaCl2/1 mM MgCl. Plates are sealed and subjected to dark adaptation at room temperature for 10 minutes before luciferase activity is quantitated on a TopCount microplate scintillation counter (Packard) using 3 seconds/well count time.

For suspension cultures, cells are propagated in Excel 301 medium containing 5% FBS and 2 mM glutamine at a minimum of 1x105- cells/ml for one week. Sixteen hours prior to an assay, cells are removed from suspension by centrifugation and resuspended in serum-free Excel 301 at a concentration of 1x106 cells/ml. At the time of assay, the cells are resuspended in serum-free DMEM/F12 at a concentration of 50,000 cells/ml. 100/-1l/well or 50 l/well of this suspension is pipetted into black 96-well or 384-well plates, respectively. The 96-well and 384-well plate contained 1 ul or 0. 5 ul ofagonist compounds in 100% DMSO at a final concentration of 10/-1M.

A Multidrop S20 cell dispenser is used to dispense cells into either 96-or 384-well plates. The reminder of the assay is the same as described for adherent culture above.

Xenopus oocyte expression Adult female Xenopus laevis (Blades Biologicals) are anaesthetised using 0.2% tricaine (3-aminobenzoic acid ethyl ester), killed and the ovaries rapidly removed. Oocytes are then de-folliculated by collagenase digestion (Sigma type I, 1.5 mg ml-') in divalent cation-free OR2 solution (82.5mM NaCl, 2. 5mM KCl, 1. 2mM NaH, PO,, 5mM HEPES; pH 7. 5 at 25 C). Single stage V and VI oocytes are transferred to ND96 solution (96mM NaCl, 2mM KCl, 1mM MgCI2, SmM HEPES, 2.5mM sodium pyruvate; pH 7.5 at 25 C) which contains 50u. g ml''gentamycin and stored at 18 C.

The EDG-like receptor (in pcDNA3, Invitrogen) is linearised and transcribed to RNA using T7 (Promega Wizard kit). m'G (5') pp (5') GTP capped cRNA is injected into oocytes (20-50ng per oocyte) and whole-cell currents are recorded using two-microelectrode voltage-clamp (Geneclamp amplifier, Axon instruments Inc. ) 3 to 7 days post-RNA injection. Microelectrodes have a resistance of 0.5 to 2MQ when filled with 3M KCl.

Melanophore screens may be carried out as follows:

Modified or unmodified receptors are expressed in melanophores using appropriate vector constructs including pJG3.6. The expressed receptors are then screened for Gs, Gq, Gi or Go activity. When a ligand binds to a Gs-coupled receptor, it activates adenylyl cyclase that in turn activates protein kinase A. This results in the initiation of phosphorylation events that cause the melanosomes to disperse. When a G,-coupled receptor is activated, it inhibits adenylyl cyclase which in turn reverses the pigment dispersion process to result in aggregation. When a Gqcoupled receptor is activated, it activates phospholipase C, which in turn activates protein kinase C. This results in the initiation of phosphorylation events to cause melanosome dispersion. The expressed receptors can be screened in agonist, antagonist or constitutive modes using bead-based lawn format or 96-well, 384-well or 1536-well formats.

Melanophores are grown in conditioned fibroblast medium (CFM) at room temperature. After harvesting the cells with trypsin/EDTA, approximately 6 to 10 million cells are electroporated with relevant receptor-expression vectors at 475 V, 425 IlFd, 720 ohms. The transfected cells are then plated into T225 flasks and are incubated for 24 hours. Cells are then harvested and plated into assay plates and incubated for 24 hours. Test substances are added to wells at 10 uM final concentration and 30-120 minutes later the dispersion or aggregation is measured using an SLT Spectra plate reader. For dispersion assays, cells are first treated with 2 nM melatonin in assay buffer (0.7X L15/0. 1% BSA) for 60 minutes before addition of test compounds. For aggregation assays, CFM is replaced with the assay buffer and cells are incubated for 60 minutes before addition of test compounds.

SEQUENCE LISTING < 110 > GLAXO GROUP LIMITED < 120 > PROTEIN < 130 > QG 1032 (P80207) < 160 > 2 < 170 > PatentIn version 3.0 < 210 > 1 < 211 > 1485 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (1).. (1485) < 400 > 1

atg gag gag ccg ccg ccg ccc cgc cca cca gcg age atg gcc tta ctg 48 Met Glu Glu Pro Pro Pro Pro Arg Pro Pro Ala Ser Met Ala Leu Leu 1 5 10 15 ggc age cag cac tec ggc gcc ccc tec gcg gcc ggc cca cct ggc ggg 96 Gly Ser Gln His Ser Gly Ala Pro Ser Ala Ala Gly Pro Pro Gly Gly 20 25 30 act tec tec gcg gcc acg gcg gcc gtg etc tec ttc age ace gtg gcg 144 Thr Ser Ser Ala Ala Thr Ala Ala Val Leu Ser Phe Ser Thr Val Ala 35 40 45 ace gcg gcg ctg ggg aac ctg age gac gca age gga ggc ggc aca get 192 Thr Ala Ala Leu Gly Asn Leu Ser Asp Ala Ser Gly Gly Gly Thr Ala 50 55 60

gcc get ccc ggt ggc ggc ggc ctt ggc ggg tec ggg gca gcg egg gag 240 Ala Ala Pro Gly Gly Gly Gly Leu Gly Gly Ser Gly Ala Ala Arg Glu 65 70 75 80 gcg ggg gcg gcg gtg agg egg ccg cta ggc ccg gag gcg gcg ccg ctg 288 Ala Gly Ala Ala Val Arg Arg Pro Leu Gly Pro Glu Ala Ala Pro Leu 85 90 ---- 95 ctg teg cac gga get gca gtg gcg gcc cag gcg etc gtc etc ctg etc 336 Leu Ser His Gly Ala Ala Val Ala Ala Gln Ala Leu Val Leu Leu Leu 100 105 110 ate ttc ctg ctg tct age ctt ggc aac tgc gcg gtg atg ggg gtg att 384 Ile Phe Leu Leu Ser Ser Leu Gly Asn Cys Ala Val Met Gly Val Ile 115 120 125 gtg aag cac egg cag etc cgc ace gtc ace aac gcc ttc ate ctg teg 432 Val Lys His Arg Gln Leu Arg Thr Val Thr Asn Ala Phe Ile Leu Ser 130 135 140 ctg tec cta teg gat ctg etc acg gcg ctg etc tgc ctg ccc gcc gcc 480 Leu Ser Leu Ser Asp Leu Leu Thr Ala Leu Leu Cys Leu Pro Ala Ala 145 150 155 160 ttc ctg gac etc ttc act ccg ccc ggg ggt teg gcg cct gcc gcc gcc 528 Phe Leu Asp Leu Phe Thr Pro Pro Gly Gly Ser Ala Pro Ala Ala Ala 165 170 175 gcg ggg ccc tgg cgc ggc ttc tgc gcc gcc age cgc ttc ttc age teg 576 Ala Gly Pro Trp Arg Gly Phe Cys Ala Ala Ser Arg Phe Phe Ser Ser 180 185 190 tgc ttc ggc ate gtg tec acg etc age gtg gcg etc ate teg ttg gac 624 Cys Phe Gly Ile Val Ser Thr Leu Ser Val Ala Leu Ile Ser Leu Asp 195 200 205 cgt tac tgc get ate gtg egg ccg ccg egg gag aag ate ggc cgc cgc 672 Arg Tyr Cys Ala Ile Val Arg Pro Pro Arg Glu Lys Ile Gly Arg Arg 210 215 220 cgc gcg ctg cag ctg ctg gcg ggc gcc tgg ctg acg gcc ctg ggc ttc 720 Arg Ala Leu Gln Leu Leu Ala Gly Ala Trp Leu Thr Ala Leu Gly Phe 225 230 235 240 tec ttg ccc tgg gag ctg etc ggg gcg ccc egg gaa etc gcg gcg gcg 768 Ser Leu Pro Trp Glu Leu Leu Gly Ala Pro Arg Glu Leu Ala Ala Ala 245 250 255 cag age ttc cac ggc tgc etc tac egg ace tec ccg gac ccc gcg cag 816 Gln Ser Phe His Gly Cys Leu Tyr Arg Thr Ser Pro Asp Pro Ala Gln 260 265 270 ctg ggc gcg gcc ttc age gtg ggg ctg gtg gtg gcc tgc tac ctg ctg 864 Leu Gly Ala Ala Phe Ser Val Gly Leu Val Val Ala Cys Tyr Leu Leu 275 280 285 ccc ttc ctg etc atg tgc ttc tgc cac tac cac ate tgc aag acg gtg 912 Pro Phe Leu Leu Met Cys Phe Cys His Tyr His Ile Cys Lys Thr Val 290 295 300 cgc ctg teg gac gtg cgc gtg egg ccg gtg aac ace tac gcg cgc gtg 960 Arg Leu Ser Asp Val Arg Val Arg Pro Val Asn Thr Tyr Ala Arg Val 305 310 315 320 ctg cgc ttc ttc age gag gtg cgc acg gcc ace ace gtc etc ate atg 1008 Leu Arg Phe Phe Ser Glu Val Arg Thr Ala Thr Thr Val Leu Ile Met 325 330 335 ate gtc ttc gtc ate tgc tgc tgg ggg ccc tac tgc ttc ctg gtg ctg 1056 Ile Val Phe Val Ile Cys Cys Trp Gly Pro Tyr Cys Phe Leu Val Leu 340 345 350 ctg gcc gcc gcc egg cag gcc cag ace atg cag gcc ccc teg etc etc 1104 Leu Ala Ala Ala Arg Gln Ala Gln Thr Met Gln Ala Pro Ser Leu Leu 355 360 365

age gtg gtg gcc gtc tgg ctg ace tgg gcc aat ggg gcc ate aac cct 1152 Ser Val Val Ala Val Trp Leu Thr Trp Ala Asn Gly Ala Ile Asn Pro 370 375 380 gtc ate tac gcc ate cgc aat ccc aac att teg atg etc cta ggg cgc 1200 Val Ile Tyr Ala Ile Arg Asn Pro Asn Ile Ser Met Leu Leu Gly Arg 385 390 395 400 aac cgc gag gag ggc tac egg act agg aat gtg gac get ttc ctg ccc 1248 Asn Arg Glu Glu Gly Tyr Arg Thr Arg Asn Val Asp Ala Phe Leu Pro 405 410 415 age cag ggc ccg ggt ctg caa gcc aga age cgc agt cgc ctt cga aac 1296 Ser Gln Gly Pro Gly Leu Gln Ala Arg Ser Arg Ser Arg Leu Arg Asn 420 425 430 cgc tat gcc aac egg ctg ggg gcc tgc aac agg atg tec tct tec aac 1344 Arg Tyr Ala Asn Arg Leu Gly Ala Cys Asn Arg Met Ser Ser Ser Asn 435 440 445 ccg gcc age gga gtg gca ggg gac gtg gcc atg tgg gcc cgc aaa aat 1392 Pro Ala Ser Gly Val Ala Gly Asp Val Ala Met Trp Ala Arg Lys Asn 450 455 460 cca gtt gta ctt ttc tgc cga gag gga cca cca gag ccg gtg acg gca 1440 Pro Val Val Leu Phe Cys Arg Glu Gly Pro Pro Glu Pro Val Thr Ala 465 470 475 480 gcg ace aaa cag cct aaa tec gaa get ggg gat ace age etc taa 1485 Ala Thr Lys Gln Pro Lys Ser Glu Ala Gly Asp Thr Ser Leu 485 490 < 210 > 2 < 211 > 494 < 212 > PRT < 213 > Homo sapiens < 400 > 2 Met Glu Glu Pro Pro Pro Pro Arg Pro Pro Ala Ser Met Ala Leu Leu 1 5 10 15 Gly Ser Gln His Ser Gly Ala Pro Ser Ala Ala Gly Pro Pro Gly Gly 20 25 30 Thr Ser Ser Ala Ala Thr Ala Ala Val Leu Ser Phe Ser Thr Val Ala 35 40 45 Thr Ala Ala Leu Gly Asn Leu Ser Asp Ala Ser Gly Gly Gly Thr Ala 50 55 60 Ala Ala Pro Gly Gly Gly Gly Leu Gly Gly Ser Gly Ala Ala Arg Glu 65 70 75 80 Ala Gly Ala Ala Val Arg Arg Pro Leu Gly Pro Glu Ala Ala Pro Leu 85 90 95 Leu Ser His Gly Ala Ala Val Ala Ala Gln Ala Leu Val Leu Leu Leu 100 105 110 Ile Phe Leu Leu Ser Ser Leu Gly Asn Cys Ala Val Met Gly Val Ile 115 120 125 Val Lys His Arg Gln Leu Arg Thr Val Thr Asn Ala Phe Ile Leu Ser 130 135 140 Leu Ser Leu Ser Asp Leu Leu Thr Ala Leu Leu Cys Leu Pro Ala Ala 145 150 155 160

Phe Leu Asp Leu Phe Thr Pro Pro Gly Gly Ser Ala Pro Ala Ala Ala 165 170 175 Ala Gly Pro Trp Arg Gly Phe Cys Ala Ala Ser Arg Phe Phe Ser Ser 180 185 190 Cys Phe Gly Ile Val Ser Thr Leu Ser Val Ala Leu Ile Ser Leu Asp 195 200 205 Arg Tyr Cys Ala Ile Val Arg Pro Pro Arg Glu Lys Ile Gly Arg Arg 210 215 220 Arg Ala Leu Gln Leu Leu Ala Gly Ala Trp Leu Thr Ala Leu Gly Phe 225 230 235 240 Ser Leu Pro Trp Glu Leu Leu Gly Ala Pro Arg Glu Leu Ala Ala Ala 245 250 255 Gln Ser Phe His Gly Cys Leu Tyr Arg Thr Ser Pro Asp Pro Ala Gln 260 265 270

Leu Gly Ala Ala Phe Ser Val Gly Leu Val Val Ala Cys Tyr Leu Leu 275 280 285 Pro Phe Leu Leu Met Cys Phe Cys His Tyr His Ile Cys Lys Thr Val 290 295 300

Arg Leu Ser Asp Val Arg Val Arg Pro Val Asn Thr Tyr Ala Arg Val 305 310 315 320 Leu Arg Phe Phe Ser Glu Val Arg Thr Ala Thr Thr Val Leu Ile Met 325 330 335

Ile Val Phe Val Ile Cys Cys Trp Gly Pro Tyr Cys Phe Leu Val Leu 340'345 350 Arg Leu Ala Ala Ala Arg G n Ala Gln Thr Met Gln Ala Pro Ser Leu Leu 355 360 365 r Ser Val Val Ala Trp Leu Thr Trp Ala Asn Gly Ala Ile Asn Pro

370 375 380 Val Ile Tyr Ala Ile Arg Asn Pro Asn Ile Ser Met Leu Leu Gly Arg 385 390 395 400 Asn Arg Glu Glu Gly Tyr Arg Thr Arg Asn Val Asp Ala Phe Leu Pro 405 410 415 Ser Gln Gly Pro Gly Leu Gln Ala Arg Ser Arg Ser Arg Leu Arg Asn 420 425 430 Arg Tyr Ala Asn Arg Leu Gly Ala Cys Asn Arg Met Ser Ser Ser Asn 435 440 445 Pro Ala Ser Gly Val Ala Gly Asp Val Ala Met Trp Ala Arg Lys Asn 450 455 460

Pro Val Val Leu Phe Cys Arg Glu Gly Pro Pro Glu Pro Val Thr Ala 465 470 475 480 Ala Thr Lys Gln Pro Lys Ser Glu Ala Gly Asp Thr Ser Leu 485 490

Claims

CLAIMS 1. An isolated histamine-like receptor polypeptide comprising (i) the amino acid sequence of SEQ ID NO : 2 or (ii) a variant thereof which is capable of binding histamine or a structurally related molecule or (iii) a fragment of (i) or (ii) which is capable of binding histamine or a structurally related molecule.
2. A polypeptide according to claim 1 wherein the variant (ii) has at least 80% identity to the amino acid sequence of SEQ ID NO : 2.
3. A polynucleotide encoding a polypeptide according to claim 1 or 2.
4. A polynucleotide according to claim 3 which is a cDNA sequence.
5. A polynucleotide encoding a histamine-like receptor polypeptide which is capable of binding histamine or a structurally related molecule which polynucleotide comprises : (a) the nucleic acid sequence of SEQ ID NO : 1 and/or a sequence complementary thereto ; (b) a sequence which hybridises under stringent conditions to a sequence as defined in (a) ; (c) a sequence that is degenerate as a result of the genetic code to a sequence as defined in (a) or (b) ; or (d) a sequence having at least 60% identity to a sequence as defined in (a), (b) or (c).
6. An expression vector comprising a polynucleotide according to any one of claims 3 to 5.
7. A host cell comprising an expression vector according to claim 6.
8. An antibody specific for a polypeptide according to claim 1 or 2.
9. A method for the identification of a substance that modulates histamine-like receptor activity and/or expression, which method comprises : (i) contacting a test substance and a polypeptide according to claim 1 or 2, a polynucleotide according to any one of claims 3 to 5, an expression vector according to claim 6 or a host cell according to claim 7, and

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(ii) determining the effect of the test substance on the activity and/or expression of the said polypeptide or the polypeptide encoded by said polynucleotide, thereby to determine whether the test substance modulates histamine-like receptor activity and/or expression.
10. A method according to claim 9 wherein the polypeptide is expressed in a cell.
11. A substance which modulates histamine receptor activity and which is identifiable by a method according to claim 9 or 10.
12. A method of treating a subject having a disorder that is responsive to histamine-like receptor modulation, which method comprises administering to said subject an effective amount of a substance according to claim 11.
13. A method according to claim 12 wherein the disorder is selected from neurodegenerative disorders, for example cerebellar and spinocerebellar disorders, ataxia, spinocerebellar ataxia and atrophy, progressive supranuclear palsy, Huntington's disease, amyotrophic lateral sclerosis, Alzheimer's disease, Bell's palsy, cerebral palsy, Parkinson's disease, tardive dyskinesia, tremor, CNS disorders, for example pain, psychogenic erectile dysfunction, seizures, migraine, attention deficit, depression, anxiety, psychosis, mania, schizophrenia, diseases related to immune function, autoimmune function, viruses, inflammation or immunodeficiency, for example asthma, COPD, Crohn's disease, Inflammatory bowel syndrome, ulcerative colitis, gastroenteritis, inflammatory pain, allergy, rhinitis, dermatitis, anaphylaxis and septic shock, viral diseases, for example HBV, HSV and HIV, general inflammation, rheumatoid arthritis, osteoarthritis, osteoporosis, osteopetrosis, male and female reproductive fertility (ovary and testes expression), pre-term labor, dysmenorrhea, induction of labor, polycystic ovary disease, ovarian cysts, urinary incontinence, for example urge, stress, or mixed incontinence, hypothyroidism, hyperthyroidism, control of metabolic rate, obesity and/or insulindependent diabetes.
14. Use of a substance as defined in claim 11 in the manufacture of a medicament for treatment or prophylaxis of a disorder that is responsive to stimulation or modulation of histamine-like receptor activity.
15. A use according to claim 14 wherein the disorder is selected from

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neurodegenerative disorders, for example cerebellar and spinocerebellar disorders, ataxia, spinocerebellar ataxia and atrophy, progressive supranuclear palsy, Huntington's disease, amyotrophic lateral sclerosis, Alzheimer's disease, Bell's palsy, cerebral palsy, Parkinson's disease, tardive dyskinesia, tremor, CNS disorders, for example pain, psychogenic erectile dysfunction, seizures, migraine, attention deficit, depression, anxiety, psychosis, mania, schizophrenia, diseases related to immune function, autoimmune function, viruses, inflammation or immunodeficiency, for example asthma, COPD, Crohn's disease, Inflammatory bowel syndrome, ulcerative colitis, gastroenteritis, inflammatory pain, allergy, rhinitis, dermatitis, anaphylaxis and septic shock, viral diseases for example HBV, HSV and HIV, general inflammation, rheumatoid arthritis, osteoarthritis, osteoporosis, osteopetrosis, male and female reproductive fertility (ovary and testes expression), pre-term labor, dysmenorrhea, induction of labor, polycystic ovary disease, ovarian cysts, urinary incontinence, for example urge, stress, or mixed incontinence, hypothyroidism, hyperthyroidism, control of metabolic rate, obesity and/or insulin-dependent diabetes.
16. A method of producing a polypeptide according to claim 1 or 2, which method comprises maintaining a host cell as defined in claim 7 under conditions suitable for obtaining expression of the polypeptide and isolating the said polypeptide.