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WO1992022667A1 - Analogues de recepteurs d'hormone glycoproteique liant les gonadotrophines chorioniques, les leutrophines et les follitrophines, et leurs procedes de preparation et d'utilisation - Google Patents

Analogues de recepteurs d'hormone glycoproteique liant les gonadotrophines chorioniques, les leutrophines et les follitrophines, et leurs procedes de preparation et d'utilisation Download PDF

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Publication number
WO1992022667A1
WO1992022667A1 PCT/US1992/004987 US9204987W WO9222667A1 WO 1992022667 A1 WO1992022667 A1 WO 1992022667A1 US 9204987 W US9204987 W US 9204987W WO 9222667 A1 WO9222667 A1 WO 9222667A1
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Prior art keywords
amino acid
acid sequence
hcg
lhr
fshr
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William R. Moyle
Michael Bernard
Rebecca Myers
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Rutgers State University of New Jersey
Rutgers Health
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University of Medicine and Dentistry of New Jersey
Rutgers State University of New Jersey
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/033—Fusion polypeptide containing a localisation/targetting motif containing a motif for targeting to the internal surface of the plasma membrane, e.g. containing a myristoylation motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"

Definitions

  • This invention relates to glycoprotein hormone receptor chimeras and other analogs and methods for preparing and using the analogs.
  • the molecules of the invention are derivatives of naturally occurring receptors found in the ovary and testes. These proteins are usually found associated with the cell surface and combine specifically with the hormones. Protein engineering techniques permit construction of novel forms of the receptors which have the ability to bind both hCG and hFSH with high affinity. These analogs are useful in assays designed to measure biologically active hCG or hLH and hFSH at the same time.
  • the glycoprotein hormone family (1) consists of three alpha, beta heterodimeric glycoproteins found in the anterior pituitary gland where they are made and includes luteinizing hormone (LH), follicle stimulating hormone (FSH), and thyroid stimulating hormone (TSH).
  • LH luteinizing hormone
  • FSH follicle stimulating hormone
  • TSH thyroid stimulating hormone
  • a glycoprotein hormone structurally similar to LH is found in the placenta wherein it is synthesized.
  • this glycoprotein hormone is called human chorionic gonadotropin (hCG).
  • hCG human chorionic gonadotropin
  • Urine from pregnant women serves as a convenient source of hCG.
  • hMG human menopausal gonadotropins
  • Gonadotropins such as CG, LH, and FSH play a major role in the reproductive process (1, 2) while the structurally related hormone, TSH, is important for thyroid function (1).
  • LH and FSH are essential for puberty and normal reproductive function. Lack of sufficient FSH, LH, or hCG at appropriate times results in infertility or termination of pregnancy (2). Excessive amounts of these hormones can result in premature puberty or hyperstimulation of the gonads.
  • FSH is essential for follicular development leading to oocyte maturation and ovulation.
  • polycystic ovarian disease a condition which is characterized by incomplete follicular development, fertility can usually be restored by administration of FSH or treatments which increase FSH secretion.
  • LH is essential for ovulation and formation of the corpus luteum.
  • hCG is important for maintenance of progesterone secretion from the corpus luteum during early pregnancy.
  • LH is required for puberty and, in its absence, there is a failure to acquire the sexual attributes and fertility of an adult.
  • FSH is needed for the onset of spermatogenesis. The clinical activities of these hormones are reviewed extensively in several standard textbooks including that by Yen and Jaffe (2).
  • LHR LH receptors
  • FSHR FSH receptors
  • LH the major role of LH is to stimulate the formation of cyclic AMP leading to increased steroid hormone synthesis.
  • the steroids made depend on the cell type and include the androgens testosterone and androstenedione (Leydig and thecal cells) and progesterone (FSH-stimulated granulosa, thecal, and luteal cells).
  • LH also alters the activity of other enzymes which leads to the ovulation of mature follicles.
  • hCG is normally produced only by the placenta during pregnancy and acts on LHR to stimulate luteal progesterone synthesis.
  • FSH FSH
  • cyclic AMP which, in granulosa cells, enhances the conversion of androgens (i.e., the steroids produced in response to LH stimulation of testicular Leydig cells and ovarian thecal cells) to estrogens.
  • FSH also promotes the synthesis of inhibin and activin, the development of Sertoli and granulosa cells, gamete maturation.
  • the cDNA for several glycoprotein hormone receptors have been cloned (6-10) and enabled the primary amino acid sequences of the receptors to be determined (c.f., Figure 1 for the sequences of the rat LH, FSH, and TSH receptors).
  • Those for FSH and LH encode proteins of approximately 700 amino acids.
  • the N-terminal halves of these proteins i.e., those residues shown in the upper panel of Figure 1) appear to be located outside the cell.
  • the C-terminal half i.e., those residues shown in the lower panel of Figure 1 contains seven hydrophobic regions which presumably span the plasma membrane and anchor the protein on the cell surface.
  • the LHR appears to have two ligand binding domains.
  • a high affinity domain is found in the extracellular N-terminus (11-13) and a low affinity domain is located in the transmembrane region (11).
  • the specificity of ligand binding appears to be a property of the extracellular N-terminus since 1) analogs containing only the N-terminus or portions of the N-terminus of the LHR bind hCG (12, 13).
  • hCG hLH
  • hFSH hCG
  • hLH hLH
  • hFSH bioimmunoassay
  • the first two depend on the use of antisera and/or monoclonal antibodies.
  • the RLA and bioassays depend on the ability of the hormones to interact with their receptors.
  • the bioimmunoassay depends on the ability of the hormones to interact with their receptor and with an antibody (14). Since the regions of the hormones which interact with most antibodies and with the LHR and FSHR are not identical, these assays give different values when they are used to measure the hormones (15, 16).
  • the immunological assays often give lower values for the amount of LH and FSH in serum and this observation has led to the concept of a biological/immunological assay ratio (B/I). There is some evidence that this ratio changes as a function of hormone secretion. Diagnosis and treatment of infertility often depend on accurate measurements of circulating levels of LH and FSH as well as on the ratio of the two hormones. Given the variability between the hormone assays and the importance of obtaining accurate measurements of biologically active hormone, assays which could monitor the biological activity of LH and FSH as well as the ratio of LH:FSH activities in an unbiased fashion would be valuable.
  • Figure 1 shows a comparison of the extracellular N-termini and the transmembrane domain of the rat LHR (1), FSHR (4), TSHR (24). Numbers correspond to those of the rat LHR and exclude the signal sequences. Amino acids identical to rat LHR are indicated by a dot (.) Absent amino acids are indicated by a dash (-).
  • FIG. 2 shows the affinity of 125 I-hCG for the LHR, LHR-B, and LHR-E forms.
  • COS-7 cells were transfected with pSVL-LHR, pSVL-LHR-B, or pSVL-LHR-E. Four days latter, the cells were scraped from the dishes into 0.9% NaCl solution, sedimented at 600 ⁇ G (10 min), homogenized in buffer containing ice cold 1% Triton X- 100, and binding of 125 I-hCG was measured as described in the Examples.
  • the average value of Ka for the LHR expressed in COS-7 cells was (3.4+/-1.7) ⁇ 10 9 M -1 .
  • the binding constants obtained in this experiment were: LHR,
  • FIG 3 shows the binding of 125 I-hCG to LLLLL-LLL-LA and LLLLL-AAA-A.
  • 125 I-hCG was added to Triton-X100 extracts of COS-7 cells in duplicate. Bound and free radiolabel was separated by immunoprecipitation using an antiserum prepared against the C-terminal 11 amino acids of the ⁇ 2 AR as described in the Examples. Binding constants were estimated to be (2.2+/-0.1) ⁇ 10 9 M -1 and (3.6+/-0.4) ⁇ 10 9 M -1 for LLLLL-LLL-LA (LLLA) and LLLLL-AAA-A (LAA), respectively.
  • Figure 4 shows the binding of 125 I-hCG to LLLLL-LLL-L and LLLLLHH ⁇ .
  • FIG. 5 depicts the competition of hCG and hFSH for various receptor chimeras.
  • Triton X-100 solubilized receptors from transfected COS-7 cells were incubated with 125 I-hCG or 125 I-hFSH and varying amounts of hCG or hFSH in duplicate as shown.
  • the maximal amounts of I-hCG bound over the blank were 20,260; 3227; and 4253 cpm for LLLLL-LLL-L, FLLFF-FFF-F, F(F/L) (L/F) FF-FFF-F.
  • the maximal amounts of 125 l-hFSH bound over the blank were 19,850; 12,790; and 12220 cpm for FFFFF-LLL-L, FFFFF-FFF-F, and F(F/L) (L/F) FF-FFF-F. Binding of 125 I-hCG to LLLFF-LLL-L and LLLFF-FFF-F was not inhibited by a 10, 000-fold excess of hFSH.
  • Figure 6 Upper panel shows the affinity of 125 I-hCG for LHR/FSHR chimeras.
  • the binding constants obtained were: LLLFF-LLL-L, 8.0 ⁇ 10 9 M -1 ; FLLLL-LLL-L, 9.7 ⁇ 10 9 M -1 ; FLLFF-FFF-F, (2.2+/-1.0) ⁇ 10 9 M -1 ; F(F/L) (L/F) FF-FFF-F, (1.8+/-0.6) ⁇ 10 9 M -1 ; LLLFF-FFF-F, 3.2 ⁇ 10 9 M -1 (curve not illustrated).
  • FIG. 6 Lower panel shows the affinity of 125 I-hFSH for the FSHR and F(F/L) (L/F) FF-FFF-F.
  • the binding constants obtained were: FSHR, (3.5+/-2.7) ⁇ 10 9 M -1 ; F(F/L) (L/F) FF-FFF- F, (4.1+/-2.2) ⁇ 10 9 M -1 .
  • Data illustrated in these panels were obtained in different experiments.
  • Figure 7 is a representation of a model of receptor binding determinants of the LHR and FSHR.
  • a schematic diagram of the LHR and FSHR is illustrated showing the extracellular N-terminus, the seven transmembrane domains, and the intracellular C-terminus. The regions of the extracellular domains that convey the ability of the LHR and FSHR to distinguish hCG and hFSH are indicated. Note that this model does not exclude a lower affinity interaction of the hormones with other portions of their receptors.
  • the numbering system employed herein corresponds to that for LHR.
  • the corresponding numbering system for FSHR may be determined by reference to Figure l.
  • Figure 8 illustrates a strategy for preparing the LHR/FSHR chimeras used in these studies.
  • the upper portion describes the PCR strategy for obtaining cDNA fragments which encode various forms of the FSHR. Primer combinations 1 & 2, 3 & 4, and 5 & 6 were used to obtain coding sequences for residues -26/201, 202/341, and 342/675, respectively.
  • restriction sites were incorporated into the cDNA fragments which permitted subcloning directly into the indicated sites of the LHR (i.e., LLLLL-LLL-L) as illustrated.
  • the lower portion illustrates the remaining LHR/FSHR chimeras used in these studies.
  • the first hyphen denotes the junction of the N-terminus and transmembrane domain and the second hyphen denotes the junction of the transmembrane domain and the C-terminus;
  • the first block (five letters) refers to the location of DNA sequences between selected restriction endonuclease sites in the N-terminus including XbaI-BglII (-26 to 58), BglII-BglII (59-123), BglII-Apal (124-202), Apal-Fspl (203-259), and FspI-Bsu36I (260-341), respectively;
  • the second block (three letters) refers to location of DNA sequences between selected restriction endonuclease sites in the transmembrane domain including Bsu36I-SphI, Sphl-Ball, and Ball-Espl; and the third block (one letter) refers to the C-terminus.
  • L residues derived from LHR;
  • FIG. 9 illustrates methods for the preparation of analogs LLLLL-LLL-L (LLL), LLLLL-LLL-LA (LLLA), LLLLL-AAA-A (LAA), A-LLL-L (ALL), A-LLL-LA (ALLA), LLLLL ⁇ (L ⁇ ), LLLLLHH ⁇ (LLLHH), and LLLLL-V-V (LVV) are described in the Examples.
  • AAA refers to the hamster ß 2 -adrenergic receptor (24).
  • the open bars refer to residues derived from the rat LHR and the solid bars refer to residues derived from the hamster ß 2 AR.
  • Residues in the bars denoted by ascending hatchmarks (////) refer to the 27 C-terminal residues of the ß 2 AR which include the 11 amino acid sequence recognized by the rabbit anti-ß 2 AR serum used to immunoprecipitate the receptor (c.f., LLLA, below). Residues in bars denoted by descending hatchmarks ( ⁇ ) are derived from the VSV-G transmembrane and C-terminus.
  • Amino acid sequences in the region of the junctions are: LLLA, -VHCQQPIPPRAFFV- NCQGTVPSLSLDSQGRNCSTNDSPL*; LAA, -DIMGYAFLRDEAWVVGMAILM- ; ALL, -TTNGSHVPDHDVTDLVLIWLINILAIFGNLTVLFVLLTS-; L ⁇ , - DIMGYAFLR*; LVV, -DIMGYAFLRSSIASFFFFIIGLIIGLFLVLR-; LHH, -DIMGYAFLRHH* where the residues derived from the LHR are underlined. Note that the corresponding junctions in related analogs (i.e., ALLA and LLLA or ALL and ALLA) are identical.
  • Figure 10 shows the binding of 125 I-hCG to LHR expressed in ldl-d cells.
  • pMB13 was expressed stably in ldl-d cells as set out in Example 27.
  • the cells were cultured in the presence or absence of galactose (Gal) and N-acetylgalactosamine (GalNac) for 4 days (26).
  • Gal galactose
  • GalNac N-acetylgalactosamine
  • the ldl-d cells cannot make Ser-linked oligosaccharides. Other oligosaccharides lack galactose and sialic acid.
  • Whole ldl-d cells were incubated with 125 I-hCG and varying amounts of unlabeled 125 I-hCG as shown on the abscissa.
  • Bound and free 125 ⁇ - hCG were separated by sedimentation. Detergent solubilized receptors from these cells were also incubated with 125 I-hCG and unlabeled hCG as shown on the abscissa. Bound and free 125 I-hCG were separated by PEI filtration (21). As illustrated in this Figure, 125 I-hCG had a lower affinity to LHR in the membrane of ldl-d cells which had been grown in the presence of galactose and N-acetylgalactosamine than those which had been grown in the absence of these sugars. This difference was less apparent for the solubilized receptors from these cells.
  • Figure 11 shows the competition of hCG and hFSH for binding of 125 I-hCG to pMB133.
  • COS-7 cells transfected with pMB133 were solubilized with buffer containing Triton X-100 and incubated with 125 I-hCG and hormones as described in Example 1. Bound and free 125 ⁇ - hCG were separated by filtration on PEI coated filters (21). Results illustrated here show that hCG and hFSH bind to pMB133. This shows that only a very small region of the receptor is responsible for ligand binding specificity and that this pMB133 analog would be useful for measurement of hormones which bind to LH and FSH receptors.
  • Figure 12 shows a comparison of the extracellular N-termini of several LHR/FSHR chimeras discussed in the Examples. Numbers correspond to those of the rat LHR and exclude the signal sequences.
  • the present invention pertains to a protein having binding affinity to human chorionic gonadotrophin (hCG), luteinizing hormone (LH), and follicle stimulating hormone (FSH), comprising an amino acid sequence of 341 amino acids, wherein the protein is a chimera having an amino acid sequence homologous to the amino acid sequence of residues 1-92 of FSHR, an amino acid sequence homologous to the amino acid sequence of residues 93-170 of LHR, and an amino acid sequence homologous to the amino acid sequence of residues 171-341 of FSHR, wherein the protein is a chain of amino acids that is not naturally occurring as a binding protein. (LH numbering system).
  • hCG human chorionic gonadotrophin
  • LH luteinizing hormone
  • FSH follicle stimulating hormone
  • the present invention pertains to a glycoprotein hormone receptor having binding affinity to human chorionic gonadotrophin (hCG), luteinizing hormone (LH), and follicle stimulating hormone (FSH), wherein the receptor comprises a transmembrane region and an extracellular N-terminus region, wherein the N-terminus region is a chimera containing an amino acid sequence of 341 amino acids comprising an amino acid sequence homologous to the amino acid sequence of residues 1-92 of FSHR, an amino acid sequence homologous to the amino acid sequence of residues 93-170 of LHR, and an amino acid sequence homologous to the amino acid sequence of residues 171-341 of FSHR, wherein the receptor is a chain of amino acids that is not naturally occurring as a receptor. (LH numbering system).
  • hCG human chorionic gonadotrophin
  • LH luteinizing hormone
  • FSH follicle stimulating hormone
  • This invention relates to glycoprotein hormone receptor chimeras and other analogs and methods for preparing and using the analogs.
  • the molecules of the present invention are derivatives of naturally occurring receptors found in the ovary and testes which are usually found associated with the cell surface and combine specifically with the hormones. These glycoprotein hormone receptor analogs are useful in assays designed to measure biologically active hCG or both hLH and hFSH at the same time.
  • the specificity of ligand binding appears to be a property of the extracellular N- terminus because analogs containing only the N-terminus or portions of the N-terminus of the LHR bind hCG (12, 13, Figures 2, 3 and 4). Accordingly, applicants have prepared analogs of the LHR and FSHR in an effort to identify the region of the N-terminus which contains the ligand binding domain.
  • vectors were prepared encoding the rat LHR N- terminal domain alone (L ⁇ ), the LHR N-terminal domain fused to the transmembrane and C-terminal domains of the vesicular stomatitis virus-G protein (LVV), the LHR N- terminal domain fused to the transmembrane and C-terminal domains of the hamster B 2 AR (LAA), and the B 2 AR N- terminal domain fused to the transmembrane and C-terminal domains of the rat LHR (ALL).
  • L ⁇ rat LHR N- terminal domain alone
  • LAA vesicular stomatitis virus-G protein
  • LAA hamster B 2 AR
  • ALL B 2 AR N- terminal domain fused to the transmembrane and C-terminal domains of the rat LHR
  • the first hyphen denotes the junction of the N-terminus and transmembrane domain and the second hyphen denotes the junction of the transmembrane domain and the C-terminus;
  • the first block (five letters) refers to the location of DNA sequences between selected restriction endonuclease sites in the N-terminus including XbaI-BglII (-26 to 58), BglII-BglII (59-123), BglII-Apal (124-202), Apal-Fspl (203-259), and FspI-Bsu36I (260-341), respectively;
  • the second block (three letters) refers to location of DNA sequences between selected restriction endonuclease sites in the transmembrane domain including Bsu36I-SphI, Sphl-Ball, and Ball-Espl; and the third block (one letter) refers to the C-terminus.
  • L refers the location of DNA sequences between selected
  • amino acid sequence variants will be substantially homologous with the relevant portion of the N-terminus of the receptor coupled to the transmembrane as set forth in Figure 1.
  • substantially homologous means that greater than about 70% of the primary amino acid sequence of the candidate polypeptide corresponds to the sequence of the relevant portion of the N-terminus polypeptide when aligned in order to maximize the number of amino acids residue matches between the two polypeptides. Alignment to maximize matches of residues includes shifting the amino and/or carboxyl terminus, introducing gaps as required or deleting residues present as inserts in the candidate polypeptide, or both. For example, see Figure 1 where the glycoprotein hormone receptors are aligned for maximum homology.
  • amino acid sequence variants will be greater than about 90% homologous with the corresponding sequences shown for the proteins in Figure 1.
  • Variants that are not hormonally-active fall within the scope of this invention, and include polypeptides that may or may not be substantially homologous with a sequence described herein but which are 1) immunologically cross-reactive with antibodies raised against the counterpart polypeptide or 2) capable of competing with such counterpart polypeptides for cell surface receptor binding.
  • Hormonally active variants are produced by the recombinant or organic synthetic preparation of fragments or by introduction of amino acid sequence variations so that the molecule no longer demonstrates hormonal activity as defined herein.
  • Immunological or receptor cross-reactivity means that the candidate polypeptide is capable of competitively inhibiting the binding of the hormonally-active analogue to polyclonal antisera raised against the hormonally-active analogue.
  • antisera are prepared in a conventional manner by injecting goats or rabbits S.C. with the hormonally active analogue or derivative in complete Freunds adjuvant, followed by booster intraperitoneal or S.C. injections in incomplete Freunds.
  • Variants that are not hormonally active but which are capable of cross-reacting with antisera to hormonally active polypeptides are useful a) as reagents in diagnostic assays for the analogues of their antibodies, b) when msolubilized in accord with known methods, as an agent for purifying anti-analogue anti- antibodies from anti-sera, and c) as an immunogen for raising antibodies to hormonally active analogues.
  • Binding affinity studies of the series of analogs of the LHR and FSHR described herein permit the deduction of the relative locations of the ligand binding sites. Some of these analogs have the unique characteristics of being able to recognize both hCG and FSH. Since hCG and LH bind to the same receptors, analogs that bind hCG will also recognize LH. One of these analogs F(F/L) (L/F) FF-FFF-F (pMB111) binds both ligands with high affinity, albeit not at the same time.
  • LLLLL-LLL-LH 2 and LLLLL-H 2 have high affinity for 125 I-hCG and FFFFF-FFF-FH 3 has high affinity for 125 I-hFSH.
  • NTA resin obtained from Qiagen, Chatsworth, CA 91311
  • COS-7 cells were used herein to express pMB111. These cells are good at transient expression using the vector described below. However, as noted in this disclosure, other eukaryotic cells have been shown to express these types of receptors and it is expected that a large variety of expression systems will work well. Indeed, since the region of the binding domain which determines ligand binding specificity has been determined in this invention, it is possible that bacterial expression systems will be useful. We have also found that the LHR can be expressed in a CHO cell line known as ldl-D (26) which can produce receptors which lack fully glycosylated proteins.
  • Useful antibodies in the bioimmunoassays of the present invention include but are not limited to antibodies having the properties of B105 (obtained from
  • the LHR coding sequences were obtained from an ovarian corpus luteum library in three segments using PCR conditions which have been described (17).
  • the PCR product encoding amino acids -26 to 315 was created using primers 5'-GGCTCAACCTCTAGAGCTCACACTCA-3' and 5'-GCAGAAGCCGTAGTCGTAGTCCCA-3' digested with XbaI and subcloned between the XbaI and Smal sites of pIBI31 (obtained from IBI) to give pMB3.
  • the sequence of the bases in the region between the XbaI and Apal sites was determined by dideoxy methods and shown to encode the same amino acids between 26 and 201 as reported for the rat LHR (6).
  • the PCR product obtained using primers 5'-CCACGGGGCCCAGCATCCTGGATATTTC-3' and 5'- TGGGATGGCATGCCTCAGTCTTAGCT-3' and which encoded amino acids 201 to 460 was digested with Apal and SphI and subcloned between the Apal and SphI sites of pIBI31 to give pMBl.
  • the sequences of the bases in the region between the Apal and SphI sites was determined by dideoxy methods and shown to encode the same amino acids reported for the rat LHR (6).
  • PCR product obtained using primers 5'-CTGAGGCATGCCATCCCAATTATGCTC-3' and 5'-ATTTTGGATCCTAGTGAGTTAACGCTCTCGGT-3' and which encoded amino acids 461 to 674 was digested with SphI and BamHI and subcloned between the SphI and BamHI sites of pIBI31 to give pMB4.
  • the coding sequence of pMB4 was found to be the same as that of the rat LHR (6). All subcloning and dideoxy sequencing used in these studies were performed by standard methods (18, 19).
  • pMB11 a vector which expressed the full length LHR, the LHR coding fragments formed upon digestion of pMB3 with XbaI/Apal (i.e., residues 26->201), digestion of pMB1 with Apal/SphI (i.e., residues 201->460), and digestion of pMB4 with Sphl/BamHI (i.e., residues 461->674) were ligated together with the large fragment released from pSVL (obtained from Pharmacia) by digestion with XbaI and BamHI.
  • pMB11 was transfected into COS-7 cells using calcium phosphate procedures (18, 19).
  • hCG and hFSH were measured using 1 25 I-hCG, 125 I-eLH, and 125 I-hFSH.
  • the cells were solubilized using a buffer containing Triton X-100 as described (21) and incubated with the radiolabels for 1-2 hours at room temperature or at 4° C. overnight.
  • the 1 25 I-hCG used in these assays was prepared using an iodogen procedure which has been described (22).
  • Preparation of 125 I-eLH and 125 I-hFSH was similar to that of 125 I-hCG except that the labeling period with Iodogen was reduced from 5 minutes to 30 seconds.
  • pMB52 is an LHR/FSHR chimera containing FSHR sequences for LHR residues -25->201.
  • the FSHR coding sequences used to make pMB52 were obtained from an ovarian corpus luteum library (17) by PCR as follows ( Figure 8, upper section). During PCR, some codons of the FSHR were modified to create endonuclease restriction sites which facilitated chimera construction and enabled the PCR products to be directionally subcloned into XbaI and Apal sites of pSVL-LHR.
  • pMB52 was expressed in COS-7 cells by methods which have been described for pMB11 in Example 1. As illustrated in Table 1, FFFLL-LLL-L did not substantially bind 125 I-hCG, 125 I-eLH, or 125 I-hFSH specifically in these conditions
  • an LHR/FSHR chimera containing FSHR sequences for LHR residues 202->341 we used primers #3 [5'-GGAGCCTCTGGGCCCGTCATTTTAGAT-3'] and #4 [5'-TATCAAGACCCTAAGGATGTTGTACCC-3'] and the ovarian corpus luteum library (17) as a template ( Figure 8, upper section).
  • the resulting PCR product contained Apal and Bsu36I endonuclease restriction sites which were used to subclone the fragment into the pMB11 vector by standard methods.
  • the base sequence of the FSHR portion of this chimera was determined using a double stranded dideoxy method (18, 19).
  • LLLLL-FFF-F the LHR/FSHR chimera which contained the transmembrane and C-terminal portions derived from the FSHR
  • primers #5 [5'- TACAACATCCTTAGGGTCTTGATATGG-3'] and #6 [5'- CTCTCTGGATCCTAATGATGATGGTTCTGGCTCGAGTGATTAAGAGGGACAAGCACG TAACT-3] and the ovarian corpus luteum library (17) as a template ( Figure 8, upper section).
  • This PCR product contained Bsu36I and BamHI sites and was cloned into the corresponding sites of pMB11 using standard methods.
  • This chimera also contained three additional His residues at the C-terminus not found in the naturally occurring FSHR.
  • the base sequence of the FSHR portion of this chimera was determined using a double stranded dideoxy method (18, 19). This was expressed in COS-7 cells using procedures described for pMB11.
  • the binding of hCG and hFSH was monitored using 125 I-hCG, 125 I-eLH, and 125 I- hFSH also as described for pMB11 in Example 1. This analog bound 125 I-hCG better than 125 I-hFSH although it did not appear to be expressed well (Table 1).
  • This analog was prepared by ligating the small XbaI/Apal, ApaI/Bsu36I, and Bsu36I/BamHI fragments of pMB52, pMB53, and pMB11 with the large XbaI/BamHI fragment of pSVL.
  • pMB55 was expressed in COS-7 cells using procedures described for pMB11. Binding of hCG and hFSH was monitored using 125 I-hCG, 125 I-eLH, and 125 ⁇ -hFSH also as described for pMB11 in Example 1. This analog was found to bind 125 I-hFSH and 125 I-eLH better than 125 I-hCG ( Figure 6, Table 1).
  • This analog was prepared by ligating the small XbaI/Apal, ApaI/Bsu36I, and Bsu36I/BamHI fragments from pMB59, pMB11, and pMB59 with the large XbaI/BamHI fragment of pSVL. This was expressed in COS-7 cells using procedures described for pMB11. Binding of hCG and hFSH was monitored using 125 I-hCG, 125 I-eLH, and 125 I- hFSH also as described for pMB11 in Example 1. This analog was found not to substantially bind any of the ligands above the level of non-specific binding (Table 1).
  • Example 10 After the region between XbaI and Apal was sequenced using dideoxy methods, pMB72 was expressed in COS-7 cells using the procedure outlined in Example 1. Binding of 125 I-hCG, 125 I-eLH, and 125 I-hFSH was also measured as described in Example 1. This analog was found to bind 125 I-hCG with high affinity ( Figure 6, Table 1). While it bound 125 l-eLH, it did not substantially bind 125 I-hFSH above the level of non-specific binding (Table 1).
  • Example 10 Example 10
  • pMB72 was expressed in COS-7 cells using the procedure outlined in Example 1. Binding of 125 I-hCG, 125 I-eLH, and 125 I-hFSH was also measured as described in Example 1. LFLLL-LLL-L did not substantially bind any of the ligands above the level of non-specific binding (Table 1).
  • pMB72 was expressed in COS-7 cells using the procedure outlined in Example 1. Binding of 1 25 I-hCG, 125 I-eLH, and 125 I-hFSH was also measured as described in Example 1. LLFLL-LLL-L did not substantially bind any of the ligands above the level of non-specific binding (Table 1).
  • This analog was prepared by ligating the small fragment produced by digestion of pMB72 with XbaI and Apal to the large fragment produced by digestion of pMB59 with XbaI and Apal.
  • FLLFF-FFF-F was expressed in COS-7 cells using the procedure outlined in Example 1. Binding of 125 I-hCG, 125 I-eLH, and 125 I-hFSH was also measured as described in Example 1. FLLFF-FFF-F had high, affinity for hCG. It also bound hFSH and was approximately 1% as good as the product of pMB59 in this regard. Both hCG and hFSH competed for binding to this analog ( Figures 5 and 6, Table 1). FLLFF-FFF-F also bound 125 I-eLH (Table 1).
  • F(F/L) (L/F) FF-FFF-F was made by in a two-step PCR reaction.
  • three separate PCR reactions were performed including 1) primers 5'-AGGATTGAAAAGGCCAACAACCTGCTATACATTGAACCTGGTG-3' and 5'-CATCTAGCTGCGTCCCATTGAATGCATGGCTTTGTACTTC-3' with pSVL-11 as template, 2) primers 5'-AGCTGTCCTGGAGCTAGGAATCTCTGTACGGAAGTGTTACTTCTGCTCT-3' and 5'-GTATAGCAGGTTGTTGGCCTTTTCAATCCTAATTTCATGC-3' with pSVL- 59 as template, and 3) primers 5'-GCATTCAATGGGACGCAGCTAGATGAACTGAATCTAAGCG-3' and 5'-CTAGCCTAGAAGCTCTGACTGTCTTTGGTGGAAGAAAATATCCAGGATGCT-3' with pSVL-59 as template.
  • analog LFLLL-LLL-L contains Cys residues which are not comparable to those in the LHR.
  • PCR was employed to change Ser 109 /Cys in analog LFLLL-LLL-L using primers 5'-GAGAGAGGAGATCTTGTGCACCGCGGGCAAGTGCTTAATGCCTGTGTTGCATATTAA CAG-3'and 5'-CTCAGTCTAGAGGGCCATGGGGCGGCGAGTCCCAG-3' with LFLLL-LLL-L as template.
  • the BglII fragment from this PCR product was used to replace the BglII/BglII fragment of LFLLL-LLL-L to form LF C LLL-LLL-L and the expected product confirmed by dideoxy DNA sequencing.
  • This analog was expressed in COS-7 cells using methods identical to those used to express pMB11.
  • LF C LLL-LLL-L did not substantially bind 125 I-hCG , 125 I-eLH, or 125 I-hFSH better than the non-specific binding controls (Table 1) .
  • TGGGATGGCATGCCTCAGTCTTAGCT-3' were mixed and a second PCR performed using primers 5'- GTTCACAAGATCTCCTCTCTGCAGAAGAACGTTCTACTAGACATTTGCGATAACATA AAC-3' and 5'-TGGGATGGCATGCCTCAGTCTTAGCT-3'.
  • the final PCR product was digested with BglII and Apal and the fragment that was produced was ligated to the large piece created by digestion of pMB11 with BglII and Apal.
  • the LHR coding sequences were obtained from an ovarian corpus luteum library in three segments using PCR conditions which have been described (17).
  • the short PCR product encoding an alternately spliced form of the LHR cDNA in the region coding amino acids 201 to 460 was digested with Apal and SphI and subcloned between the Apal and SphI sites of pIBI31 to give pMB2 which had the sequence reported (17).
  • pMB10 a vector which expressed the B-form of the LHR, the small fragments formed upon digestion of pMB3 with Xbal/Apal, digestion of pMB2 with Apal/SphI, and digestion of pMB4 with Sphl/BamHI were ligated together with the large fragment released from pSVL (obtained from Pharmacia) by digestion with Xbal and BamHI.
  • pMB10 was transfected into COS-7 cells using calcium phosphate procedures which have been described (20). Triton X-100 extracts of cells transfected with pMB10 bound 125 I-hCG with high affinity ( Figure 2).
  • Example 20 Example 20
  • the LHR coding sequences were obtained from an ovarian corpus luteum library in three segments using PCR conditions which have been described (17).
  • the intermediate PCR product encoding an alternately spliced form of the LHR cDNA in the region coding amino acids 201 to 460 was obtained using primers 5'-GGCTTTGGGCCCAGCATCCTGCAGAATTTTTCATTTTCCATTTTTGA-3' and 5'-TGGGATGGCATGCCTCAGTCTTAGCT-3', digested with Apal and SphI, and ligated with the coding sequence released by XbaI/Apal digestion of pMB3, the coding sequence released by Sphl/BamHI digestion of pMB4, and the large fragment released by XbaI/BamHI digestion of pIBI31 to give pMB33.
  • This vector encodes an LHR analog in which residues Leu 672 Thr 673 His 674 are replaced by the sequence
  • pMB9 was made by ligating the coding sequences from XbaI/Apal digestion of pMB3, Apal/SphI digestion of pMBl, and Sphl/BamHI digestion of pMB4 with the large fragment produced by digesting pIBI31 with XbaI and BamHI.
  • pMB40 was made by digesting pMB9 with Hpal and BamHI and ligating the large fragment with the AluI/HamHI fragment of the product of a PCR reaction which employed primers 5'-GACGGAAAGCTTTGTGAACTGT-3' and 5'- GAAAGCGGATCCTACAGCGGTGAGTCATTTGT-3' and the hamster ß 2 - adrenergic receptor cDNA (25) as template.
  • the coding sequence released from pMB40 by XbaI/BamHI digestion was ligated to the large fragment obtained by digesting pSVL with XbaI and BamHI to give pMB46.
  • This vector was expressed in COS-7 cells using methods described in Example 1. LLLLL-LLL-LA bound 125 I-hCG with high affinity and the complex was readily immunoprecipitated using the antisera to the C-terminus of the ß 2 -adrenergic receptor ( Figure 3, Table 3).
  • This vector encodes an LHR analog in which residues of the LHR transmembrane and C-terminal domains are replaced with those of the hamster ß 2 -adrenergic receptor ( Figure 8).
  • the DNA- sequence encoding the transmembrane and C-terminal sections of the ß 2 -adrenergic receptor was amplified by PCR with primers 5'-GTCACTGACCTTAGGGACGAAGCATGGGTG-3' and 5'- GAAAGCGGATCCTACAGCGGTGAGTCATTTGT-3' and the ß 2 -adrenergic receptor cDNA as a template.
  • This vector encodes an LHR analog in which the seven membered LHR transmembrane domain and C-terminal domain are replaced with the single transmembrane domain and the C-terminal domain of the vesicular stomatis G protein, respectively ( Figure 8).
  • LLLLL-V-V was prepared from an analog of the hCG ß-subunit which contained the VSV-G transmembrane and C-terminal domains.
  • oligonucleotides 5'-GGGCTCTATTGCCTCTTTTTTCTTTATCAT-3' and 5'-GGATCCGAGTTACTTTCCAAGTCGGTTCAT-3' to insert Smal and BamHI restriction sites into the region of the VSV-G cDNA encoding the transmembrane and C-terminal domains.
  • the PCR product was digested with Mbol and cloned into the Smal-BamHI sites of hCG ß-subunit cDNA which had previously been inserted into pSVL as described (20).
  • Preparation of LLLLL-V-V was performed by PCR of the hCG-VSV-G construct in pSVL using primers S'-GCATCCC-GCCTTAGGAGCTCTATTGCCTCT-3' and 5'-GTCCAAACTCATCAATG3' , digesting the PCR product with Mstll and BamHI, and subcloning it into the Mstll-BamHI sites of the vector encoding the LHR. When made in COS-7 cells, this analog had high affinity for 125 I-hCG (Table 2).
  • This vector encodes an LHR analog in which the transmembrane and C-terminal domains were replaced by His residues (Figure 8).
  • LLLLLHH— the analog truncated after amino acid residue 341 was prepared by inserting a termination codon at the Mstll site.
  • pMB11 was digested with Mstll and BamHI and the large fragment obtained was ligated with a cassette prepared by annealing oligos 5'- TTAGGCATCATTAG-3' and 5'-GATCCTAATGATGCC-3'. When made in COS-7 cells, this analog had high affinity for 125 l- hCG (Table 2). This analog also binds to an NTA resin.
  • This vector encodes an LHR analog in which the N-terminal domain was replaced with that derived from the hamster ß 2 -adrenergic receptor ( Figure 8).
  • Figure 8 To prepare A- LLL-L, we used PCR to insert an Mstll site into the region which encodes the junction of the N-terminus and the transmembrane domains of the ß 2 AR using primers 5'- TTCGTCCCTAAGGGTCAGTGACATCGTGGTC-3' and 5'- GGCTCAACCTCTAGAGCTCACACTCA-3'.
  • This vector encodes an LHR analog which two additional His residues at the C-terminus.
  • pMB9 was digested with Hpal and BamHI and the large piece which remained was ligated with the cassette prepared by annealing oligos 5'-GACTCACCACCACTAG-3' and 5'-GATCCTAGTGGTGGTGAGTC-3' to make pMB21.
  • the 2.1Kb fragment released on digestion with XbaI and BamHI was ligated into the XbaI/BamHI sites of pSVL.
  • this receptor analog When expressed in COS-7 cells, this receptor analog had high affinity for hCG. This analog was adsorbed to an NTA resin and enables 125 I-hCG to bind to this resin (Table 4).
  • pMB13 a vector used to stably express an analog of the LHR lacking galactose and sialic acid residues.
  • the LHR coding sequence was removed from pMB11 by XbaI/BamHI digestion and ligated into the large fragment obtained after XbaI and BamHI digestion of expression vector pLEN'-hCGß'.
  • pLEN'-hCGß' is a vector derived from pLEN (25) which was obtained from P.J. Kushner, UCSF, San Francisco, CA 94143.
  • pLEN contains the sequences of pUC8, the 1.12Kbp Malawi fragment of SV40 as an enhancer, bases -771->70 of the human metalothionein IIA gene as a promoter, a unique BamHI site, and approximately 0.3 Kbp of the 3' end of the human growth hormone gene. Constructs cloned into the BamHI site in the correct orientation can be expressed stably in CHO cells (25).
  • pLEN'-hCGß' was made from pLEN as follows. pKBM-hCGß' (20) was digested with Hindlll and ligated to the cassette formed by annealing the oligonucleotide 5'-AGCTAGATCT-3' to itself.
  • pLEN'-hCGß' This vector was then termed pLEN'-hCGß' and contained the hCG ⁇ ' cDNA flanked on the 5' side by Xhol and XbaI restriction endonuclease sites and on the 3' side by Sad and BamHI restriction sites in place of the original BamHI site.
  • pLEN'-hCGß' was digested with XbaI and BamHI and the large fragment ligated to the small fragment obtained from pMB11 by XbaI/BamHI digestion.
  • pMB13 was cotransfected into ldl-d CHO cells (26) obtained from M.
  • pMB13 and pSV2Neo were transfected into Y-1 adrenal cells obtained from the American Type Culture Collection using a calcium phosphate procedure (18, 19). One day later, 250 ug/ml G418 was added and the cells which survived were tested for their abilities to bind 125 I-hCG. Clones of cells were obtained which were resistant to G418 and which expressed LHR. These cells became round shortly after exposure to hCG.
  • the DNA region encoding the receptor construct was excised by digestion of pMB21 with XbaI and BamHI and ligated to the large fragment obtained from pLEN'-hCGß' by digestion with XbaI and BamHI to give pMB26.
  • pMB26 and pSV40Neo were cotransfected into CHO cells and stable clones were obtained using G418 selection as described in Example 27. Triton X-100 extracts of these cells bound to the NTA resin (Table 4).
  • pMB120 a vector which encodes FFFEE-FFF- F, an analog of the FSHR analogous to the LHR-E form.
  • pMB120 was created by ligating the fragment resulting from Apal and Bsu36I digestion of the PCR product obtained using primers 5'-AGTAGCAAGTAGATGCC-3' and 5'-GCCTCTGGGCCCGTCATTTTAATCTCTGAACTTCATCCAATTTGCA-3' with pMB59 as template to the small fragment created by digesting pMB52 with XbaI and Apal, the small fragment created by digestion of pMB54 with Bsu36I and BamHI, and the large fragment created by digestion of pSVL with XbaI and BamHI.
  • pMB120 was expressed in COS-7 cells similar to the procedure noted in Example 1.
  • the FSHR-E form did not substantially bind 125 I-hCG or 125 I-hFSH (Table 1).
  • This vector was prepared by ligating the small fragment resulting from XbaI and Bsu36I digestion of pMB22 with the large fragment obtained from XbaI and Bsu36I digestion of pMB40 described in Example 21. This chimera did not substantially bind 125 I-hCG.
  • This vector was prepared by ligating the smal fragment resulting from XbaI and Bsu36I digestion of pMB22 with the large fragment obtained from XbaI and Bsu36 digestion of pMB40 described in Example 21. This chimera di not substantially bind 125 I-hCG.
  • Example 32
  • pMB132 a vector which encodes F(F/L)LFF-FFF-F, a chimera of the LHR and FSHR.
  • pMB111 was digested with BglII and the small fragment was ligated with the large fragment which resulted from BglII digestion of pMB88.
  • the insert with the correct orientation was confirmed by a dideoxysequencmg procedure (18, 19).
  • F(F/L)LFF-FFF-F was found to have low ability to bind 125 I-hCG and 125 I-hFSH (Table 1).
  • Example 33 Preparation of pMB136, a vector which encodes F(F/FL) (LL/F) FF- FFF-F, a chimera of the LHR and FSHR.
  • pMB136/pMB139 were made in an effort to further localize the smallest number of amino acid residues derived from the LHR that are needed to confer 125 I-hCG binding to pMB111. These are all derivatives of pMB111 in which the region derived from the LHR (i.e., corresponding to LHR residues 93/170) was divided into four sections and each was replaced by residues from the FSHR.
  • pMB136 was made in a two step PCR reaction.
  • Step Ia employed primers 5'- CTAGCCTAGAAGCTCTGACTGTCTGTCCTTGAGATATCTAAAATGAC-3' and 5'-ATCTGTTAATATGTAACACAGGCATCCGAACCCTT-3' and pMB111 as templat and step lb employed primers 5'-GCCTGTGTTACATATTAACAGATATCTGAGACTGG-3' and 5'-AGCTGTCCTG GAGCTAGGAATCTCTGTACGGAAGTGTTACTTCTGCTCT-3' and pMB59 a template.
  • Step II employed primers 5'- AGCTGTCCTGGAGCTAGGAATCT-3' and 5'-CTAGCCTAGAAGCTCTGACTGTC-3' and the products of steps Ia and lb as template.
  • pMB137 a vector which encodes F(F/LF) (LL/F) FF- FFF-F, a chimera of the LHR and FSHR.
  • pMB137 was made in a two step PCR reaction.
  • Step Ia employed primers and 5'- CTAGCCTAGAAGCTCTGACTGTCTGTCCTTGAGATATCTAAAATGAC-3' 5'- CACAAGATCCAGTCTCTCCCAAAAGGTTCTACTAGACATCTGTGATAACTTACACATAACCA CC-3' and pMB111 as template
  • step lb employed primers 5'-GGAGAGACTGGATCTTGTGAACAGCTGGCAAGTGCTTGATGCCTGTGTTACAGATGC-3' and 5'-AGCTGTCCTGGAGCTAGGAATCTCTGTACGGAAGTGTTACTTCTGCTCT-3 ' and pMB111 as template.
  • Step II employed primers 5'-AGCTGTCCTGGAGCTAGGAATCT-3 ' and 5'-CTAGCCTAGAAGCTCTGACTGTC-3' and the products of steps Ia and lb as template.
  • the resulting product was digested with XbaI and Apal and ligated to the large fragment resulting from XbaI and Apal digestion of pMB59.
  • F(F/LF) (LL/F) FF-FFF-F had low ability to bind 125 I-hCG and 125 I-hFSH (Table 1).
  • Step Ia employed primers 5'- CTAGCCTAGAAGCTCTGACTGTCTGTCCTTGAGATATCTAAAATGAC-3' and 5'- GCTTGCCAGGAACTCCTTCATGGGGCTGAGTTTTGAGTCTGTCACACTAAAACTG-3' an pMB111 as template and step lb employed primers 5'- GAAGGAGTTCCTGGCAACGATGTGTATGTTTATGTTATCACAGATTTCCAG-3' and 5'- AGCTGTCCTGGAGCTAGGAATCTCTGTACGGAAGTGTTACTTCTGCTCT-3' an pMB111 as template.
  • Step II employed primers 5'- AGCTGTCCTGGAGCTAGGAATCT-3' and 5'-CTAGCCTAGAAGCTCTGACTGTC-3' and the products of steps Ia and lb as template.
  • the resulting product was digested with XbaI and Apal and ligated to the large fragment resulting from XbaI and Apal digestion of pMB59.
  • F(F/LL) (FL/F)FF-FFF- F had higher ability to bind 125I-hCG than 1251-hFSH (Table 1). This binding ability shows that only a portion of the region between residues 93/170 needs to be derived from the LHR to insure that the receptor binds 125I-hCG.
  • pMB139 a vector which encodes F(F/LL) (LF/F) FF- FFF-F, a chimera of the LHR and FSHR.
  • pMB139 was made in a two step PCR reaction.
  • Step Ia employed primers 5'- CTAGCCTAGAAGCTCTGACTGTCTGTCCTTGAGATATCTAAAATGAC-3' and 5'-AAGAATGGGATTGAAGAAATACACAACCATGCATTCAATGGGACGC-3' and pMB111 as template and step lb employed primers 5'-GTGTATTTCTTCAATCCCATTCTTACTCAGCCATAAAATGACAGACTCGTTATTCATCCCTT
  • Step II employed primers 5'-AGCTGTCCTGGAGCTAGGAATCT-3' and 5'-CTAGCCTAGAAGCTCTGACTGTC-3' and the products of steps Ia and lb as template.
  • the resulting product was digested with XbaI and Apal and ligated to the large fragment resulting from XbaI and Apal digestio of pMB59.
  • F(F/LL) (LF/F)FF-FFF-F had low ability to bind 125I-hCG and 125I-hFSH (Table 1).
  • pMB133 a vector which encodes FL(L/F) FF- FFF-F, a chimera of the LHR and FSHR.
  • pMB111 was digested with BglII and the large fragment was ligated with the small fragment which resulted from BglII digestion of pMB88. The insert with the correct orientation was confirmed by a dideoxysequencing procedure (18, 19).
  • FL(L/F) FF-FFF-F was found to have high ability to bind 125 I-hCG and 125 I-hFSH ( Figure 11, Table 1).
  • Binding of 125 I-hCG, 125 I-eLH, and 125 I-hFSH to COS-7 cells transfected with LHR/FSHR chimeras was measured in duplicate or triplicate. Values shown are pooled from several experiments and are the average mean counts per minute over the non-specific blank, the standard error of the mean, and the number of independent experiments used to calculate the mean. When only one experiment was performed, the error values indicate the standard error of the replicates within the experiment.
  • Non-specific binding was determined as: 1) the CPM bound in the presence of lug unlabeled ligand, 2) the CPM bound to extracts of cells that had not been transfected, and/or 3) the CPM bound to extracts of cells that had been transfected with an analog that had been prematurely truncated at residue 145.
  • Binding was carried out of 125 I-hCG to Triton X-100 solubilized COS-7 cells which had been transfected with various receptor constructs.
  • the bound and free 125 I-hCG were separated by PEG precipitation or PEI filtration as described (21).
  • IgGsorb obtained from The Enzyme Center, Maiden, MA
  • IgGsorb obtained from The Enzyme Center, Maiden, MA
  • 2 mis of 0.9% NaCl solution containing 1 mg/ml bovine serum albumin sedimented the IgGsorb by centrifugation (2000 ⁇ g - 15 min), aspirated the supernate, and analyzed the bound 125 I-hCG in a gamma-counter.
  • 125 I-hCG was added to the column and allowed to bind to the receptors for 2 hours at room temperature. Bound and free 125 I-hCG were separated by washing the column with the detergent extract. Specifically bound 125 I-hCG was eluted from the column using pH 5.0 buffer. These data show that the presence of the His residues on the receptor do not interfere with its ability to bind 125 I- hCG and enable the 125 I-hCG to become specifically bound to the receptor. In addition they demonstrate that the unoccupied receptor can adsorb to the resin and capture 1 25 I-hCG.

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Abstract

Dans un mode de réalisation, on a prévu une protéine présentant une affinité de liaison à la gonadotrophine chorionique humaine (hCG), à l'hormone lutéinisante (LH) et à l'hormone folliculostimulante (FSH), et comportant une séquence de 341 acides aminés. La protéine est une chimère possédant une séquence d'acides aminés homologue à celle des restes 1 à 92 de FSHR, une séquence d'acides aminés homologue à celle des restes 93 à 170 de LHR, ainsi qu'une séquence d'acides aminés homologue à celle des restes 171 à 341 de FSHR, la protéine étant une chaîne d'acides aminés ne se trouvant pas dans la nature sous forme de protéine liante. Dans un autre mode de réalisation, on a prévu un récepteur d'hormone glycoprotéique possédant une affinité de liaison à la gonadotrophine chorionique humaine (hCG), à l'hormone lutéinisante (LH) et à l'hormone folliculostimulante (FSH). Le récepteur comporte une région transmembranaire et une région N-terminale extracellulaire, cette dernière étant une chimère contenant une séquence de 341 acides aminés homologue à celle des restes 1 à 92 de FSHR, une séquence d'acides aminés homologue à celle des restes 93 à 170 de LHR, ainsi qu'une séquence d'acides aminés homologue à celle des restes 171 à 341 de FSHR, le récepteur étant une chaîne d'acides aminés ne se trouvant pas dans la nature sous forme de récepteur (système de numérotation LH).
PCT/US1992/004987 1991-06-14 1992-06-12 Analogues de recepteurs d'hormone glycoproteique liant les gonadotrophines chorioniques, les leutrophines et les follitrophines, et leurs procedes de preparation et d'utilisation Ceased WO1992022667A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5925549A (en) * 1996-04-15 1999-07-20 The Board Of Trustees Of The Leland Stanford Junior University Soluble 7-transmembrane domain G-protein-coupled receptor compositions and methods
EP0950711A3 (fr) * 1998-02-06 2003-09-17 Akzo Nobel N.V. Récepteurs de Gonadotropine
CN104569441A (zh) * 2015-01-19 2015-04-29 成都大熊猫繁育研究基地 大熊猫尿液促黄体素的酶联检测方法及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4560649A (en) * 1981-10-15 1985-12-24 Cornell Research Foundation Assaying for hLH or hCG with immobilized hormone receptors
US4774180A (en) * 1986-02-26 1988-09-27 Massachusetts Institute Of Technology Construction and application of polyproteins
US4981784A (en) * 1987-12-02 1991-01-01 The Salk Institute For Biological Studies Retinoic acid receptor method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4560649A (en) * 1981-10-15 1985-12-24 Cornell Research Foundation Assaying for hLH or hCG with immobilized hormone receptors
US4774180A (en) * 1986-02-26 1988-09-27 Massachusetts Institute Of Technology Construction and application of polyproteins
US4981784A (en) * 1987-12-02 1991-01-01 The Salk Institute For Biological Studies Retinoic acid receptor method

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MOLECULAR ENDOCRINOLOGY, Volume 4(4), issued 1990, R. SPRENGEL et al., "The Testicular Receptor for Follicle Stimulating Hormone: Structure and Functional Expression of Cloned cDNA", pages 525-530. *
THE EMBO JOURNAL, Volume 10(7), issued 1991, T. BRAUN et al., "Amino-Terminal Leucine-Rich Repeats in Gonadotropin Receptors Determine Hormone Selectivity", pages 1885-1890. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5925549A (en) * 1996-04-15 1999-07-20 The Board Of Trustees Of The Leland Stanford Junior University Soluble 7-transmembrane domain G-protein-coupled receptor compositions and methods
EP0950711A3 (fr) * 1998-02-06 2003-09-17 Akzo Nobel N.V. Récepteurs de Gonadotropine
CN104569441A (zh) * 2015-01-19 2015-04-29 成都大熊猫繁育研究基地 大熊猫尿液促黄体素的酶联检测方法及其应用
CN104569441B (zh) * 2015-01-19 2016-02-03 成都大熊猫繁育研究基地 大熊猫尿液促黄体素的酶联检测方法及其应用

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