FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1981984074> ?p ?o ?g. }

• W1981984074 endingPage "30271" @default.
• W1981984074 startingPage "30264" @default.
• W1981984074 abstract "Glycoprotein hormone receptors are G protein-coupled receptors with ligand-binding ectodomains consisting of leucine-rich repeats. The ectodomain is connected by a conserved cysteine-rich hinge region to the seven transmembrane (TM) region. Gain-of-function mutants of luteinizing hormone (LH) and thyroid-stimulating hormone receptors found in patients allowed identification of residues important for receptor activation. Based on constitutively active mutations at Ser-281 in the hinge region of the thyroid-stimulating hormone receptor, we mutated the conserved serine in the LH (S277I) and follicle-stimulating hormone receptors (S273I) and observed increased basal cAMP production and ligand affinity by mutant receptors. For the LH receptor, conversion of Ser-277 to all natural amino acids led to varying degrees of receptor activation. Hydropathy index analysis indicated that substitution of neutral serine with selective nonpolar hydrophobic residues (Leu>Val>Met>Ile) confers constitutive receptor activation whereas serine deletion or substitution with charged Arg, Lys, or Asp led to defective receptor expression. Furthermore, mutation of the angular proline near Ser-273 to flexible Gly also led to receptor activation. The findings suggest the ectodomain of glycoprotein hormone receptors constrain the TM region. Point mutations in the hinge region of these proteins, or ligand binding to these receptors, could cause conformational changes in the TM region that result in Gsactivation. Glycoprotein hormone receptors are G protein-coupled receptors with ligand-binding ectodomains consisting of leucine-rich repeats. The ectodomain is connected by a conserved cysteine-rich hinge region to the seven transmembrane (TM) region. Gain-of-function mutants of luteinizing hormone (LH) and thyroid-stimulating hormone receptors found in patients allowed identification of residues important for receptor activation. Based on constitutively active mutations at Ser-281 in the hinge region of the thyroid-stimulating hormone receptor, we mutated the conserved serine in the LH (S277I) and follicle-stimulating hormone receptors (S273I) and observed increased basal cAMP production and ligand affinity by mutant receptors. For the LH receptor, conversion of Ser-277 to all natural amino acids led to varying degrees of receptor activation. Hydropathy index analysis indicated that substitution of neutral serine with selective nonpolar hydrophobic residues (Leu>Val>Met>Ile) confers constitutive receptor activation whereas serine deletion or substitution with charged Arg, Lys, or Asp led to defective receptor expression. Furthermore, mutation of the angular proline near Ser-273 to flexible Gly also led to receptor activation. The findings suggest the ectodomain of glycoprotein hormone receptors constrain the TM region. Point mutations in the hinge region of these proteins, or ligand binding to these receptors, could cause conformational changes in the TM region that result in Gsactivation. G protein-coupled receptor transmembrane luteinizing hormone follicle stimulating hormone thyroid stimulating hormone human chorionic gonadotropin Dulbecco's modified Eagle's medium leucine-rich repeat-containing G protein-coupled receptor inositol phosphate Dulbecco's phosphate-buffered saline quantitative structure-activity relationship The large G protein-coupled receptor (GPCR)1 superfamily of proteins plays an important role in virtually every physiological process and represents the largest group of genes in nature, accounting for 1–2% of the entire human genome (1Fraser C.M. Lee N.H. Pellegrino S.M. Kerlavage A.R. Prog. Nucleic Acid Res. Mol. Biol. 1994; 49: 113-156Crossref PubMed Scopus (26) Google Scholar). These seven transmembrane (TM) proteins are capable of transducing messages as different as photons, organic odorants, nucleotides, nucleosides, peptides, lipids, and proteins. After ligand binding, GPCRs interact with a specific subset of heterotrimeric G-proteins that can in the activated forms inhibit or activate various effector enzymes and/or ion channels (2Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1233) Google Scholar). One subfamily of GPCRs has a large amino-terminal ectodomain containing leucine-rich repeats connected to the seven TM region. The leucine-rich repeat-containing G protein-coupled receptors (LGRs) consist of the classic gonadotropin and thyrotropin (TSH) receptors (3Ji T.H. Grossmann M. Ji I. J. Biol. Chem. 1998; 273: 17299-17302Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar) together with the recently identified mammalian orphan receptors, LGR4 and LGR5/Fex/HG38 (4Hsu S.Y. Liang S.G. Hsueh A.J. Mol. Endocrinol. 1998; 12: 1830-1845Crossref PubMed Google Scholar, 5Hermey G. Methner A. Schaller H.C. Hermans-Borgmeyer I. Biochem. Biophys. Res. Commun. 1999; 254: 273-279Crossref PubMed Scopus (28) Google Scholar, 6McDonald T. Wang R. Bailey W. Xie G. Chen F. Caskey C.T. Liu Q. Biochem. Biophys. Res. Commun. 1998; 247: 266-270Crossref PubMed Scopus (71) Google Scholar). Constitutively activated TSH and luteinizing hormone (LH) receptors have been identified in patients with nonimmune hyperthyroidism (7Vassart G. Horm. Res. (Basel). 1997; 48: 47-50Crossref PubMed Scopus (21) Google Scholar) and male-limited precocious puberty (8Shenker A. Laue L. Kosugi S. Merendino Jr., J.J. Minegishi T. Cutler Jr., G.B. Nature. 1993; 365: 652-654Crossref PubMed Scopus (661) Google Scholar, 9Laue L. Chan W.Y. Hsueh A.J. Kudo M. Hsu S.Y. Wu S.M. Blomberg L. Cutler Jr., G.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1906-1910Crossref PubMed Scopus (223) Google Scholar, 10Kudo M. Osuga Y. Kobilka B.K. Hsueh A.J.W. J. Biol. Chem. 1996; 271: 22470-22478Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), respectively. These gain-of-function receptor mutants not only allow the understanding of the etiology of different pathological states but also provide the unique opportunity to investigate the molecular mechanisms by which GPCRs are activated. The allosteric ternary complex model proposes the isomerization of GPCRs from an inactive to an active state capable of coupling to the G proteins (11Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (757) Google Scholar). This isomerization involves conformational changes that may occur spontaneously or be induced by agonists or appropriate mutations, which abrogate the normal constraining function of the receptor, allowing it to ‘relax’ into the active conformation. Although the majority of gain-of-function mutations in LH and TSH receptors, like most other GPCRs, are present in the TM region (9Laue L. Chan W.Y. Hsueh A.J. Kudo M. Hsu S.Y. Wu S.M. Blomberg L. Cutler Jr., G.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1906-1910Crossref PubMed Scopus (223) Google Scholar, 12Shenker A. Baillieres Clin. Endocrinol. Metab. 1995; 9: 427-451Abstract Full Text PDF PubMed Scopus (77) Google Scholar,13Parma J. Duprez L. Van Sande J. Hermans J. Rocmans P. Van Vliet G. Costagliola S. Rodien P. Dumont J.E. Vassart G. J. Clin. Endocrinol. Metab. 1997; 82: 2695-2701Crossref PubMed Scopus (251) Google Scholar), recent studies in patients with toxic thyroid nodules and congenital hyperthyroidism indicate that point mutations of a serine residue in the ectodomain of the TSH receptor lead to constitutive receptor activation (14Kopp P. Muirhead S. Jourdain N. Gu W.X. Jameson J.L. Rodd C. J. Clin. Invest. 1997; 100: 1634-1639Crossref PubMed Scopus (122) Google Scholar, 15Duprez L. Parma J. Costagliola S. Hermans J. Van Sande J. Dumont J.E. Vassart G. FEBS Lett. 1997; 409: 469-474Crossref PubMed Scopus (131) Google Scholar, 16Gruters A. Schoneberg T. Biebermann H. Krude H. Krohn H.P. Dralle H. Gudermann T. J. Clin. Endocrinol. Metab. 1998; 83: 1431-1436Crossref PubMed Scopus (117) Google Scholar). These findings are consistent with the hypothesis that the ectodomain of the glycoprotein hormone receptors could constrain the TM region. Because the key serine residue in the hinge region between leucine-rich repeats and the TM region of the TSH receptor is conserved in the homologous LH and follicle stimulating hormone (FSH) receptors, we performed mutagenesis analysis to test the constitutive activation of LH and FSH receptors following serine substitutions. Taking advantage of the observation that the human LH receptor is more constrained than the TSH receptor, we further substituted Ser-277 of this receptor with all natural amino acids and demonstrated the importance of a nonpolar hydrophobic residue in this position for receptor activation. In addition, substitution of an angular proline immediately adjacent to Ser-277 with a flexible glycine but not a more rigid alanine also led to receptor activation. Purified human CG (hCG, CR-129) was supplied by the National Hormone and Pituitary Program (NIDDK, National Institutes of Health, Bethesda, MD) and human recombinant FSH (Org32489) was from Organon (Oss, The Netherlands). Anti-FLAG M1 monoclonal antibody and FLAG peptide were purchased from Sigma.125I-sodium iodine and myo-[3H]inositol were purchased from Amersham Pharmacia Biotech. Polymerase chain reaction-based mutagenesis was performed to generate mutant LH and FSH receptor cDNAs as described previously (10Kudo M. Osuga Y. Kobilka B.K. Hsueh A.J.W. J. Biol. Chem. 1996; 271: 22470-22478Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) using cDNA encoding human LH receptor (17Jia X.C. Oikawa M. Bo M. Tanaka T. Ny T. Boime I. Hsueh A.J. Mol. Endocrinol. 1991; 5: 759-768Crossref PubMed Scopus (139) Google Scholar) or human FSH receptor (10Kudo M. Osuga Y. Kobilka B.K. Hsueh A.J.W. J. Biol. Chem. 1996; 271: 22470-22478Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Polymerase chain reaction was performed with Vent DNA polymerase (New England BioLabs, Inc., Beverly, MA) in accordance with manufacturer's instructions. All cDNAs were subcloned into the expression vector pcDNA3 (Invitrogen Corp., Carlsbad, CA) and the plasmids were purified using the Maxi plasmid preparation kit (Qiagen, Inc., Valencia, CA). Fidelity of the polymerase chain reaction products was confirmed by sequencing on both strands of the final constructs before use in expression studies. Human 293T cells derived from embryonic kidney fibroblast were maintained in Dulbecco's modified Eagle's medium/Ham's F-12 (DMEM/F12) supplemented with 10% fetal bovine serum, 100 μg/ml penicillin, 100 μg/ml streptomycin, and 2 mml-glutamine. Before transfection, cells (2 × 106/culture) were seeded in 10-cm dishes (Nalge, Nunc International, Naperville, IL). When cells were 70–80% confluent, transient transfection was performed with 10 μg of plasmid using the calcium phosphate precipitation method (18Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4824) Google Scholar) following replacement of culture medium. After 18–24 h of incubation with the calcium phosphate-DNA precipitates, medium was replaced with DMEM/F12 containing 10% fetal bovine serum. Forty-eight hours after transfection, cells were washed twice with Dulbecco's phosphate-buffered saline (D-PBS), harvested from culture dishes, and centrifuged at 400 × g for 5 min. Cell pellets were then resuspended in DMEM/F12 supplemented with 1 mg/ml bovine serum albumin. Cells (2 × 105/ml) were placed on 24-well tissue culture plates (Corning, Inc., Corning, NY) and preincubated at 37 °C for 30 min in the presence of 0.25 mm3-isobutyl-1-methylxanthine (IBMX, Sigma) before treatment with or without hormones for 16 h. For studies using increasing amounts of plasmids, transfection was also performed in 12-well culture plates using calcium phosphate precipitation. Each well was transfected separately with different amounts of plasmids. Forty-eight hours after transfection, each well was washed once with D-PBS, replaced with DMEM/F12 supplemented with 1 mg/ml bovine serum albumin and 0.25 mm 3-isobutyl-1-methylxanthine, and incubated for 16 h. Total cAMP in each well was measured in triplicate by a specific radioimmunoassay as described previously (19Davoren J.B. Hsueh A.J. Biol. Reprod. 1985; 33: 37-52Crossref PubMed Scopus (123) Google Scholar). For inositol phosphate (IP) measurement, transfected cells were labeled for 24 h with myo-[3H]inositol (4 μCi/ml) in inositol-free DMEM supplemented with 5% fetal bovine serum in a 10-cm dish. After washing cells three times with D-PBS, 2 × 105 cells were preincubated for 30 min in D-PBS containing 20 mmLiCl, and treated with or without hormones at 37 °C for 45 min. Total IPs were extracted and separated as described previously (20Hirsch B. Kudo M. Naro F. Conti M. Hsueh A.J. Mol. Endocrinol. 1996; 10: 1127-1137PubMed Google Scholar). All experiments were repeated at least three times using cells from independent transfections. To monitor transfection efficiency, 0.5 μg of the Rous sarcoma virus-β-galactosidase (RSV·β-gal) plasmid (21Hall C.V. Jacob P.E. Ringold G.M. Lee F. J. Mol. Appl. Genet. 1983; 2: 101-109PubMed Google Scholar) was routinely included in the transfection mixture, and β-galactosidase activity in cell lysate was measured as described previously (22Su J.G. Hsueh A.J. Biochem. Biophys. Res. Commun. 1992; 186: 293-300Crossref PubMed Scopus (48) Google Scholar). Statistical analysis was performed using Student's t test. Human CG (CR-129) and recombinant human FSH were iodinated by the lactoperoxidase method (23Miyachi Y. Vaitukaitis J.L. Nieschlag E. Lipsett M.B. J. Clin. Endocrinol. Metab. 1972; 34: 23-28Crossref PubMed Scopus (401) Google Scholar) and characterized by radioligand receptor assay using recombinant human LH or FSH receptors expressed in 293T cells. Specific activity and maximal binding of the labeled hCG was 150,000 cpm/ng and 30%, respectively. These values for labeled FSH were 220,000 cpm/ng and 15%, respectively. To estimate ligand binding to the cell surface, transfected cells were washed two times with D-PBS and collected in D-PBS before centrifugation at 400 × g for 5 min. Pellets were resuspended in D-PBS containing 1 mg/ml bovine serum albumin (binding assay buffer). Resuspended cells (2 × 105/tube) were incubated with increasing doses or a saturating dose of 125I-hCG or 125I-FSH at room temperature for 22 h in the presence or absence of unlabeled hCG (Pregnyl, Organon, 100 IU/tube) or FSH (Pergonal, Organon, 50 IU/tube), respectively. At the end of the incubation, cells were centrifuged and washed twice with the binding assay buffer. Radioactivity in the pellets was determined in a gamma spectrometer. Data from saturation binding studies were used to derive equilibrium dissociation constant (Kd) values based on Scatchard plot analysis. In addition to multiple gain-of-function mutations found in the TM regions of LH or TSH receptors, recent studies further indicated that replacement of Ser-281 in the human TSH receptor with isoleucine, threonine, or asparagine is associated with constitutive activation of the receptor, thereby leading to nonimmune hyperthyroidism (14Kopp P. Muirhead S. Jourdain N. Gu W.X. Jameson J.L. Rodd C. J. Clin. Invest. 1997; 100: 1634-1639Crossref PubMed Scopus (122) Google Scholar, 15Duprez L. Parma J. Costagliola S. Hermans J. Van Sande J. Dumont J.E. Vassart G. FEBS Lett. 1997; 409: 469-474Crossref PubMed Scopus (131) Google Scholar, 16Gruters A. Schoneberg T. Biebermann H. Krude H. Krohn H.P. Dralle H. Gudermann T. J. Clin. Endocrinol. Metab. 1998; 83: 1431-1436Crossref PubMed Scopus (117) Google Scholar). As shown in Fig.1 A, this serine is situated in a stretch of residues (YPSHCCAF XN) conserved in all three glycoprotein hormone receptors. This conserved region is present in the hinge between the nine leucine-rich repeats in the ectodomain and the seven TM bundles (Fig. 1 B); the corresponding positions for Ser-281 in the TSH receptor are Ser-277 for the LH receptor and Ser-273 for the FSH receptor, respectively. Based on observed structural conservation, we mutated Ser-277 and Ser-273 in the LH and FSH receptors, respectively, to test for possible constitutive activation of these homologous receptors. As shown in Fig.2 A, transfection of 293T cells with increasing amounts of plasmid encoding the S277I LH receptor mutant but not the wild-type receptor led to dose-dependent increases in basal cAMP production. As expected, transfection with plasmids encoding another mutant LH receptor (D564G) known to have constitutive activity (10Kudo M. Osuga Y. Kobilka B.K. Hsueh A.J.W. J. Biol. Chem. 1996; 271: 22470-22478Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) also led to increases in basal cAMP production. Furthermore, treatment with increasing doses of hCG further increased cAMP production by both the wild-type LH receptor and the D564G mutant but was minimally effective on the highly activated S277I mutant (Fig. 2 B). As shown in Fig. 2 C and Table I, Scatchard plot analysis further demonstrated that the S277I LH receptor, like the D564G mutant, showed increased ligand-binding affinity.Table IEquilibrium dissociation constants (K d), maximal binding, and basal and maximal cAMP production for wild-type and mutant LH receptorsKdMaximal bindingcAMP productionBasalMaximalpmng/2 × 10 5 cellpmol/ng hCG bindingLHR wild type164.1 ± 15.02.13 ± 0.562.6 ± 0.2208.2 ± 2.1LHR S277A122.9 ± 12.61.48 ± 0.404.1 ± 0.3389.9 ± 8.7LHR S277H118.9 ± 17.81.55 ± 0.388.6 ± 0.3268.0 ± 9.3LHR S277Y118.9 ± 3.91.51 ± 0.3612.6 ± 0.2438.7 ± 70.8LHR S277F34.1 ± 6.91-aSignificantly different from the wild-type receptor in Kd values (p < 0.05).0.65 ± 0.1526.9 ± 1.4463.0 ± 22.2LHR S277C35.9 ± 8.71-aSignificantly different from the wild-type receptor in Kd values (p < 0.05).0.46 ± 0.1149.5 ± 1.1608.3 ± 28.8LHR S277T53.3 ± 7.41-aSignificantly different from the wild-type receptor in Kd values (p < 0.05).0.88 ± 0.2051.7 ± 1.5460.7 ± 66.8LHR S277W37.0 ± 4.21-aSignificantly different from the wild-type receptor in Kd values (p < 0.05).0.56 ± 0.1274.2 ± 3.0436.2 ± 24.9LHR S277G41.1 ± 9.91-aSignificantly different from the wild-type receptor in Kd values (p < 0.05).0.49 ± 0.1279.4 ± 2.3721.5 ± 63.0LHR S277E27.4 ± 6.51-aSignificantly different from the wild-type receptor in Kd values (p < 0.05).0.76 ± 0.19108.2 ± 4.6349.3 ± 14.5LHR S277Q38.2 ± 5.71-aSignificantly different from the wild-type receptor in Kd values (p < 0.05).1.11 ± 0.29138.8 ± 0.8825.0 ± 75.3LHR S277N41.2 ± 10.51-aSignificantly different from the wild-type receptor in Kd values (p < 0.05).0.66 ± 0.16182.0 ± 10.6897.3 ± 39.0LHR S277P45.5 ± 2.81-aSignificantly different from the wild-type receptor in Kd values (p < 0.05).0.44 ± 0.11182.4 ± 13.5869.8 ± 56.0LHR S277I41.8 ± 1.71-aSignificantly different from the wild-type receptor in Kd values (p < 0.05).0.66 ± 0.16513.4 ± 33.4455.4 ± 31.6LHR S277M46.9 ± 4.01-aSignificantly different from the wild-type receptor in Kd values (p < 0.05).0.28 ± 0.07712.2 ± 41.91166.8 ± 51.3LHR S277V19.0 ± 5.01-aSignificantly different from the wild-type receptor in Kd values (p < 0.05).0.75 ± 0.18846.1 ± 75.4629.0 ± 40.7LHR S277L55.5 ± 26.00.08 ± 0.021755.1 ± 125.12513.8 ± 126.9LHR P276G53.7 ± 6.91-aSignificantly different from the wild-type receptor in Kd values (p < 0.05).0.41 ± 0.10457.8 ± 30.41409.3 ± 48.1LHR P276A56.9 ± 12.81-aSignificantly different from the wild-type receptor in Kd values (p < 0.05).0.59 ± 0.1416.3 ± 0.7956.7 ± 60.5LHR D564G45.4 ± 3.81-aSignificantly different from the wild-type receptor in Kd values (p < 0.05).0.57 ± 0.13225.9 ± 8.2471.6 ± 11.7LHR D578Y93.0 ± 4.51-aSignificantly different from the wild-type receptor in Kd values (p < 0.05).1.21 ± 0.29196.9 ± 11.2236.2 ± 17.3Data are shown as mean ± S.D. (n = 3).1-a Significantly different from the wild-type receptor in Kd values (p < 0.05). Open table in a new tab Data are shown as mean ± S.D. (n = 3). We further monitored cAMP production in cells transfected with plasmids encoding the wild-type FSH receptor or the FSH receptor hinge mutant. As shown in Fig. 2 D, basal cAMP production was increased in cells that expressed increasing amounts of the S273I FSH receptor mutant but not the wild-type receptor. Also, treatment with increasing doses of FSH further stimulated cAMP mediated by either the wild-type or mutant FSH receptor (Fig. 2 E). Similar to the LH receptor mutants, Scatchard plot analysis indicated that the mutant FSH receptor exhibited increased affinity to its ligand as reflected by a decrease in the Kd value (Fig. 2 F,Kd values: wild-type receptor, 666 pm; S273I mutant, 71 pm). To elucidate the molecular basis of the constitutive activation of the LH receptor at residue 277, random mutagenesis was performed to change Ser-277 to each of all natural amino acids or to delete Ser-277. The deletion of Ser-277 or its substitution into charged Arg, Lys, or Asp resulted in defective expression of these mutants as reflected by negligible125I-hCG binding and the lack of cAMP production in response to hCG by transfected cells. However, the remaining 16 mutants showed varying degrees of constitutive activation based on basal cAMP production by cells transfected with expression plasmids encoding each mutant construct (Fig. 3 A). To correct for varying levels of receptor expression, the cAMP results were normalized based on cell surface 125I-hCG binding. When challenged with a saturating dose (100 ng/ml) of hCG, all but two highly constitutively activated mutant receptors (S277V and S277I) showed further increases in cAMP production (Fig. 3 B). With the exception of the four most active mutants (S227L, S277V, S277M, and S277I), these mutant receptors showed maximal cAMP production within 2- to 3-fold of the levels found for the wild-type LH receptor. As shown in Table I, most mutant receptors also showed increased binding affinity to the hCG ligand as reflected by decreases in their Kd values. We plotted the degree of constitutive activation of different LH receptor mutants in relation to the hydropathy index (24Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17296) Google Scholar) of the individual amino acid residue that was replaced. As shown in Fig.3 C, four mutants with a substitution of Ser-277 by residues with the highest hydropathy values (Leu, Val, Met, Ile) also showed the highest constitutive activity. We further subgrouped the receptor mutants into six categories based on a quantitative structure-activity relationship (QSAR) analysis (25Hellberg S. Sjostrom M. Skagerberg B. Wold S. J. Med. Chem. 1987; 30: 1126-1135Crossref PubMed Scopus (462) Google Scholar) of the substituted residue. The variation in individual amino acid residue is described by three principal properties, z1, z2, and z3, derived from a principal component analysis of a matrix of 29 physicochemical variables for all amino acids. Again, the four mutants with highest basal cAMP production belonged to the same category. Secondary structure analysis (26Garnier J. Biochimie (Paris). 1990; 72: 513-524Crossref PubMed Scopus (39) Google Scholar) of the hinge region of the LH receptor indicated that the conserved YPSHCCAF region is situated in a structure bend contributed by proline and is preceded by an α-helix and followed by a β-sheet structure. We, therefore, tested whether replacement of the more fixed Pro-276 by a flexible glycine could lead to constitutive activation of the LH receptor. As shown in Fig.4 A, the P276G mutant showed increases in basal cAMP production comparable with that of the S277I mutant. In addition, the P276G mutant showed a higher affinity to hCG (Fig. 4 B and Table I). We further tested whether replacement of Pro-276 with alanine to allow the continuation of the α-helical structure could alter receptor function. As shown in Fig.4 A, P276A substitution only led to minimal increases in basal cAMP production. However, the agonist affinity for this mutant was increased as compared with the wild-type LH receptor (Fig.4 B and Table I). Earlier studies have indicated that treatment of wild-type LH receptors with LH/hCG led to increases in PI turnover, whereas some mutant LH receptors are also associated with increases in basal PI turnover (20Hirsch B. Kudo M. Naro F. Conti M. Hsueh A.J. Mol. Endocrinol. 1996; 10: 1127-1137PubMed Google Scholar, 27Kosugi S. Mori T. Shenker A. J. Biol. Chem. 1996; 271: 31813-31817Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 28Gilchrist R.L. Ryu K.S. Ji I. Ji T.H. J. Biol. Chem. 1996; 271: 19283-19287Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). We further tested whether point mutations in the hinge region of the LH receptor are associated with increases in basal PI turnover in transfected cells. As shown in Fig. 5, treatment with hCG led to time-dependent increases in PI turnover in cells expressing wild-type LH receptors or three different mutants (S277I, P276G, or D578Y). In contrast, basal levels of PI turnover were not different between wild-type and mutant receptors, suggesting a preferential activation of the protein kinase A pathway by these mutants. Likewise, basal IP turnover was found to be comparable between the wild-type FSH receptor and the S273I mutant with -fold increases (0 versus 45 min of incubation) of 1.84 ± 0.10 and 1.68 ± 0.09, respectively. Pathological studies in patients provided unique opportunities to investigate the molecular mechanisms of glycoprotein hormone receptor activation. Based on findings of gain-of-function mutants found in the hinge region of TSH receptors in hyperthyroid patients, we have established the importance of this region in the maintenance of an inactive conformation in all three glycoprotein hormone receptors for Gs coupling but not for IP turnover. For the LH receptor, substitution of Ser-277 with a subgroup of hydrophobic residues confers a constitutively active phenotype together with higher agonist-binding affinity. In addition, replacement of the neighboring Pro-276 to a flexible glycine also led to receptor activation. These findings highlight the importance of the hinge region in the maintenance of an inactive protein conformation in this subgroup of leucine-rich repeat-containing G protein-coupled receptors. It is likely that agonist binding, like hinge region mutations, could lead to changes in receptor conformation important for Gs activation. The observed constitutive activity of various gain-of-function mutants of the LH receptor is closely related to their increased binding affinity to the labeled hCG ligand. Increased agonist-binding affinity is a common observation for constitutively activated GPCRs (29Ren Q. Kurose H. Lefkowitz R.J. Cotecchia S. J. Biol. Chem. 1993; 268: 16483-16487Abstract Full Text PDF PubMed Google Scholar, 30Perez D.M. Hwa J. Gaivin R. Mathur M. Brown F. Graham R.M. Mol. Pharmacol. 1996; 49: 112-122PubMed Google Scholar, 31Dufau M.L. Annu. Rev. Physiol. 1998; 60: 461-496Crossref PubMed Scopus (308) Google Scholar). Because Ser-277 is not situated within the leucine-rich repeats of the LH receptor that are postulated to be important for ligand binding, the observed enhancement in agonist binding likely reflects a conformational shift of the mutant receptor toward a more active state. Furthermore, substitution of an angular proline residue adjacent to Ser-277 of the LH receptor to a flexible glycine also led to constitutive receptor activation and higher agonist affinity, further confirming the importance of the conformation of the hinge region in the activation of the glycoprotein hormone receptors. It is also interesting to note that observed increases in basal cAMP production mediated by different LH and FSH receptor mutants were not accompanied by increases in PI turnover, consistent with an earlier report demonstrating that the activation of adenylate cyclase and phospholipase C by these receptors is mediated through different receptor conformational states (28Gilchrist R.L. Ryu K.S. Ji I. Ji T.H. J. Biol. Chem. 1996; 271: 19283-19287Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Mutagenesis of Ser-277 to all possible natural amino acids further indicated that the most active gain-of-function mutants belong to the same subgroup of amino acids with similar properties based on QSAR analysis (25Hellberg S. Sjostrom M. Skagerberg B. Wold S. J. Med. Chem. 1987; 30: 1126-1135Crossref PubMed Scopus (462) Google Scholar). In this analysis, the variation in individual amino acid residue was described by three principal properties, z1, z2, and z3, derived from a principal components analysis of a matrix of 29 physicochemical variables for all amino acids. z1 is mainly related to hydrophilicity, z2 is additionally influenced by size and some hydrophobicity/hydrophilicity scales, and z3 contains information from the analysis of p Ka , pI, and 1H NMR values. A similar approach has been used to analyze the role of an aspartate residue in the conserved (E/D)RY sequence for the activation of the α1B-adrenergic receptor (32Scheer A. Fanelli F. Costa T. De Benedetti P.G. Cotecchia S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 808-813Crossref PubMed Scopus (200) Google Scholar). Among the three glycoprotein hormone receptors, we selected the LH receptor for extensive mutagenesis analysis based on its unique activation property. As reported earlier (33Cetani F. Tonacchera M. Vassart G. FEBS Lett. 1996; 378: 27-31Crossref PubMed Scopus (77) Google Scholar), the unliganded TSH receptor is less constrained than its homologs and more susceptible to activation by a wide spectrum of mutations. Following overexpression in transfected mammalian cells, the wild-type TSH (but not LH) receptor showed constitutive activity, thus rendering it difficult to analyze minimally active TSH receptor mutants. In contrast, the FSH receptor appears to be highly constrained. Although gain-of-function mutations have been found in patients for both TSH and LH receptors, few cases of constitutively activated mutations were identified for the FSH receptor. Indeed, studies using chimeric LH/FSH receptors have indicated that interactions between TM V and VI of the FSH receptor maintain this receptor in a more constrained state (10Kudo M. Osuga Y. Kobilka B.K. Hsueh A.J.W. J. Biol. Chem. 1996; 271: 22470-22478Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). As shown in Fig. 2, the hinge region mutation (S273I) in the FSH receptor does lead to constitutive activation; however, the basal cAMP production of the FSH receptor mutant is lower than that of the comparable mutation in the homologous LH receptor. Although LH receptor mutants with Ser-277 substituted by Leu, Val, Met, or Ile residues showed the highest constitutive activity, it is interesting to note that only the Ser to Ile mutation of the TSH receptor was found in patients with toxic thyroid nodules. This is probably because of the fact that only one nucleotide alteration is involved for the Ser to Ile codon switch whereas substitution to the remaining three residues involves two nucleotide changes. Based on studies on LH receptor mutants, it is possible that additional gain-of-function mutations with substitution of these hydrophobic residues could be identified in the future in hyperthyroid patients. Although LH receptor mutations found in patients with male-limited precocious puberty have only been localized to the TM region, it is also of interest to screen for gain-of-function mutations in the hinge region of the LH receptor gene. The GPCRs usually exist in an inactive state in the absence of ligands whereas ligand binding converts the receptor into an active conformation capable of coupling to the G protein. In the “allosteric ternary complex model,” this isomerization of receptor proteins involves conformational changes that may occur spontaneously or be induced by agonists or appropriate mutations that abrogate the normal constraining function of the receptor, allowing it to relax into the active conformation (34Lefkowitz R.J. Nature. 1993; 365: 603-604Crossref PubMed Scopus (87) Google Scholar). It appears that at least three regions are important to maintain the LH receptor in an inactive state. Earlier studies analyzed two prominent LH receptor point mutations in greater detail. The Asp-578 side chain in the TM VI serves as a properly positioned hydrogen bond acceptor that is important for stabilizing the inactive state of the LH receptor. A bulky aromatic side chain at this position rather than the negative charge destabilizes the inactive receptor conformation (27Kosugi S. Mori T. Shenker A. J. Biol. Chem. 1996; 271: 31813-31817Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In addition, studies on the point mutant in Asp-546 of intracellular loop 3 indicated that a negative charge at this position might interact with an unidentified cationic residue to maintain receptor constraint (35Kosugi S. Mori T. Shenker A. Mol. Pharmacol. 1998; 53: 894-901PubMed Google Scholar, 36Schulz A. Schoneberg T. Paschke R. Schultz G. Gudermann T. Mol. Endocrinol. 1999; 13: 181-190Crossref PubMed Scopus (66) Google Scholar). The present study documents the amino-terminal hinge region as a third region important in maintaining all three glycoprotein hormone receptors in an inactive conformation. Most constitutively active mutants for GPCRs have been found in the TM domains or connecting loop sequences. To our knowledge, gain-of-function mutations in the amino-terminal sequences have only been observed in glycoprotein hormone receptors and calcium-sensing receptors (37Mancilla E.E. De Luca F. Baron J. Mol. Gen. Metab. 1998; 64: 198-204Crossref PubMed Scopus (23) Google Scholar). Unlike the majority of GPCRs, the mammalian LGR subfamily of proteins, including glycoprotein hormone receptors and the orphan LGR4 and LGR5, all have large ectodomain-containing leucine-rich repeats presumably important for ligand binding. Of interest, a conserved hinge region between the leucine-rich repeats and the TM domain is also found in the only LGR identified in the completely sequenced genome of Caenorhabditis elegans. Although the nematode LGR showed constitutive activity, its TM region is mainly responsible for its activation (38Kudo M. Chen T. Nakabayashi K. Hsu S.Y. Hsueh A.J. Mol. Endocrinol. 2000; 14: 272-284Crossref PubMed Scopus (57) Google Scholar). The conserved serine found in the hinge region of mammalian glycoprotein hormone receptors (YPSHCCAF) is substituted by a histidine in the worm receptor; however, similar substitution in the LH receptor did not lead to increases in basal cAMP production (Fig. 3 A). It is interesting to note that the hinge region of two other known LGRs from lower species (fly LGR, HSFHCCAF; sea anemone LGR, NGFLCCEF) (39Hauser F. Nothacker H.P. Grimmelikhuijzen C.J. J. Biol. Chem. 1997; 272: 1002-1010Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 40Nothacker H.P. Grimmelikhuijzen C.J. Biochem. Biophys. Res. Commun. 1993; 197: 1062-1069Crossref PubMed Scopus (74) Google Scholar), and the two mammalian orphan LGRs (LGR4, YAYQCCAF; LGR5, YAYQCCAF) (4Hsu S.Y. Liang S.G. Hsueh A.J. Mol. Endocrinol. 1998; 12: 1830-1845Crossref PubMed Google Scholar) are not conserved despite the preservation of two downstream cysteine residues. Future studies are needed to investigate the importance of the hinge region for the activation of these LGRs. At least three different but nonexclusive models could explain the activation of glycoprotein hormone receptors. First, based on studies on hinge mutants of gonadotropin receptors (present study) and the TSH receptor (15Duprez L. Parma J. Costagliola S. Hermans J. Van Sande J. Dumont J.E. Vassart G. FEBS Lett. 1997; 409: 469-474Crossref PubMed Scopus (131) Google Scholar), one can propose that the large ectodomain of these proteins directly constrains the TM region, whereas ligand binding or hinge mutations relax the receptor (Fig.6, R to R*). An earlier study proposed that the ectodomain of the LH receptor makes several contacts with the TM domain (41Moyle W.R. Campbell R.K. Rao S.N. Ayad N.G. Bernard M.P. Han Y. Wang Y. J. Biol. Chem. 1995; 270: 20020-20031Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Ligand binding to the curved portion of the U- or J-shaped ectodomain of the receptor leads to steric influences on the distances between the “arms” of the ectodomain of the receptor. Although the exact contact sites between the ectodomain and the TM region are still unclear, key residues in exoloops 2 and 3 have been shown to be involved in the modulation of ligand binding by the ectodomain of the LH and FSH receptor, respectively (42Ryu K. Lee H. Kim S. Beauchamp J. Tung C.S. Isaacs N.W. Ji I. Ji T.H. J. Biol. Chem. 1998; 273: 6285-6291Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 43Ryu K. Gilchrist R.L. Tung C.S. Ji I. Ji T.H. J. Biol. Chem. 1998; 273: 28953-28958Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). It is likely that ligand binding to the leucine-rich repeats in the ectodomain leads to conformational alterations in the hinge region, followed by the relaxation of the TM bundles leading to Gs activation. Second, in the “two-step activation” model (44Jiang X. Dreano M. Buckler D.R. Cheng S. Ythier A. Wu H. Hendrickson W.A. el Tayar N. Structure. 1995; 3: 1341-1353Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), agonist binding to the ectodomain brings the hormone close to the TM region. After binding mediated by the hormone-specific β-subunit, the common α-subunit directly interacts with the TM domain (45Braun T. Schofield P.R. Sprengel R. EMBO J. 1991; 10: 1885-1890Crossref PubMed Scopus (314) Google Scholar, 46Kajava A.V. Vassart G. Wodak S.J. Structure. 1995; 3: 867-877Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 47Nagayama Y. Rapoport B. Mol. Endocrinol. 1992; 6: 145-156Crossref PubMed Scopus (151) Google Scholar), leading to signal transduction (Fig. 6, AR*). This model is supported by the findings that a mutant LH receptor containing mainly the TM region can mediate hCG stimulation of signal transduction at extremely high ligand concentrations (48Ji I.H. Ji T.H. J. Biol. Chem. 1991; 266: 13076-13079Abstract Full Text PDF PubMed Google Scholar, 49Ji I. Ji T.H. Endocrinology. 1991; 128: 2648-2650Crossref PubMed Scopus (120) Google Scholar). Modifications at key residues of the α-subunit of hCG (50Ji I. Zeng H. Ji T.H. J. Biol. Chem. 1993; 268: 22971-22974Abstract Full Text PDF PubMed Google Scholar, 51Ji I. Ji T.H. J. Biol. Chem. 1993; 268: 20851-20854Abstract Full Text PDF PubMed Google Scholar) and the first extracellular loop of the LH receptor also suggest direct counterionic interactions between the α-subunit of hCG and the receptor. Although this model does not take into account the hinge region in receptor activation, hinge mutations could independently cause conformational changes in the receptor resembling those induced by the α-subunit following its interaction with the TM bundles. For several LH hinge mutants with the highest constitutive activity (Fig. 3), hCG treatment did not lead to further increases in cAMP production. In addition, basal cAMP production produced by these mutants is higher than the maximal cAMP production following ligand stimulation of the wild-type LH receptor. For the more constrained FSH receptor, point mutation in the hinge region is less effective in inducing constitutive activation, thus allowing further increases in cAMP production induced by FSH. Thus, ligand interaction with the TM region could be important in eliciting and maintaining full signal transduction by the FSH receptor. Finally, several studies have suggested the importance of receptor dimerization or oligomerization in the signal transduction of different GPCRs (52Overton M.C. Blumer K.J. Curr. Biol. 2000; 10: 341-344Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar), including the LH receptor (53Dufau M.L. Minegishi T. Buczko E.S. Delgado C.J. Zhang R. J. Steroid Biochem. 1989; 33: 715-720Crossref PubMed Scopus (10) Google Scholar, 54Grewal N. Nagpal S. Chavali G.B. Majumdar S.S. Pal R. Salunke D.M. Biophys. J. 1997; 73: 1190-1197Abstract Full Text PDF PubMed Scopus (10) Google Scholar, 55Podesta E.J. Solano A.R. Attar R. Sanchez M.L. Molina y Vedia L. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3986-3990Crossref PubMed Scopus (44) Google Scholar). Ligand binding could confer a conformation that allows the receptors to form dimers. For the LH receptor hinge mutants, one could speculate that substitution of the conserved serine by hydrophobic residues also facilitates receptor dimerization. Future studies on the hinge region of the glycoprotein hormone receptors could elucidate their ligand signaling mechanisms and allow the design of small molecular weight molecules capable of modifying the conformation of these constrained proteins. We thank Caren Spencer for editorial assistance. We also thank the National Pituitary and Hormone Distribution Program for the cAMP antiserum and hCG preparations." @default.
• W1981984074 created "2016-06-24" @default.
• W1981984074 creator A5006681163 @default.
• W1981984074 creator A5013252958 @default.
• W1981984074 creator A5061090554 @default.
• W1981984074 creator A5079682203 @default.
• W1981984074 date "2000-09-01" @default.
• W1981984074 modified "2023-10-18" @default.
• W1981984074 title "Activation of the Luteinizing Hormone Receptor Following Substitution of Ser-277 with Selective Hydrophobic Residues in the Ectodomain Hinge Region" @default.
• W1981984074 cites W1481709903 @default.
• W1981984074 cites W1514627997 @default.
• W1981984074 cites W1607832506 @default.
• W1981984074 cites W1900995848 @default.
• W1981984074 cites W1968331360 @default.
• W1981984074 cites W1970953993 @default.
• W1981984074 cites W1973197643 @default.
• W1981984074 cites W1975304761 @default.
• W1981984074 cites W1981236576 @default.
• W1981984074 cites W1983434985 @default.
• W1981984074 cites W1985937682 @default.
• W1981984074 cites W1998541756 @default.
• W1981984074 cites W2002221745 @default.
• W1981984074 cites W2005241408 @default.
• W1981984074 cites W2005417512 @default.
• W1981984074 cites W2006969256 @default.
• W1981984074 cites W2012054995 @default.
• W1981984074 cites W2017132134 @default.
• W1981984074 cites W2023306209 @default.
• W1981984074 cites W2024554932 @default.
• W1981984074 cites W2026005641 @default.
• W1981984074 cites W2029999222 @default.
• W1981984074 cites W2031604686 @default.
• W1981984074 cites W2033388874 @default.
• W1981984074 cites W2034105003 @default.
• W1981984074 cites W2047009435 @default.
• W1981984074 cites W2048120663 @default.
• W1981984074 cites W2050890804 @default.
• W1981984074 cites W2050922757 @default.
• W1981984074 cites W2062778364 @default.
• W1981984074 cites W2063547305 @default.
• W1981984074 cites W2064323950 @default.
• W1981984074 cites W2064552105 @default.
• W1981984074 cites W2071587854 @default.
• W1981984074 cites W2078190500 @default.
• W1981984074 cites W2086149638 @default.
• W1981984074 cites W2086319805 @default.
• W1981984074 cites W2088098601 @default.
• W1981984074 cites W2089578429 @default.
• W1981984074 cites W2091020913 @default.
• W1981984074 cites W2105605178 @default.
• W1981984074 cites W2109245267 @default.
• W1981984074 cites W2122158500 @default.
• W1981984074 cites W2134874967 @default.
• W1981984074 cites W2154590674 @default.
• W1981984074 cites W2157959080 @default.
• W1981984074 cites W3849076 @default.
• W1981984074 doi "https://doi.org/10.1074/jbc.m005568200" @default.
• W1981984074 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10889210" @default.
• W1981984074 hasPublicationYear "2000" @default.
• W1981984074 type Work @default.
• W1981984074 sameAs 1981984074 @default.
• W1981984074 citedByCount "101" @default.
• W1981984074 countsByYear W19819840742012 @default.
• W1981984074 countsByYear W19819840742013 @default.
• W1981984074 countsByYear W19819840742014 @default.
• W1981984074 countsByYear W19819840742015 @default.
• W1981984074 countsByYear W19819840742016 @default.
• W1981984074 countsByYear W19819840742018 @default.
• W1981984074 countsByYear W19819840742019 @default.
• W1981984074 countsByYear W19819840742021 @default.
• W1981984074 countsByYear W19819840742022 @default.
• W1981984074 countsByYear W19819840742023 @default.
• W1981984074 crossrefType "journal-article" @default.
• W1981984074 hasAuthorship W1981984074A5006681163 @default.
• W1981984074 hasAuthorship W1981984074A5013252958 @default.
• W1981984074 hasAuthorship W1981984074A5061090554 @default.
• W1981984074 hasAuthorship W1981984074A5079682203 @default.
• W1981984074 hasBestOaLocation W19819840741 @default.
• W1981984074 hasConcept C1009742 @default.
• W1981984074 hasConcept C121332964 @default.
• W1981984074 hasConcept C170493617 @default.
• W1981984074 hasConcept C185592680 @default.
• W1981984074 hasConcept C199360897 @default.
• W1981984074 hasConcept C2778220771 @default.
• W1981984074 hasConcept C2778575703 @default.
• W1981984074 hasConcept C41008148 @default.
• W1981984074 hasConcept C44000306 @default.
• W1981984074 hasConcept C55493867 @default.
• W1981984074 hasConcept C71240020 @default.
• W1981984074 hasConcept C71315377 @default.
• W1981984074 hasConcept C74650414 @default.
• W1981984074 hasConceptScore W1981984074C1009742 @default.
• W1981984074 hasConceptScore W1981984074C121332964 @default.
• W1981984074 hasConceptScore W1981984074C170493617 @default.
• W1981984074 hasConceptScore W1981984074C185592680 @default.
• W1981984074 hasConceptScore W1981984074C199360897 @default.
• W1981984074 hasConceptScore W1981984074C2778220771 @default.
• W1981984074 hasConceptScore W1981984074C2778575703 @default.

• next