Mediation of Cyclic AMP Signaling by the First Intracellular Loop of the Gonadotropin-releasing Hormone Receptor

Abstract

The gonadotropin-releasing hormone (GnRH) receptor, which is a unique G protein-coupled receptor without a C-terminal cytoplasmic domain, activates both inositol phosphate (InsP) and cAMP signaling responses. The function of the highly basic first intracellular (1i) loop of the GnRH receptor in signal transduction was evaluated by mutating selected residues located in its N and C termini. Replacements of Leu58, Lys59, Gln61, and Lys62 at the N terminus, and Leu73, Ser74, and Leu80 at the C terminus, caused no change in binding affinity. The agonist-induced InsP and cAMP responses of the Q61E and K59Q,K62Q receptors were also unaffected, but the L58A receptor showed a normal InsP response and an 80% decrease in cAMP production. At the C terminus, the InsP response of the L73R receptor was normal, but cAMP production was reduced by 80%. The EC50 for GnRH-induced InsP responses of the S74E and L80A receptors was increased by about one order of magnitude, and the cAMP responses were essentially abolished. These findings indicate that cAMP signaling from the GnRH receptor is dependent on specific residues in the 1i loop that are not essential for activation of the phosphoinositide signaling pathway. The gonadotropin-releasing hormone (GnRH) receptor, which is a unique G protein-coupled receptor without a C-terminal cytoplasmic domain, activates both inositol phosphate (InsP) and cAMP signaling responses. The function of the highly basic first intracellular (1i) loop of the GnRH receptor in signal transduction was evaluated by mutating selected residues located in its N and C termini. Replacements of Leu58, Lys59, Gln61, and Lys62 at the N terminus, and Leu73, Ser74, and Leu80 at the C terminus, caused no change in binding affinity. The agonist-induced InsP and cAMP responses of the Q61E and K59Q,K62Q receptors were also unaffected, but the L58A receptor showed a normal InsP response and an 80% decrease in cAMP production. At the C terminus, the InsP response of the L73R receptor was normal, but cAMP production was reduced by 80%. The EC50 for GnRH-induced InsP responses of the S74E and L80A receptors was increased by about one order of magnitude, and the cAMP responses were essentially abolished. These findings indicate that cAMP signaling from the GnRH receptor is dependent on specific residues in the 1i loop that are not essential for activation of the phosphoinositide signaling pathway. G protein-coupled receptor gonadotropin-releasing hormone cyclic AMP 4,5)P3, inositol 1,4,5-trisphosphate inositol bisphosphate thyroid stimulating hormone transmembrane domain wild-type cholecystokinin. A wide variety of neurotransmitters, peptide and protein hormones, chemokines, growth factors, and other ligands elicit specific cellular responses by binding to plasma membrane receptors that are coupled to one or more heterotrimeric guanine nucleotide binding/regulatory proteins (G proteins). The primary signaling pathways for many of these receptors have been elucidated (1Strader C.D. Fong T.M. Tota M.R. Underwood D. Dixon R.A.F. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (996) Google Scholar, 2Gudermann T. Schoneberg T. Schultz G. Annu. Rev. Neurosci. 1997; 20: 399-427Crossref PubMed Scopus (252) Google Scholar). Agonist binding to a specific receptor on the cell surface causes a conformational change in the receptor that allows it to interact with its cognate G protein, stimulating guanine nucleotide exchange on the α subunit of the G protein. The release of the α subunit-GTP and βγ subunits from the receptor-G protein complex, and the activation of effector systems including phospholipase C, adenylyl cyclase, and ion channels, regulate the intracellular levels of inositol phosphate, calcium, cyclic AMP, and other second messengers. Although most G protein-coupled receptors (GPCRs)1 share a common structure, based on seven membrane-spanning domains, relatively little is known about the functional significance of this arrangement. It probably provides structural and functional integrity to the receptor, and the presence of several conserved amino acid residues in the transmembrane regions and cytoplasmic loops may reflect their role in agonist-induced G protein coupling and signal generation. Mutagenesis and chimeric studies with several receptor-G protein pairs, including the β-adrenergic-Gs, muscarinic acetylcholine-Gi, angiotensin II-Gq, and rhodopsin-Gt, have implicated the N- and C-terminal portions of the third intracellular (3i) loop of the receptors in G protein activation and signal transduction (3Kobilka B.K. Kobilka T.S. Daniel K. Regan J.W. Caron M.G. Lefkowitz R.J. Science. 1988; 240: 1310-1316Crossref PubMed Scopus (609) Google Scholar, 4O'Dowd B.F. Hnatowich M. Regan J.W. Leader W.M. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1988; 263: 15985-15992Abstract Full Text PDF PubMed Google Scholar, 5Weiss E.R. Kelleher D.J. Johnson G.L. J. Biol. Chem. 1988; 263: 6150-6154Abstract Full Text PDF PubMed Google Scholar, 6Franke R.R. Konig B. Sakmar T.P. Khorana H.G. Hofmann K.P. Science. 1990; 250: 123-125Crossref PubMed Scopus (306) Google Scholar, 7Kubo T. Bujo H. Akiba I. Nakai J. Mishina M. Numa S. FEBS Lett. 1988; 241: 119-125Crossref PubMed Scopus (126) Google Scholar, 8Luttrell L.M. Ostrowski J. Cotecchia S. Kendall H. Lefkowitz R.J. Science. 1993; 259: 1453-1457Crossref PubMed Scopus (89) Google Scholar, 9Ohyama K. Yamano Y. Chaki S. Kondo T. Inagami T. Biochem. Biophys. Res. Commun. 1992; 189: 677-683Crossref PubMed Scopus (128) Google Scholar, 10Hunyady L. Baukal A.J. Balla T. Catt K.J. J. Biol. Chem. 1994; 269: 24798-24804Abstract Full Text PDF PubMed Google Scholar, 11Hedin K.E. Duerson K. Clapham D.E. Cell. Signalling. 1993; 5: 505-518Crossref PubMed Scopus (96) Google Scholar). In the case of the thyroid stimulating hormone (TSH) receptor-Gs, dopamine (D1) receptor-Gs, and rhodopsin-Gt, regions within the cytoplasmic tail have also been found to interact with G proteins (12Konig B. Gratzel M. Biochim. Biophys. Acta. 1994; 1223: 261-266Crossref PubMed Scopus (30) Google Scholar, 13Kosugi S. Mori T. Biochem. Biophys. Res. Commun. 1994; 200: 401-407Crossref PubMed Scopus (10) Google Scholar, 14Phillips W.J. Cerione R.A. Biochem. J. 1994; 299: 351-357Crossref PubMed Scopus (41) Google Scholar). An analysis of the structural determinants in the IGF-II receptor, and GPCRs that are associated with Gi and Gsactivation (15Okamoto T. Nishimoto I. J. Biol. Chem. 1992; 267: 8342-8346Abstract Full Text PDF PubMed Google Scholar) revealed the following criteria: (a) at least two basic residues at the N terminus; (b) either a BBXXB or BBXB motif (where B is a basic amino acid and X is any amino acid) at the C terminus; and (c) a sequence length of 10–26 amino acids. The structural motifs satisfying G protein activation requirements for several GPCRs were conserved in the 2i and 3i loops, and in the C-terminal tail (15Okamoto T. Nishimoto I. J. Biol. Chem. 1992; 267: 8342-8346Abstract Full Text PDF PubMed Google Scholar). Using synthetic peptides with these structural characteristics, multiple Gi-activating regions were identified in M4 muscarinic cholinergic and α2-adrenergic receptors (15Okamoto T. Nishimoto I. J. Biol. Chem. 1992; 267: 8342-8346Abstract Full Text PDF PubMed Google Scholar). Recently, a synthetic peptide corresponding to the cytoplasmic tail of the rat testicular follicle-stimulating hormone receptor that satisfied these criteria was shown to modulate G protein signaling (16Grasso P. Leng N. Reichert Jr., L.E. Mol. Cell. Endocrinol. 1995; 108: 43-50Crossref PubMed Scopus (22) Google Scholar). However, these studies did not identify functionally important specific residues within the structural motifs. The gonadotropin-releasing hormone (GnRH) receptor is a member of the GPCR superfamily and has several unique structural features. These include (a) the absence of a cytoplasmic C-terminal tail; (b) the replacement of Tyr by Ser in the conserved GPCR “signature” motif DRY located at the junction of transmembrane domain (TMD) III and the 2i loop; (c) the interchange of conserved Asp and Asn residues in TMDs II and VII; and (d) the presence of a relatively long and highly basic 1i loop. These characteristics are conserved in the GnRH receptors of all mammalian species sequenced to date (17Stojilkovic S.S. Reinhart J. Catt K.J. Endocr. Rev. 1994; 15: 462-499Crossref PubMed Scopus (429) Google Scholar, 18Sealfon S.C. Weinstein H. Millar R.P. Endocr. Rev. 1997; 18: 180-205Crossref PubMed Scopus (388) Google Scholar). The primary structure of the 1i loop of the GnRH receptor, which is coupled to both phosphoinositide hydrolysis and cAMP generation, meets the criteria for G protein activation by other receptors that are coupled to Gs and Gi (15Okamoto T. Nishimoto I. J. Biol. Chem. 1992; 267: 8342-8346Abstract Full Text PDF PubMed Google Scholar, 16Grasso P. Leng N. Reichert Jr., L.E. Mol. Cell. Endocrinol. 1995; 108: 43-50Crossref PubMed Scopus (22) Google Scholar). The 18 amino acid 1i loop of the GnRH receptor contains two basic amino acid residues at the N terminus (Lys59 and Lys62) and a BBXXB motif at the C terminus (Lys71-Lys-Ser-Glu-Lys75) (see Fig. 1). The present study was performed to determine whether these regions of the 1i loop are involved in GnRH-induced signal transduction and receptor internalization. The roles of the N and C termini of the 1i loop of the mouse GnRH receptor in signal transduction mediated by Gq and Gs proteins were evaluated by introducing positive or negative charges into these sequences, or eliminating positive charges. Specifically, residues Lys59, Gln61, and Lys62 at the N terminus of the loop, and Leu73 and Ser74 at the C terminus, were mutated. In addition, the role of two leucine residues located at the junction of the 1i loop and TMDs I and II (Leu58 and Leu80) was assessed. The mutant receptors expressed in COS-7 cells were analyzed for ligand binding, GnRH-induced inositol phosphate and cAMP production, and agonist-induced internalization. The results of this study have demonstrated that specific residues in the 1i loop are required for agonist-induced cAMP responses but are not essential for activation of the phosphoinositide/calcium signaling pathway. GnRH and its agonist (des-Gly10-[d-Ala6]GnRHN-ethylamide, GnRH-Ag) and antagonist ([d-pGlu1,d-Phe2,d-Trp3,6]GnRH) analogs were obtained from Peninsula Laboratories, Inc. (Belmont, CA). LipofectAMINE and Opti-MEM media were purchased from Life Technologies, Inc., cell culture related products were from Biofluids (Rockville, MD), restriction and DNA-modifying enzymes were from New England BioLabs (Beverly, MA), and Sequenase II from United States Biochemical Corp. Oligonucleotide primers for site-directed mutagenesis were synthesized in a Beckman Oligo 1000 DNA synthesizer. The Muta-Gene phagemid in vitro mutagenesis kit (Version 2) was obtained from Bio-Rad and was used according to the instructions of the manufacturer. AG-1-X8 resin (100–200 mesh formate form) and Poly-Prep chromatography columns for anion exchange chromatography were obtained from Bio-Rad. All other reagents were of high performance liquid chromatography or analytical grade quality.myo-[3H]Inositol (80–100 Ci/mmol) was from Amersham Pharmacia Biotech.125I-des-Gly10-[d-Ala6]GnRHN-ethylamide (125I-GnRH-Ag) and125I-S-cAMP-TME were prepared by Covance Laboratories Inc. (Vienna, VA). The 1220-base pair GnRH receptor cDNA subcloned into pcDNAI/Amp at the XbaI site (19Arora K.K. Sakai A. Catt K.J. J. Biol. Chem. 1995; 270: 22820-22826Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) was used as a template for creating site-directed mutations according to the method of Kunkel et al. (20Kunkel T.A. Roberts J.D. Zakour R.A. Meth. Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4560) Google Scholar) using a Muta-Gene phagemid in vitromutagenesis kit. Mutations were identified by the dideoxy sequencing method of Sanger et al. (21Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52758) Google Scholar) using Sequenase II. Wild-type and mutant GnRH receptors were transiently expressed in COS-7 cells. To measure inositol phosphate and cAMP responses, internalization kinetics, or125I-GnRH-Ag binding to intact cells, cultures were seeded in 24-well plates (Costar Corp.) at a density of 4 × 104 cells/well. The plated cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum containing 100 units/ml penicillin and 100 μg/ml streptomycin (Pen-Strep) at 37 °C in an atmosphere consisting of 5% CO2, 95% humidified air. At 70–80% confluence, the cells were transfected in 0.5 ml of serum-free Opti-MEM I medium with 1 μg of wild-type or mutant plasmid DNA and 6–8 μg of LipofectAMINE/well. Six h later, the medium was replaced with fresh medium and cultures were maintained for 48 h before use in ligand binding and functional assays. The binding affinity and abundance of the mutant receptors were determined in transfected COS-7 cells incubated with 2 nm125I-GnRH-Ag in binding medium (M199 containing 25 mm HEPES and 0.1% bovine serum albumin) in the absence or presence of increasing concentrations of unlabeled peptide for 4 h at 4 °C. The cells were then rapidly washed twice with ice-cold phosphate-buffered saline (pH 7.4) and solubilized in 0.2 mNaOH, 1% SDS solution. The cell-associated radioactivity was measured by γ-spectrometry. All time studies were performed in duplicate on at least three occasions, and displacement curves were analyzed for binding affinity and capacity by the LIGAND program using a one-site model (22Munson P.J. Rodbard D. Anal. Biochem. 1980; 107: 220-239Crossref PubMed Scopus (7772) Google Scholar). Surface expression of most of the mutant receptors ranged from 50 to 82% of that of the wild-type (WT) receptor, except that L73R receptors were better expressed (150% of WT) and S74E receptors were poorly expressed (30% of WT). The expression levels for the L58A, Q61E, K59Q,K62Q, and L80A receptors were 82, 51, 44, and 71%, respectively, of the wild-type. The 100% level of wild-type receptor expression corresponded to 710 fmol/mg protein. Irrespective of differences in plasma membrane binding sites, the mutant receptors had similar binding affinities with apparent K in the of to for the of the wild-type receptor was 0.2 The binding for determined in the presence of unlabeled agonist for wild-type or mutant receptors was 5% of the internalization transfected COS-7 cells were washed with binding medium before the of 2 GnRH binding was determined in the presence of a of the unlabeled GnRH at 37 °C for the the cells were washed twice with ice-cold phosphate-buffered saline (pH 7.4) and incubated with 1 ml of 50 mm mm (pH for to The radioactivity was to determine the and the radioactivity was the cells in solution. were measured by and the at time was expressed as a of the COS-7 cells were h by in Dulbecco's modified Eagle's medium containing as (19Arora K.K. Sakai A. Catt K.J. J. Biol. Chem. 1995; 270: 22820-22826Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). h of cells were washed with medium and in the medium containing mm for at 37 °C and then with increasing of GnRH to for were by the of ice-cold acid The inositol were and by anion exchange chromatography as K.K. Catt K.J. Mol. Endocrinol. 1997; PubMed Scopus Google and their were measured by liquid 48 h of cells or mutant receptors were incubated in the presence of increasing concentrations of GnRH to in serum-free medium Dulbecco's modified Eagle's containing 0.1% and 1 mm for at 37 °C. were by the medium and the cells in 0.5 ml of The were for and intracellular cAMP was measured by N. Catt K.J. PubMed Scopus Google Scholar) using a specific cAMP at a of showed no with or The were analyzed by the program using analysis and the Six mutations were in the 1i loop of the mouse GnRH receptor, as shown in Fig. at the N terminus of the loop of residues to and and Lys62 to At the C terminus, residues Leu73 and Ser74 in the BBXXB motif were to and In addition, leucine residues (Leu58 and Leu80) located at the portions of the N and C termini of the 1i loop, respectively, were replaced with of binding in intact COS-7 cells transfected with or mutant GnRH to determine the expression and the functional integrity of the expressed that the and modified receptors the with high affinity. analysis of the binding a of with similar (see The of the mutant receptors to to phospholipase C proteins was determined by the inositol phosphate (InsP) responses of transfected COS-7 cells with GnRH in the presence of mm these the products of phosphoinositide hydrolysis in GnRH cells are and (19Arora K.K. Sakai A. Catt K.J. J. Biol. Chem. 1995; 270: 22820-22826Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The GnRH-induced InsP responses mediated by receptors with mutations at the N terminus Q61E, and were similar to of the and their were the EC50 for the receptor was 0.2 2 the C-terminal mutant receptors the InsP response of the L73R receptor was normal, with EC50 of the S74E and L80A agonist was as by of EC50 to and 2 The of the InsP responses by a of GnRH for the mutant receptors were of that for the wild-type the plasma membrane binding of cells the mutant GnRH receptors showed differences (see for in expression levels of the mutant receptors were by their signaling responses to the of binding measured in the have shown a the measured receptor and the InsP responses to GnRH in cells K.K. Catt K.J. Mol. Endocrinol. 1997; PubMed Scopus Google Scholar). of the based on receptor showed that the InsP responses mediated by the L73R and L80A receptors were similar to that of the The InsP responses of the L58A, Q61E, K59Q,K62Q, and S74E were to the receptor, that the of these receptors more to Gq the wild-type These results that at the N and C termini of the first loop of the GnRH receptor not the of the receptors to phospholipase The of the wild-type GnRH receptor to adenylyl is shown in Fig. with increasing concentrations of GnRH a in cAMP with a The response was at least with an EC50 of transfected with showed no in GnRH-induced cAMP production The receptor of the cAMP response was demonstrated by the of a GnRH antagonist to with to cAMP production the antagonist no cAMP These results that the GnRH receptors expressed in COS-7 cells are functionally coupled not to the C but also activation of the adenylyl signaling pathway. The of the 1i loop mutant receptors to GnRH-induced activation of adenylyl cyclase, as by cAMP production, was also in transfected COS-7 cells. The responses are shown in A and and the based on receptor are shown in B and B. the the cAMP response of cells the L58A receptor was with of 80% from that of the wild-type receptors in response to a of GnRH In the cAMP response for the Q61E receptor did not from that of the receptor, and K59Q,K62Q receptors were about the The of the C-terminal on the cAMP response were more as shown in Fig. cAMP production for the L73R and S74E receptors was reduced by of the and the L80A receptors to adenylyl in cells These results that of the first loop, at its C terminus, the of the GnRH receptors to adenylyl of agonist-induced cAMP response by a GnRH to with cells were with a GnRH antagonist The shown are of three similar of site-directed mutations in the N terminus of the 1i loop of the GnRH receptor on GnRH-induced cAMP responses. COS-7 cells and mutant GnRH receptors were with GnRH cAMP curves of and mutant These are of three similar performed in the cAMP responses to the of binding These were the levels in cells from the response obtained with 1 GnRH and then to the of expressed receptor are expressed as of the wild-type the were 10% of the of site-directed mutations in the C-terminal of 1i loop of GnRH receptor on GnRH-induced cAMP responses. the to Fig. The of mutations on internalization were evaluated by the a of at 37 °C in cells wild-type or mutant A the wild-type and mutant receptors was by the of that was with increasing time of A and of the mutant receptors at the N terminus, and L73R at the C terminus, showed internalization similar to that of the wild-type. However, internalization of the S74E and L80A was reduced by about 1 h To into the of the GnRH receptor, a of GnRH receptors with mutations in the first intracellular loop and their binding characteristics and second signaling with of wild-type receptors transiently expressed in COS-7 cells. These revealed that mutations of residues Leu58, Lys59, Gln61, and Lys62 at the N terminus, and Leu73, Ser74, and Leu80 at the C terminus, of the 1i loop had no on the inositol phosphate production. However, adenylyl cAMP production was by at the of the loop. These findings that residues in the 1i loop are required for coupling to adenylyl but are not essential for coupling to the phospholipase C pathway. The similar binding of the mutant receptors to of the wild-type receptor indicate that the did not the structural integrity of the receptor and that of the cAMP response was not of of binding affinity. and the capacity of the mutant receptors to normal GnRH-induced InsP responses, that the structure of the receptors was A of the primary sequence of the GnRH receptor for G based on conserved sequence motifs with G protein coupling Gs and within the of other that the 1i loop may as a receptor-G protein activation of the structural criteria used to identify G protein in a of coupled receptors (15Okamoto T. Nishimoto I. J. Biol. Chem. 1992; 267: 8342-8346Abstract Full Text PDF PubMed Google Scholar) to the GnRH receptor revealed that the 1i loop has at least two basic residues at the N terminus and a BBXXB sequence motif at the C In other this motif is present in the 2i or 3i loop, or in the C-terminal cytoplasmic tail (15Okamoto T. Nishimoto I. J. Biol. Chem. 1992; 267: 8342-8346Abstract Full Text PDF PubMed Google Scholar, 16Grasso P. Leng N. Reichert Jr., L.E. Mol. Cell. Endocrinol. 1995; 108: 43-50Crossref PubMed Scopus (22) Google Scholar). However, in the GnRH receptor, it is located in the 1i loop, which is and in basic residues most other receptor loops in this The present studies on the GnRH receptor the first analysis of this structural motif identified in other GPCRs by the synthetic peptide (15Okamoto T. Nishimoto I. J. Biol. Chem. 1992; 267: 8342-8346Abstract Full Text PDF PubMed Google Scholar, 16Grasso P. Leng N. Reichert Jr., L.E. Mol. Cell. Endocrinol. 1995; 108: 43-50Crossref PubMed Scopus (22) Google Scholar). Although it is that most of the of GnRH are mediated by to of and intracellular (17Stojilkovic S.S. Reinhart J. Catt K.J. Endocr. Rev. 1994; 15: 462-499Crossref PubMed Scopus (429) Google increased cAMP signaling may also responses in have a role of cAMP as a of GnRH in the J. 1987; Google Scholar). in cells, the of intracellular cAMP levels by or of was found to GnRH receptor activation by stimulating N. Catt K.J. PubMed Scopus Google Scholar). have shown that GnRH the adenylyl pathway. cAMP production in cells the rat GnRH receptor was found to release a G protein D. 1994; PubMed Scopus Google Scholar). In addition, activation of GnRH receptors in cells not phospholipase C but also increased cAMP production in a that the GnRH receptor interact with both adenylyl and G N. K. K. and K. J. The present results that the structural determinants that are associated with activation are not essential for inositol phosphate but are important for cAMP production. In this results are in with on and receptors by synthetic peptide (15Okamoto T. Nishimoto I. J. Biol. Chem. 1992; 267: 8342-8346Abstract Full Text PDF PubMed Google Scholar, 16Grasso P. Leng N. Reichert Jr., L.E. Mol. Cell. Endocrinol. 1995; 108: 43-50Crossref PubMed Scopus (22) Google Scholar). In addition, the present study that these structural criteria are not involved in the activation of phosphoinositide/calcium signaling and specific amino acid residues involved in Gs positive charges are important for the protein the is by an or basic at the C terminus of the 1i loop, the adenylyl cAMP response is it is that these structural to the receptor-G protein activation agonist binding, GnRH receptor activation causes a in protein C and of intracellular It is that the activation of adenylyl in GnRH COS-7 cells and is caused by Gq increased protein C or levels Endocrinol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the of InsP signaling for of the the of these coupling to multiple G coupling or has been in several receptors for A and to both phospholipase C and adenylyl pathways A. B. A. Schultz G. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (126) Google Scholar, A. S. K. J. Schultz G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, M. J. N. M. M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In coupling has been to differences in their intracellular loops, or to the presence of specific regions in the a receptor containing amino acid residues from the 1i loop of the receptor to was to cAMP that the 1i loop of receptor is essential for G protein coupling and effector activation M. J. N. M. M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In mutations in the 2i loop of the rat hormone receptor were found to in of agonist-induced phospholipase C the capacity of these mutant receptors to adenylyl was A. J. M. Jr., A. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In residues in both the 2i and 3i loops have been implicated in coupling to Gs and Gq A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the receptor, a amino acid in the 3i loop to or InsP but did not the activation of adenylyl S. F. T. A. A. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). It is that of in the receptor at the as in the with any other amino acid caused activation of the receptor Cotecchia S. Ostrowski J. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). all three intracellular loops, in 2i and as as the of the tail (3Kobilka B.K. Kobilka T.S. Daniel K. Regan J.W. Caron M.G. Lefkowitz R.J. Science. 1988; 240: 1310-1316Crossref PubMed Scopus (609) Google Scholar, 4O'Dowd B.F. Hnatowich M. Regan J.W. Leader W.M. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1988; 263: 15985-15992Abstract Full Text PDF PubMed Google Scholar, 5Weiss E.R. Kelleher D.J. Johnson G.L. J. Biol. Chem. 1988; 263: 6150-6154Abstract Full Text PDF PubMed Google Scholar, 6Franke R.R. Konig B. Sakmar T.P. Khorana H.G. Hofmann K.P. Science. 1990; 250: 123-125Crossref PubMed Scopus (306) Google Scholar, 7Kubo T. Bujo H. Akiba I. Nakai J. Mishina M. Numa S. FEBS Lett. 1988; 241: 119-125Crossref PubMed Scopus (126) Google Scholar, 8Luttrell L.M. Ostrowski J. Cotecchia S. Kendall H. Lefkowitz R.J. Science. 1993; 259: 1453-1457Crossref PubMed Scopus (89) Google Scholar, 9Ohyama K. Yamano Y. Chaki S. Kondo T. Inagami T. Biochem. Biophys. Res. Commun. 1992; 189: 677-683Crossref PubMed Scopus (128) Google Scholar, 10Hunyady L. Baukal A.J. Balla T. Catt K.J. J. Biol. Chem. 1994; 269: 24798-24804Abstract Full Text PDF PubMed Google Scholar, 11Hedin K.E. Duerson K. Clapham D.E. Cell. Signalling. 1993; 5: 505-518Crossref PubMed Scopus (96) Google Scholar, B. Gratzel M. Biochim. Biophys. Acta. 1994; 1223: 261-266Crossref PubMed Scopus (30) Google Scholar, 13Kosugi S. Mori T. Biochem. Biophys. Res. Commun. 1994; 200: 401-407Crossref PubMed Scopus (10) Google Scholar, 14Phillips W.J. Cerione R.A. Biochem. J. 1994; 299: 351-357Crossref PubMed Scopus (41) Google have been implicated in receptor activation by However, the the Recently, the receptor was found to to both Gs and and protein of the was shown to as a to regulate the G protein coupling of the receptor Y. L.M. Lefkowitz R.J. 1997; PubMed Scopus Google Scholar). It is that a similar found to in other In results that activation of cAMP signaling by the GnRH receptor is dependent on specific residues in the 1i loop that are not required for the of inositol phosphate production. In addition, the presence of basic residues in the BBXXB motif at the C terminus of the 1i loop, or a of positive is important for coupling the GnRH receptor to Gs and adenylyl However, these residues not to required for internalization of the