The Agonist-binding Domain of the Calcium-sensing Receptor Is Located at the Amino-terminal Domain

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that displays 19–25% sequence identity to the γ-aminobutyric acid type B (GABAB) and metabotropic glutamate (mGlu) receptors. All three groups of receptors have a large amino-terminal domain (ATD), which for the mGlu receptors has been shown to bind the endogenous agonist. To investigate whether the agonist-binding domain of the CaR also is located in the ATD, we constructed a chimeric receptor named Ca/1a consisting of the ATD of CaR and the seven transmembrane region and C terminus of mGlu1a. The Ca/1a receptor stimulated inositol phosphate production when exposed to the cationic agonists Ca2+, Mg2+, and Ba2+ in transiently transfected tsA cells (a transformed HEK 293 cell line). The pharmacological profile of Ca/1a (EC50 values of 3.3, 2.6, and 3.9 mm for these cations, respectively) was very similar to that of the wild-type CaR (EC50 values of 3.2, 4.7, and 4.1 mm, respectively). For the mGlu1a receptor, it has been shown that Ser-165 and Thr-188, which are located in the ATD, are involved in the agonist binding. An alignment of CaR with the mGlu receptors showed that these two amino acid residues have been conserved in CaR as Ser-147 and Ser-170, respectively. Each of these residues was mutated to alanines and tested pharmacologically using the endogenous agonist Ca2+. CaR-S147A showed an impaired function as compared with wild-type CaR both with respect to potency of Ca2+(4-fold increase in EC50) and maximal response (79% of wild-type response). CaR-S170A showed no significant response to Ca2+ even at 50 mm concentration. In contrast, each of the two adjacent mutations, S169A and S171A, resulted in pharmacological profiles almost identical to that of the wild-type receptor. These data demonstrate that Ser-170 and to some extent Ser-147 are involved in the Ca2+ activation of the CaR, and taken together, our results reveal a close resemblance of the activation mechanism between the CaR and the mGlu receptors. The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that displays 19–25% sequence identity to the γ-aminobutyric acid type B (GABAB) and metabotropic glutamate (mGlu) receptors. All three groups of receptors have a large amino-terminal domain (ATD), which for the mGlu receptors has been shown to bind the endogenous agonist. To investigate whether the agonist-binding domain of the CaR also is located in the ATD, we constructed a chimeric receptor named Ca/1a consisting of the ATD of CaR and the seven transmembrane region and C terminus of mGlu1a. The Ca/1a receptor stimulated inositol phosphate production when exposed to the cationic agonists Ca2+, Mg2+, and Ba2+ in transiently transfected tsA cells (a transformed HEK 293 cell line). The pharmacological profile of Ca/1a (EC50 values of 3.3, 2.6, and 3.9 mm for these cations, respectively) was very similar to that of the wild-type CaR (EC50 values of 3.2, 4.7, and 4.1 mm, respectively). For the mGlu1a receptor, it has been shown that Ser-165 and Thr-188, which are located in the ATD, are involved in the agonist binding. An alignment of CaR with the mGlu receptors showed that these two amino acid residues have been conserved in CaR as Ser-147 and Ser-170, respectively. Each of these residues was mutated to alanines and tested pharmacologically using the endogenous agonist Ca2+. CaR-S147A showed an impaired function as compared with wild-type CaR both with respect to potency of Ca2+(4-fold increase in EC50) and maximal response (79% of wild-type response). CaR-S170A showed no significant response to Ca2+ even at 50 mm concentration. In contrast, each of the two adjacent mutations, S169A and S171A, resulted in pharmacological profiles almost identical to that of the wild-type receptor. These data demonstrate that Ser-170 and to some extent Ser-147 are involved in the Ca2+ activation of the CaR, and taken together, our results reveal a close resemblance of the activation mechanism between the CaR and the mGlu receptors. The cloning of the gene coding for the calcium-sensing receptor (CaR 1The abbreviations used are: CaR, calcium-sensing receptor; ATD, amino-terminal domain; DMEM, Dulbecco's modified Eagle's medium; GPCR, G-protein-coupled receptor; HBSS, Hanks' balanced saline solution; HEK, human embryonic kidney; IP, inositol phosphate; LEUBP, leucine-binding protein; LIVBP, Leu/Ile/Val-binding protein from Escherichia coli ; LIVPA, Leu/Ile/Val-binding protein from Pseudomonas aeruginosa ; mGlu, metabotropic glutamate; GABAB, γ-aminobutyric acid type B; PBP, periplasmic binding protein.1The abbreviations used are: CaR, calcium-sensing receptor; ATD, amino-terminal domain; DMEM, Dulbecco's modified Eagle's medium; GPCR, G-protein-coupled receptor; HBSS, Hanks' balanced saline solution; HEK, human embryonic kidney; IP, inositol phosphate; LEUBP, leucine-binding protein; LIVBP, Leu/Ile/Val-binding protein from Escherichia coli ; LIVPA, Leu/Ile/Val-binding protein from Pseudomonas aeruginosa ; mGlu, metabotropic glutamate; GABAB, γ-aminobutyric acid type B; PBP, periplasmic binding protein.(1Brown E.M. Gamba G. Riccardi D. Lombardi M. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2323) Google Scholar, 2Ruat M. Molliver M.E. Snowman A.M. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3161-3165Crossref PubMed Scopus (342) Google Scholar)) has revealed that it together with the γ-aminobutyric acid type B (GABAB) (3Kaupmann K. Huggel K. Heid J. Flor P.J. Bischoff S. Mickel S.J. McMaster G. Angst C. Bittiger H. Froestl W. Bettler B. Nature. 1997; 386: 239-246Crossref PubMed Scopus (870) Google Scholar, 4White J.H. Wise A. Main M.J. Green A. Fraser N.J. Disney G.H. Barnes A.A. Emson P. Foord S.M. Marshall F.H. Nature. 1998; 396: 679-682Crossref PubMed Scopus (992) Google Scholar, 5Jones K.A. Borowsky B. Tamm J.A. Craig D.A. Durkin M.M. Dai M. Yao W.J. Johnson M. Gunwaldsen C. Huang L.Y. Tang C. Shen Q. Salon J.A. Morse K. Laz T. Smith K.E. Nagarathnam D. Noble S.A. Branchek T.A. Gerald C. Nature. 1998; 396: 674-679Crossref PubMed Scopus (906) Google Scholar, 6Kaupmann K. Malitschek B. Schuler V. Heid J. Froestl W. Beck P. Mosbacher J. Bischoff S. Kulik A. Shigemoto R. Karschin A. Bettler B. Nature. 1998; 396: 683-687Crossref PubMed Scopus (998) Google Scholar) and metabotropic glutamate (mGlu) receptors (7Nakanishi S. Neuron. 1994; 13: 1031-1037Abstract Full Text PDF PubMed Scopus (641) Google Scholar, 8Conn P.J. Pin J.-P. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 205-237Crossref PubMed Scopus (2690) Google Scholar) form a separate family (family 3) within the superfamily of seven transmembrane G-protein-coupled receptors (GPCR). A distinctive property of this receptor family is the unusually large amino-terminal domain (ATD) on the extracellular side of the membrane. However, the amino acid sequence identities between the three receptor classes within family 3 is relatively low as exemplified by the CaR, which shows approximately 25% sequence identity to the mGlu1areceptor (2Ruat M. Molliver M.E. Snowman A.M. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3161-3165Crossref PubMed Scopus (342) Google Scholar). Previously we have shown that the ATD of mGlu1a is related to bacterial periplasmic binding proteins (PBP), and based on the crystal structure of the Leu/Ile/Val binding protein (LIVBP) we developed a molecular model of the glutamate-binding domain of the mGlu1a receptor (9O'Hara P.J. Sheppard P.O. Thøgersen H. Venezia D. Haldeman B.A. McGrane V. Houamed K.M. Thomsen C. Gilbert T.L. Mulvihill E.R. Neuron. 1993; 11: 41-52Abstract Full Text PDF PubMed Scopus (615) Google Scholar). As predicted by this model, we have shown that glutamate binding was significantly reduced by mutations of Ser-165 and/or Thr-188 in mGlu1a, which align with Ser-79 and Thr-102, respectively, which bind the amino acid ligand in LIVBP (9O'Hara P.J. Sheppard P.O. Thøgersen H. Venezia D. Haldeman B.A. McGrane V. Houamed K.M. Thomsen C. Gilbert T.L. Mulvihill E.R. Neuron. 1993; 11: 41-52Abstract Full Text PDF PubMed Scopus (615) Google Scholar, 10Sack J.S. Saper M.A. Quiocho F.A. J. Mol. Biol. 1989; 206: 171-191Crossref PubMed Scopus (222) Google Scholar). The glutamate binding to the ATD has been confirmed by a series of other experiments. Thus, it has been shown that agonist selectivity can be interchanged by generating chimeric receptors within the mGlu receptor family in which the ATDs are interchanged (11Takahashi K. Tsuchida K. Tanabe Y. Masu M. Nakanishi S. J. Biol. Chem. 1993; 268: 19341-19345Abstract Full Text PDF PubMed Google Scholar, 12Tones M.A. Bendali H. Flor P.J. Knöpfel T. Kuhn R. Neuroreport. 1995; 7: 117-120Crossref PubMed Google Scholar). Furthermore, it has been shown that antibodies raised against the ATD antagonize agonist responses (13Shigemoto R. Abe T. Nomura S. Nakanishi S. Hirano T. Neuron. 1994; 12: 1245-1255Abstract Full Text PDF PubMed Scopus (222) Google Scholar), and finally it has recently been shown that the purified soluble ATD of a truncated mGlu1areceptor displays pharmacological characteristics similar to the wild-type receptor (14Okamoto T. Sekiyama N. Otsu M. Shimada Y. Sato A. Nakanishi S. Jingami H. J. Biol. Chem. 1998; 273: 13089-13096Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Recently, mGlu receptors have been shown to be modulated by Ca2+ and other polyvalent cations (15Kubo Y. Miyashita T. Murata Y. Science. 1998; 279: 1722-1725Crossref PubMed Scopus (193) Google Scholar, 16Saunders R. Nahorski S.R. Challiss R.A. Neuropharmacology. 1998; 37: 273-276Crossref PubMed Scopus (49) Google Scholar). By use of chimeric receptors, it was shown that this modulatory site is situated in the ATD, and point mutations located the site of action to Ser-166 in mGlu1a (15Kubo Y. Miyashita T. Murata Y. Science. 1998; 279: 1722-1725Crossref PubMed Scopus (193) Google Scholar). Although it is at present unclear whether Ca2+ acts as a direct agonist (15Kubo Y. Miyashita T. Murata Y. Science. 1998; 279: 1722-1725Crossref PubMed Scopus (193) Google Scholar) or a co-agonist (16Saunders R. Nahorski S.R. Challiss R.A. Neuropharmacology. 1998; 37: 273-276Crossref PubMed Scopus (49) Google Scholar), it is interesting to note the pharmacological similarity to the CaR. However, in contrast to the mGlu receptors very little is known about the site of action of agonists on the CaR, and the aim of the present study was to investigate whether the results of structure-function studies on the mGlu receptors could be applied to the CaR. All chemicals were obtained from Sigma. Culture media, serum, antibiotics, and buffers for cell culture were obtained from Life Technologies, Inc. (Paisley, United Kingdom). The plasmids CaR-pRK5 and pmGR1 were generous gifts from Professor Solomon H. Snyder (The Johns Hopkins University School of Medicine, Baltimore, MD) and Professor Shigetada Nakanishi (Kyoto University, Kyoto, Japan). The pSI vector was obtained from Promega (Madison, WI). The tsA cells were a generous gift from Dr. Penelope S. V. Jones (University of Vermont, Burlington, VT). The rat CaR was transferred from the pRK5 vector to the pSI vector using the unique flanking restriction sites XhoI andXbaI. Similarly, the rat mGlu1a receptor was transferred to the pSI vector by a partial digest using the flanking restriction sites EcoRI and XbaI. A silentDraIII site was engineered into mGlu1a-pSI at amino acid 577 and into CaR-pSI at amino acid 597. TheseDraIII sites are placed 16 and 17 amino acids upstream of the predicted first transmembrane region of the CaR and mGlu1a receptors, respectively (1Brown E.M. Gamba G. Riccardi D. Lombardi M. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2323) Google Scholar, 17Houamed K.M. Kuijper J.L. Gilbert T.L. Haldeman B.A. O'Hara P.J. Mulvihill E.R. Almers W. Hagen F.S. Science. 1991; 252: 1318-1321Crossref PubMed Scopus (451) Google Scholar). The chimeric receptor named Ca/1a was generated by cutting out theDraIII-XbaI fragment of CaR-pSI and replacing it with the DraIII-XbaI fragment of mGlu1a-pSI. By this strategy all cysteines of the cysteine-rich domain in the ATD were left intact. The point mutations S147A, S169A, S170A, and S171A were introduced in CaR-pRK5, and theXhoI-EcoRI fragments containing the mutations were transferred to CaR-pSI. Mutations were made using the Quickchange mutagenesis kit according to the manufacturer's instructions (Stratagene, La Jolla, CA), and all amplified receptor DNAs were sequenced on an ABI 310 using the Big Dye Terminator Cycle Sequencing kit (Perkin-Elmer, Warrington, UK). tsA cells (a transformed HEK 293 cell line (18Chahine M. Bennett P.B. George Jr., A.L. Horn R. Pflügers Arch. 1994; 427: 136-142Crossref PubMed Scopus (108) Google Scholar)) were maintained at 37 °C in a humidified 5% CO2 incubator in Dulbecco's modified Eagle's medium (DMEM) supplemented with penicillin (100 units/ml), streptomycin (100 mg/ml), and 10% fetal calf serum. One million cells were split into a 10-cm tissue culture plate and transfected with 5 μg of plasmid the following day using SuperFect as a DNA carrier according to the protocol by the manufacturer (Qiagen, Hilden, Germany). The day after transfection, cells were split into a poly-d-lysine-coated 24-well tissue culture plate in inositol-free DMEM containing a reduced concentration of CaCl2 (0.9 mm) and supplemented with penicillin (100 units/ml), streptomycin (100 mg/ml), 10% fetal calf serum, and 2 μCi/ml myo-2-3Hinositol (Amersham, Buckinghamshire, UK). 16–20 h later, cells were washed with Hanks' balanced saline solution (HBSS) and incubated at 37 °C for 20 min in HBSS containing 0.5 mm CaCl2, 0.5 mm MgCl2, and 10 mm LiCl. Buffer was removed, and cells were incubated for 20 min in the same buffer containing the indicated ions. The reactions were stopped by the buffer with 0.5 of 20 mm and of was out by as T. M. M. S.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). All were in and the results are as of at three experiments. were to the action is the concentration of is the and is were generated by using the As shown in the cationic agonists Ca2+, Mg2+, and Ba2+ were in tsA cells transiently transfected with CaR-pSI. In with was the Ca2+ and Ba2+ (1Brown E.M. Gamba G. Riccardi D. Lombardi M. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2323) Google Scholar, M. Snowman A.M. Snyder S.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and Ba2+ have been shown to be partial and respectively, as compared with Ca2+ M. Snowman A.M. Snyder S.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In with this we to be a partial agonist with an of in the present Ba2+ was shown to also as a partial agonist with an of Ca2+ and Ba2+ both of approximately shows a significantly of as has been shown by M. Snowman A.M. Snyder S.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). transfected with the pSI vector response to the applied maximal concentration of agonists for cationic agonists at wild-type CaR and the chimeric Ca/1a receptor in tsA were generated as in the for and and values and maximal compared to a maximal Ca2+ were shown are of three to seven experiments. in a were generated as in the for and and values and maximal compared to a maximal Ca2+ were shown are of three to seven experiments. To the binding domain of cationic agonists on CaR, we the ATD of mGlu1a with the ATD of CaR the chimeric receptor named tsA cells were transiently transfected with the cells stimulated production when exposed to of the agonists Ca2+, Mg2+, and Ba2+ in a very similar to cells transfected with wild-type CaR of the pharmacological of the three agonists tested using the chimeric Ca/1a and wild-type CaR a very similar pharmacological in of potency and the for Ba2+ were reduced to and respectively, showed a similar to the CaR For the it has been shown that Ser-165 and Thr-188 a in the binding of acid (9O'Hara P.J. Sheppard P.O. Thøgersen H. Venezia D. Haldeman B.A. McGrane V. Houamed K.M. Thomsen C. Gilbert T.L. Mulvihill E.R. Neuron. 1993; 11: 41-52Abstract Full Text PDF PubMed Scopus (615) Google Scholar) and that Ser-166 is involved in binding of the modulatory Ca2+ (15Kubo Y. Miyashita T. Murata Y. Science. 1998; 279: 1722-1725Crossref PubMed Scopus (193) Google Scholar). the ATD of the rat CaR is with the and the rat mGlu receptors, it can be that Ser-147 and Ser-170 in rat CaR to Ser-165 and Thr-188, respectively, of on the of a in the binding of the modulatory Ca2+ in we each of the point mutations and in the rat CaR to whether these amino acid residues are involved in the activation of CaR by Ca2+. In we made each of the adjacent CaR point mutations, S169A and The for the are shown in and the pharmacological are shown in The the receptor, the potency of the endogenous agonist Ca2+ approximately and the maximal response to of the wild-type A was in the CaR, which was to to Ca2+ even at a concentration of 50 In contrast, the adjacent S169A a CaR with a pharmacological profile identical with that of the wild-type CaR. The other adjacent S171A, also resulted in a CaR with a pharmacological profile close to the wild-type CaR, with a of Ca2+ potency of the agonist Ca2+ on wild-type and of as in tsA cells transfected with wild-type CaR CaR-S147A and CaR-S170A or were with 2 washed with HBSS, and incubated with HBSS supplemented with 0.5 0.5 mm Mg2+, and 10 mm for 20 Buffer was and cells were incubated for 20 min in the same buffer with the indicated of Ca2+ for 20 and was by are the of in for the agonist Ca2+ at wild-type and in tsA were generated as in the for and and values and maximal compared to a maximal Ca2+ response at wild-type CaR, were shown are of three to seven experiments. in a were generated as in the for and and values and maximal compared to a maximal Ca2+ response at wild-type CaR, were shown are of three to seven experiments. The ATD of mGlu receptors has been to structure-function which have that for the endogenous agonist glutamate to Ser-165 and Thr-188 in the ATD (9O'Hara P.J. Sheppard P.O. Thøgersen H. Venezia D. Haldeman B.A. McGrane V. Houamed K.M. Thomsen C. Gilbert T.L. Mulvihill E.R. Neuron. 1993; 11: 41-52Abstract Full Text PDF PubMed Scopus (615) Google Scholar). Furthermore, it has recently been shown that Ca2+ the receptor (15Kubo Y. Miyashita T. Murata Y. Science. 1998; 279: 1722-1725Crossref PubMed Scopus (193) Google Scholar, 16Saunders R. Nahorski S.R. Challiss R.A. Neuropharmacology. 1998; 37: 273-276Crossref PubMed Scopus (49) Google Scholar) and that the modulatory site is also located in the ATD at Ser-166 (15Kubo Y. Miyashita T. Murata Y. Science. 1998; 279: 1722-1725Crossref PubMed Scopus (193) Google Scholar). the other very little is known about the site of action of the cationic agonists the CaR. In this we have used chimeric and receptors to study whether the results of structure-function studies on the mGlu receptors could be transferred to the CaR. As of these we generated a chimeric receptor named Ca/1a consisting of the ATD of CaR and the transmembrane region and of mGlu1a. this chimeric receptor a pharmacological profile almost identical to that of the wild-type CaR. that the ATD is the site of action of cationic agonists at the CaR in with results from the mGlu receptors (11Takahashi K. Tsuchida K. Tanabe Y. Masu M. Nakanishi S. J. Biol. Chem. 1993; 268: 19341-19345Abstract Full Text PDF PubMed Google Scholar, 12Tones M.A. Bendali H. Flor P.J. Knöpfel T. Kuhn R. Neuroreport. 1995; 7: 117-120Crossref PubMed Google Scholar). In of the relatively low sequence identity of 19–25% between the CaR and the mGlu receptors (1Brown E.M. Gamba G. Riccardi D. Lombardi M. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2323) Google Scholar, 2Ruat M. Molliver M.E. Snowman A.M. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3161-3165Crossref PubMed Scopus (342) Google Scholar), it is that the chimeric receptor is this it is known the from the first is from the ATD to the that are for activation J. C. Kuhn R. Knöpfel T. J. Pin J.-P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In our results that this mechanism of of has been conserved even between related of family 3 of by large agonist binding The significant between CaR and Ca/1a was the for Ca2+ and Ba2+ at the chimeric receptor compared with the wild-type receptor. have been for the unusually for Ca2+ and other cations at CaR M. Snowman A.M. Snyder S.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Ca2+ binding between as or 3) between CaR and it was shown that a region of the C terminus of CaR close to the transmembrane domain acids is for the for Ca2+ of this region significantly the J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The chimeric receptor we constructed the of mGlu1a and is no sequence between CaR and mGlu1a in the region close to the transmembrane domain J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) this could an for the for Ca2+ and Ba2+ that we for Ca/1a compared with CaR. To study the site of action of the endogenous agonist the ATD of the CaR, we an alignment of the CaR with the and the mGlu receptors. Ser-165 and Thr-188, which have been shown to bind glutamate to (9O'Hara P.J. Sheppard P.O. Thøgersen H. Venezia D. Haldeman B.A. McGrane V. Houamed K.M. Thomsen C. Gilbert T.L. Mulvihill E.R. Neuron. 1993; 11: 41-52Abstract Full Text PDF PubMed Scopus (615) Google Scholar), have been conserved as Ser-147 and as the Ser-170 in CaR, respectively. In of the on the of a for the Ca2+ on Y. Miyashita T. Murata Y. Science. 1998; 279: 1722-1725Crossref PubMed Scopus (193) Google Scholar), we to whether Ser-147 and/or Ser-170 is involved in the receptor activation in CaR by these residues to the results from the mutations on we that CaR-S147A is CaR-S170A is to to the endogenous agonist Ca2+. also mutated each of the adjacent at and in CaR to These mutated receptors have pharmacological profiles identical or to the wild-type receptor, respectively. The of of the mutated CaR could be by of the protein the other the similar amino acid of the adjacent amino acid no on the function of the receptor, we it very that of a in this region be for of the Thus, our is that Ser-170 to some Ser-147 are for agonist activation of CaR by the endogenous agonist Ca2+. A of proteins for which the structure has been by bind Ca2+ has a been shown to as a Ca2+ ligand in these to the of groups in the side as in the of Chem. 1991; PubMed Scopus Google Scholar). In this it is interesting to note the of residues in the of Ca2+ on both and CaR. In the protein by the Ca2+ are Thus, it be that residues other Ser-147 and Ser-170 are as Ca2+ in the CaR. Furthermore, as Ca2+ bind to the receptor, which even increase the of residues as Ca2+ in the receptor. has recently been shown that in the receptor to Ser-147 and Ser-170 in CaR and to Ser-165 and Thr-188 in are for binding of to the receptor T. C. Malitschek B. K. Kuhn R. Bittiger H. Froestl W. Bettler B. Pin J.-P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, in it that all of the receptors that to family 3 of with a large ATD bind agonists in a similar in the ATD, and that the is to the site of the by an as mechanism also to be conserved within this of Professor Snyder and Professor Shigetada Nakanishi for generous gifts of CaR and mGlu1a, respectively. also for