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• W2126653660 abstract "The Ca2+-sensing receptor (CaSR) belongs to the class III G-protein-coupled receptors (GPCRs), which include receptors for pheromones, amino acids, sweeteners, and the neurotransmitters glutamate and γ-aminobutyric acid (GABA). These receptors are characterized by a long extracellular amino-terminal domain called a Venus flytrap module (VFTM) containing the ligand binding pocket. To elucidate the molecular determinants implicated in Ca2+ recognition by the CaSR VFTM, we developed a homology model of the human CaSR VFTM from the x-ray structure of the metabotropic glutamate receptor type 1 (mGluR1), and a phylogenetic analysis of 14 class III GPCR VFTMs. We identified critical amino acids delineating a Ca2+ binding pocket predicted to be adjacent to, but distinct from, a cavity reminiscent of the binding site described for amino acids in mGluRs, GABA-B receptor, and GPRC6a. Most interestingly, these Ca2+-contacting residues are well conserved within class III GPCR VFTMs. Our model was validated by mutational and functional analysis, including the characterization of activating and inactivating mutations affecting a single amino acid, Glu-297, located within the proposed Ca2+ binding pocket of the CaSR and associated with autosomal dominant hypocalcemia and familial hypocalciuric hypercalcemia, respectively, genetic diseases characterized by perturbations in Ca2+ homeostasis. Altogether, these data define a Ca2+ binding pocket within the CaSR VFTM that may be conserved in several other class III GPCRs, thereby providing a molecular basis for extracellular Ca2+ sensing by these receptors. The Ca2+-sensing receptor (CaSR) belongs to the class III G-protein-coupled receptors (GPCRs), which include receptors for pheromones, amino acids, sweeteners, and the neurotransmitters glutamate and γ-aminobutyric acid (GABA). These receptors are characterized by a long extracellular amino-terminal domain called a Venus flytrap module (VFTM) containing the ligand binding pocket. To elucidate the molecular determinants implicated in Ca2+ recognition by the CaSR VFTM, we developed a homology model of the human CaSR VFTM from the x-ray structure of the metabotropic glutamate receptor type 1 (mGluR1), and a phylogenetic analysis of 14 class III GPCR VFTMs. We identified critical amino acids delineating a Ca2+ binding pocket predicted to be adjacent to, but distinct from, a cavity reminiscent of the binding site described for amino acids in mGluRs, GABA-B receptor, and GPRC6a. Most interestingly, these Ca2+-contacting residues are well conserved within class III GPCR VFTMs. Our model was validated by mutational and functional analysis, including the characterization of activating and inactivating mutations affecting a single amino acid, Glu-297, located within the proposed Ca2+ binding pocket of the CaSR and associated with autosomal dominant hypocalcemia and familial hypocalciuric hypercalcemia, respectively, genetic diseases characterized by perturbations in Ca2+ homeostasis. Altogether, these data define a Ca2+ binding pocket within the CaSR VFTM that may be conserved in several other class III GPCRs, thereby providing a molecular basis for extracellular Ca2+ sensing by these receptors. The class III G-protein-coupled receptors (GPCRs) 5The abbreviations used are: GPCRG-protein-coupled receptorCaSRCalcium sensing receptorGABAγ-aminobutyric acidVFTMVenus flytrap module, mGluR1, metabotropic glutamate receptor type 1LBlobeGBR1GABA-B receptor type 1T1R1, T1R2, T1R3sweet taste receptors type 1, type 2, type 3ADHautosomal dominant hypocalcemiaFHHfamilial hypocalciuric hypercalcemiaIPinositol phosphateWTwild type 5The abbreviations used are: GPCRG-protein-coupled receptorCaSRCalcium sensing receptorGABAγ-aminobutyric acidVFTMVenus flytrap module, mGluR1, metabotropic glutamate receptor type 1LBlobeGBR1GABA-B receptor type 1T1R1, T1R2, T1R3sweet taste receptors type 1, type 2, type 3ADHautosomal dominant hypocalcemiaFHHfamilial hypocalciuric hypercalcemiaIPinositol phosphateWTwild type are activated by a variety of ligands, including calcium (Ca2+), pheromones, l-amino acids, diverse natural sweeteners, and the major neurotransmitters glutamate and γ-aminobutyric acid (GABA). These receptors are characterized by a large amino-terminal extracellular domain reminiscent of bacterial periplasmic binding proteins. This domain is formed by two lobes (LB1 and LB2) separated by a cavity delineating the ligand-binding site and called a Venus flytrap module (VFTM) (1Hofer A.M. Brown E.M. Nat. Rev. Mol. Cell. Biol. 2003; 4: 530-538Crossref PubMed Scopus (515) Google Scholar, 2Pin J.P. Kniazeff J. Goudet C. Bessis A.S. Liu J. Galvez T. Acher F. Rondard P. Prezeau L. Biol. Cell. 2004; 96: 335-342Crossref PubMed Google Scholar). The crystal structure of the glutamate-bound form of the extracellular domain of the metabotropic glutamate receptor type 1 (mGluR1) revealed key residues located at the interface of LB1 and LB2 and that were involved in glutamate binding (3Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1096) Google Scholar). Homology modeling of other class III GPCRs, including mGluRs and GABA-B type 1 (GBR1) receptors, the recently deorphanized receptor for basic amino acids (GPRC6a) and its goldfish relative, and the sweet taste receptors T1R1, T1R2, and T1R3, have helped identify the ligand binding pocket for these receptor ligands and the receptor activation process (4Bessis A.S. Rondard P. Gaven F. Brabet I. Triballeau N. Prezeau L. Acher F. Pin J.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11097-11102Crossref PubMed Scopus (108) Google Scholar, 5Xu H. Staszewski L. Tang H. Adler E. Zoller M. Li X. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14258-14263Crossref PubMed Scopus (411) Google Scholar, 6Liu J. Maurel D. Etzol S. Brabet I. Ansanay H. Pin J.P. Rondard P. J. Biol. Chem. 2004; 279: 15824-15830Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 7Wellendorph P. Hansen K.B. Balsgaard A. Greenwood J.R. Egebjerg J. Brauner-Osborne H. Mol. Pharmacol. 2005; 67: 589-597Crossref PubMed Scopus (173) Google Scholar).The Ca2+-sensing receptor (CaSR) expressed in the parathyroid glands senses minor changes in ionized plasma Ca2+ and by controlling parathyroid hormone secretion is the major molecular determinant of Ca2+ homeostasis (8Brown E.M. MacLeod R.J. Physiol. Rev. 2001; 81: 239-297Crossref PubMed Scopus (1216) Google Scholar, 9Hu J. Spiegel A.M. Trends Endocrinol. Metab. 2003; 14: 282-288Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Initially cloned from the parathyroid glands (10Brown 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 (2345) Google Scholar), the CaSR has been subsequently isolated from various tissues, including kidney and brain, where it is proposed to mediate diverse physiologic effects in response to variations in extracellular Ca2+ (11Ruat M. Molliver M.E. Snowman A.M. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3161-3165Crossref PubMed Scopus (343) Google Scholar, 12Garrett J.E. Capuano I.V. Hammerland L.G. Hung B.C. Brown E.M. Hebert S.C. Nemeth E.F. Fuller F. J. Biol. Chem. 1995; 270: 12919-12925Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 13Riccardi D. Park J. Lee W.S. Gamba G. Brown E.M. Hebert S.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 131-135Crossref PubMed Scopus (435) Google Scholar). The cloning of the CaSR made it possible to demonstrate directly that the CaSR is activated not only by Ca2+ but also by Mg2+ and other divalent cations (10Brown 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 (2345) Google Scholar, 14Ruat M. Snowman A.M. Hester L.D. Snyder S.H. J. Biol. Chem. 1996; 271: 5972-5975Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 15Chang W.H. Pratt S. Chen T.H. Nemeth E. Huang Z.M. Shoback D. J. Bone Miner. Res. 1998; 13: 570-580Crossref PubMed Scopus (78) Google Scholar, 16Coulombe J. Faure H. Robin B. Ruat M. Biochem. Biophys. Res. Commun. 2004; 323: 1184-1190Crossref PubMed Scopus (120) Google Scholar). Molecules aimed at modulating the activity of the CaSR and acting at the level of the transmembrane domains have been characterized (17Nemeth E.F. Curr. Pharm. Des. 2002; 8: 2077-2087Crossref PubMed Scopus (77) Google Scholar, 18Petrel C. Kessler A. Dauban P. Dodd R.H. Rognan D. Ruat M. J. Biol. Chem. 2004; 279: 18990-18997Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Indeed, targeting the parathyroid CaSR by a positive allosteric modulator has been proposed recently for the treatment of secondary hyperparathyroidism linked to renal disease (19Block G.A. Martin K.J. de Francisco A.L. Turner S.A. Avram M.M. Suranyi M.G. Hercz G. Cunningham J. Abu-Alfa A.K. Messa P. Coyne D.W. Locatelli F. Cohen R.M. Evenepoel P. Moe S.M. Fournier A. Braun J. McCary L.C. Zani V.J. Olson K.A. Drueke T.B. Goodman W.G. N. Engl. J. Med. 2004; 350: 1516-1525Crossref PubMed Scopus (937) Google Scholar), and antagonizing the CaSR activity might be of benefit for treating female osteoporosis (17Nemeth E.F. Curr. Pharm. Des. 2002; 8: 2077-2087Crossref PubMed Scopus (77) Google Scholar).Naturally occurring activating and inactivating CaSR mutations are responsible for autosomal dominant hypocalcemia (ADH) and familial hypocalciuric hypercalcemia (FHH), genetic diseases linked to perturbations in Ca2+ homeostasis (9Hu J. Spiegel A.M. Trends Endocrinol. Metab. 2003; 14: 282-288Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 20Hendy G.N. D'Souza-Li L. Yang B. Canaff L. Cole D.E. Hum. Mutat. 2000; 16: 281-296Crossref PubMed Scopus (234) Google Scholar). The CaSR VFTM has been shown to contain the Ca2+-binding site (21Brauner-Osborne H. Jensen A.A. Sheppard P.O. Hara P.O Krogsgaard-Larsen P. J. Biol. Chem. 1999; 274: 18382-18386Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 22Reyes-Cruz G. Hu J. Goldsmith P.K. Steinbach P.J. Spiegel A.M. J. Biol. Chem. 2001; 276: 32145-32151Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Among the 13 residues shown to be involved in glutamate binding to the mGluR1 VFTM, 6 are identical or conservatively substituted in the human CaSR (see supplemental Tables 3 and 4), and the analysis of variants produced by site-directed mutagenesis has shown that three of these residues impair Ca2+ activation when changed to alanine (21Brauner-Osborne H. Jensen A.A. Sheppard P.O. Hara P.O Krogsgaard-Larsen P. J. Biol. Chem. 1999; 274: 18382-18386Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 23Zhang Z. Qiu W. Quinn S.J. Conigrave A.D. Brown E.M. Bai M. J. Biol. Chem. 2002; 277: 33727-33735Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). However, the Ca2+-binding amino acids delineating the Ca2+ binding pocket within the CaSR VFTM have not been identified, nor is it known if this Ca2+ binding cavity is conserved within class III receptors.In this study, we describe a homology model of the VFTM of the human CaSR built from the x-ray structure of the rat mGluR1 and a phylogenetic analysis of the ligand binding pocket of class III GPCRs. This model, which predicts the residues contributing to Ca2+ recognition, indicates that Ca2+ does not interact with amino acids homologous to those delineating the glutamate binding cavity but to a site adjacent to this cavity. We used mutational analysis, including the description and characterization of a novel mutation associated with ADH, and data from the literature to validate our model. In addition, our data are consistent with the presence of a conserved Ca2+ binding pocket within the VFTM of other class III GPCRs, adding a molecular basis for the previously observed effects of extracellular Ca2+ on the action of these receptors.EXPERIMENTAL PROCEDURESIdentification of E297D Mutation in the CaSR Associated with ADH—Genomic DNA from the proband and family members was isolated from peripheral leukocytes using standard methods. CaSR exons 2-7 (proband) and exon 4 (all family members) and the corresponding intron-exon junctions were amplified using intronic primers (sequences available on request). PCR-amplified products were sequenced as described (24Prie D. Huart V. Bakouh N. Planelles G. Dellis O. Gerard B. Hulin P. Benque-Blanchet F. Silve C. Grandchamp B. Friedlander G. N. Engl. J. Med. 2002; 347: 983-991Crossref PubMed Scopus (284) Google Scholar).Site-directed Mutagenesis—Wild-type (WT) cDNA encoding the human CaSR inserted in pcDNA3.1/Hygro plasmid was a kind gift of E. F. Nemeth and P. Jacobson (NPS Pharmaceuticals Inc., Salt Lake City, UT). Missense mutations were introduced in the WT cDNA construct using the Quick Change Site-directed Mutagenesis kit (Stratagene) and were confirmed by complete sequencing of cDNA inserts (Genome Express, Meylan, France) (oligonucleotide sequences available on request).Cell Culture, Transient Transfection, and Western Blot Analysis—Experiments were performed in HEK293 cells. Techniques used for cell culture, transient transfection by electroporation, and the detection of WT and mutant CaSRs expression by Western blot analysis have been described (25Petrel C. Kessler A. Maslah F. Dauban P. Dodd R.H. Rognan D. Ruat M. J. Biol. Chem. 2003; 278: 49487-49494Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar).[3H]IP Formation—Cells were cultured in the presence of 0.5 μCi/well myo-[3H]inositol (Amersham Biosciences) for 20 h. The measurement of [3H]IP accumulation was performed in freshly prepared buffer (125 mm NaCl; 4.0 mm KCl; 0.5 mm MgCl2; 20 mm Hepes; 0.1% d-glucose, pH 7.4) containing the indicated CaCl2 concentration (25Petrel C. Kessler A. Maslah F. Dauban P. Dodd R.H. Rognan D. Ruat M. J. Biol. Chem. 2003; 278: 49487-49494Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Two to five independent experiments were performed in duplicate or triplicate using the same conditions. The data were fitted to a sigmoid doseresponse curve to determine EC50 values by using the GraphPAD Prism. Values for each curve were normalized to the maximal activation of each receptor, obtained by stimulation with 20 mm Ca2+. Significance was assayed by Excel 2000 Student's t test.Homology Modeling of the Amino-terminal Domain of the Calciumsensing Receptor—A multiple alignment of 14 class III GPCR extracellular domains (shown in supplemental Table 3) was obtained with the T-Coffee program (26Notredame C. Higgins D.G. Heringa J. J. Mol. Biol. 2000; 302: 205-217Crossref PubMed Scopus (5385) Google Scholar) Homology modeling of the human CaSR was performed with the SYBYL6.91 package (TRIPOS Associates Inc., St. Louis, MO) starting from monomer A of the mGluR1 x-ray structure (Protein Data Bank code 1ewk) (3Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1096) Google Scholar). The insertion of residues in loops between helix A and strand B, helix B and strand D, and helix L and helix M (3Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1096) Google Scholar) was accommodated by a knowledge-based loop search procedure as described previously (27Bissantz C. Bernard P. Hibert M. Rognan D. Proteins. 2003; 50: 5-25Crossref PubMed Scopus (306) Google Scholar). Further energy refinement of the model was achieved by a standard minimization protocol using the AMBER 8 program (University of California, San Francisco). Mapping of the Ca2+-binding site was performed using the GRID version 20 software (28Goodford P.J. J. Med. Chem. 1985; 28: 849-857Crossref PubMed Scopus (2439) Google Scholar) using a Ca2+ probe and standard settings for the grid definition. The most energetically favored location of the calcium ion was then used to minimize (by 1,000 steps of steepest descent followed by 1,000 steps of conjugate gradient refinement) the receptor model in the presence of Ca2+ and within a box of 23,844 TIP3P water molecules, placed automatically with the leap module of AMBER 8.0.Neighbor-joining Tree—19 residues lining the binding pocket of glutamate in the mGluR1 structure (3Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1096) Google Scholar) were extracted from 14 human class III GPCRs, concatenated into ungapped sequences, and were used to calculate a distance matrix with the MEGA2 software (29Kumar S. Tamura K. Jakobsen I.B. Nei M. Bioinformatics. 2001; 17: 1244-1245Crossref PubMed Scopus (4545) Google Scholar). A neighborjoining tree was calculated out of 100 bootstrap replicas using the γ correction for estimating pairwise protein distances.RESULTSIdentification of E297D Mutation in the CaSR Associated with ADH—The three generation pedigree of the family affected with ADH is shown in Fig. 1A. The index case was diagnosed during the 1st month of life because of severe hypocalcemic symptoms requiring calcium infusion. Biochemical features of the proband and family members at the time of diagnosis of hypocalcemia and identification of the activating mutation in the CaSR are shown in supplemental Table 5.Direct sequence analysis of the PCR-amplified CaSR exons led to the identification of a heterozygous G to C nucleotide substitution in exon 4 of the CaSR gene at position 889 of the CaSR cDNA sequence (Fig. 1B). This base change resulted in the substitution of glutamate for aspartate at position 297 (E297D) located in the extracellular domain. The E297D mutation was present in all affected family members and absent in others (data not shown). Most interestingly, a missense E297K mutation has been identified in subjects with FHH (30Pollak M.R. Brown E.M. Chou Y.H. Hebert S.C. Marx S.J. Steinmann B. Levi T. Seidman C.E. Seidman J.G. Cell. 1993; 75: 1297-1303Abstract Full Text PDF PubMed Scopus (897) Google Scholar, 31Bai M. Quinn S. Trivedi S. Kifor O. Pearce S.H.S. Pollak M.R. Krapcho K. Hebert S.C. Brown E.M. J. Biol. Chem. 1996; 271: 19537-19545Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar).Phylogenetic Tree of the Ligand Binding Pocket of Family 3 GPCRs—The Glu-297 residue is structurally homologous to an aspartate residue conserved in all mGluRs that is located within the mGluR VFTM (TABLE ONE and supplemental Table 4) and has been implicated in ligand binding and receptor activation (3Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1096) Google Scholar, 4Bessis A.S. Rondard P. Gaven F. Brabet I. Triballeau N. Prezeau L. Acher F. Pin J.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11097-11102Crossref PubMed Scopus (108) Google Scholar). This suggests that Glu-297 is part of the Ca2+ binding pocket of the CaSR. In an attempt to delineate the putative ligand binding pocket of this receptor, we first constructed a phylogenetic tree for 14 human class III GPCRs based on 19 residues (TABLE ONE) lining the glutamate binding cavity identified in the mGluR1 structure (3Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1096) Google Scholar). This phylogenetic tree indicates that the CaSR-binding site is not closely related to that of any other receptors (Fig. 2A), including the GPRC6a receptor recognizing basic amino acids (7Wellendorph P. Hansen K.B. Balsgaard A. Greenwood J.R. Egebjerg J. Brauner-Osborne H. Mol. Pharmacol. 2005; 67: 589-597Crossref PubMed Scopus (173) Google Scholar). This latter receptor shows the highest overall homology with the CaSR (TABLE ONE and supplemental Table 4), but its ligand-binding site segregates more closely with that of the mGluRs (Fig. 2A). Most interestingly, the binding sites of the CaSR, T1R1, T1R2, T1R3, and GBR1 form a cluster distinct from that of mGluRs (Fig. 2A).TABLE ONEAlignment of 19 amino acids lining the orthosteric binding site of 14 human class III GPCRsReceptorBinding site residuesmGluR1Y74R78W110S164S165S166S186T188D208Q211Y236F290E292G293S317D318G319R323K409mGluR2R57R61S93Y144S145D146A166T168D188Q191Y226F269R271S272S294D295G296L300K377mGluR3R67R71S103Y153S154S155A175T177D197Q200Y225F278R280S281S303D304G305Q309K392mGluR4K74R78S110G158S159S160A180T182D202Q205Y230F284N286E287S311D312S313K317K405mGluR5Y64R68W100S151S152S153S173T175D195Q198Y223F277E279G280S304D305G306R310K396mGluR6Q64R68S100A153S154S155A175T177D197Q200Y225F279N281E282S306D307S308K312K400mGluR7N64R78S110G158S159S160A180T182D202Q205Y230F286N288D289S313D314S315K319K407mGluR8K71R75S107A155S156S160A177T179D199Q202Y227F281N283E284S308D309S310K314R401GPRC6aS69Q73T104Y148S149E150E170T172D192Q195Y220F277R279Q280S302D303N304A308E408GBR1G184C188D221C246S247S248G268S270A290H293V318L365Y367E368A397D398N399I403E466CaSRR66W70N102G146S147G148A168S170D190Q193Y218F270S272G273S296E297A298S302H413T1R1H71L75S107S148T149N150A170S172D192Q195Y220F274S226R227S300E301A302S306M383T1R2167L71Y103N143S144E145S165I167A187H190Y215F275P277D278S301E302S303D307E382T1R3N68W72S104S146S147E148G168S170D190Q193Y218F274S276V277S300E301A302S306H387 Open table in a new tab FIGURE 2Phylogenetic tree of class III GPCR ligand binding pocket (A) and molecular modeling of the human mGluR1 (B) and CaSR (C) ligand binding pockets.A, neighbor-joining tree of human class III GPCRs. The 19 residues lining the binding pocket of human class III GPCRs (supplemental Table 4) were extracted to calculate a distance matrix with the MEGA2 software (29Kumar S. Tamura K. Jakobsen I.B. Nei M. Bioinformatics. 2001; 17: 1244-1245Crossref PubMed Scopus (4545) Google Scholar). A neighbor-joining tree was calculated out of 100 bootstrap replicas using the γ correction for estimating pairwise protein distances. Bootstrap values are indicated in italics. B and C, ligand binding pocket of mGluR1 (B) and CaSR (C). Receptor atoms are shown as capped sticks, whereas ligand atoms are shown in ball and sticks (white, receptor carbon atom; green, l-glutamate carbon atoms; red, oxygen; blue, nitrogen). Main chain atoms of the receptor participating in H-bonds to the ligands are displayed in yellow. Residues from the ligand binding domains 1 and 2 (LB1 and LB2) are labeled at the C-α atoms in white and cyan, respectively. Water molecules mediating ligand binding are displayed as cyan balls. Pink dashed lines indicate electrostatic (H-bonds, ion coordination) intermolecular interactions. B, the amino-terminal mGluR1-binding site residues were taken from the glutamate-liganded x-ray structure of rat mGluR1 (Protein Data Bank code 1ewk) (3Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1096) Google Scholar). C, CaSR Ca2+-binding site was modeled from the rat mGluR1 structure. Coordinates of the calcium ion are displayed by a green ball. A water molecule (cyan ball) completes the pentagonal bispyramidal coordination of the calcium ion.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Molecular Modeling of the Ca2+ -binding Site of the CaSR—We next developed a homology model of the extracellular domain of the human CaSR built from the x-ray structure of the mGluR1 and according to a multiple alignment of the above-mentioned receptor sequences (supplemental Table 4). Comparison of the molecular models of the human mGluR1 and CaSR ligand binding pockets shows that the overall three-dimensional structure of the CaSR amino-terminal tail is very similar to the x-ray structure of the rat mGluR1 described by Kunishima et al. (3Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1096) Google Scholar) (Fig. 2, B and C). Only three insertions in loop regions occur (see “Experimental Procedures”). The multiple sequence alignment used to thread the CaSR coordinates onto the mGluR1 structure (TABLE ONE and supplemental Table 4) shows only a single difference with that published (3Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1096) Google Scholar) and occurs just before the beginning of helix M at Lys-409, which is a glutamate-anchoring residue. We propose a shorter insertion (three amino acids instead of four) and a single-residue upward shift aligning His-413, and not Thr-412 (CaSR sequence), with Lys-409 (mGluR1 sequence). However, both residues present a side chain pointing inward toward the binding cavity and are putative anchoring residues for a ligand (Fig. 2, B and C). For the other 18 of the 19 residues lining the glutamate binding cavity, the multiple sequence alignment is unambiguous and identical to that proposed previously (3Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1096) Google Scholar).Our model shows that the CaSR presents a binding cavity between the two lobes of the amino-terminal domain, which is reminiscent of an amino acid-binding site but of smaller dimensions than that observed for the mGluR1 (47 versus 68 Å3, respectively) (Fig. 2, B and C). More importantly, however, Ca2+ is not predicted to bind within this pocket homologous to the glutamate binding cavity. Rather, the Ca2+ binding is predicted to occur within an adjacent site formed by a set of polar residues directly involved in Ca2+ coordination (Ser-170, Asp-190, Gln-193, Ser-296, and Glu-297) (Fig. 2C) with an additional set of residues contributing to complete the coordination sphere of the cation (Phe-270, Tyr-218, and Ser-147) through water interactions. A pentagonal bipyramidal coordination of metal is proposed, as is observed in many calcium-binding proteins (32Chattopadhyaya R. Meador W.E. Means A.R. Quiocho F.A. J. Mol. Biol. 1992; 228: 1177-1192Crossref PubMed Scopus (614) Google Scholar). Our present model identifies the presence of a Ca2+ binding pocket with the identification of Glu-193, Phe-270, Ser-296, and Glu-297 as novel potential residues involved in Ca2+ binding.The residues involved in this putative Ca2+-binding site are all conserved in T1R3 and all but one in T1R1 (Ser-147 in CaSR substituted by Thr-149 in T1R1) (TABLE TWO). They are also conserved in mGluRs and GPRC6a except for two residues (Ser-170 and Glu-297 in CaSR substituted by Thr and Asp, respectively, in mGluRs and GPRC6a). The lowest homology is found with T1R2 and GBR1 (TABLE TWO).TABLE TWOAmino acids lining the putative Ca2+-binding site of human class III GPCRsReceptorBinding residuesmGluR1S165T188D208Q211Y236F290S317D318mGluR2S145T168D188Q191Y226F269S294D295mGluR3S154T177D197Q200Y225F278S303D304mGluR4S159T182D202Q205Y230F284S311D312mGluR5S152T175D195Q198Y223F277S304D305mGluR6S154T177D197Q200Y225F279S306D307mGluR7S159T182D202Q205Y230F286S313D314mGluR8S156T179D199Q202Y227F281S308D309GPRC6aS149T172D192Q195Y220F277S302D303GBR1S247S270A290H293V318L365A397D398CaSRS147S170D190Q193Y218F270S296E297T1R1T149S172D192Q195Y220F274S300E301T1R2S144I167A187H190Y215F275S301E302T1R3S147S170D190Q193Y218F274S300E301 Open table in a new tab Functional Characterization of the Putative Ligand Contacting Residues—Four of the putative ligand contacting residues, Ser-147, Ser-170, Asp-190, and Tyr-218, have been shown previously to impair human CaSR activation when mutated to alanine (23Zhang Z. Qiu W. Quinn S.J. Conigrave A.D. Brown E.M. Bai M. J. Biol. Chem. 2002; 277: 33727-33735Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) (TABLE THREE). Similar results were obtained for Ser-147 and Ser-170 for the rat CaSR (21Brauner-Osborne H. Jensen A.A. Sheppard P.O. Hara P.O Krogsgaard-Larsen P. J. Biol. Chem. 1999; 274: 18382-18386Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). In addition, Tyr-218 mutations (Y218C and Y218S) have been identified in humans with familial hypocalciuric hypercalcemia (33Pearce S.H. Trump D. Wooding C. Besser G.M. Chew S.L. Grant D.B. Heath D.A. Hughes I.A. Paterson C.R. Whyte M.P. J. Clin. Investig. 1995; 96: 2683-2692Crossref PubMed Scopus (326) Google Scholar, 34Cetani F. Pardi E. Borsari S. Tonacchera M. Morabito E. Pinchera A. Marcocci C. Dipollina G. Clin. Endocrinol. 2003; 58: 199-206Crossref PubMed Scopus (32) Google Scholar). To assess the functional importance of the remaining putative Ca2+- contacting residues, we measured PI hydrolysis as a function of extracellular Ca2+ concentration in HEK293 cells transfected with the WT receptor and CaSR constructs harboring the naturally occurring E297D and E297K mutations or the artificial Q193A, F270A, and S296A mutations. The functional characterization of the E297D mutation identified in the family described above indicated that this receptor shows a left-shifted concentration-response curve to Ca2+ compared with the WT receptor (EC50 = 2.70 ± 0.30 mmversus 4.30 ± 0.20 mm, mean ± S.E., n = 4, p < 0.001) (Fig. 3A and TABLE TWO). In contrast, the E297K receptor was accompanied by a complete loss of Ca2+ sensitivity (Fig. 3A and TAB" @default.
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