The Venus Fly Trap Domain of the Extracellular Ca²⁺-sensing Receptor Is Required for l-Amino Acid Sensing

Abstract

We previously demonstrated that the human calcium-sensing receptor (CaR) is allosterically activated by l-amino acids (Conigrave, A. D., Quinn, S. J., and Brown, E. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4814–4819). However, the domain-based location of amino acid binding has been uncertain. We now show that the Venus Fly Trap (VFT) domain of CaR, but none of its other major domains, is required for amino acid sensing. Several constructs were informative when expressed in HEK293 cells. First, the wild-type CaR exhibited allosteric activation by l-amino acids as previously observed. Second, two CaR-mGlu chimeric receptor constructs that retained the VFT domain of CaR, one containing the extracellular Cys-rich region of CaR and the other containing the Cys-rich region of the rat metabotropic glutamate type-1 (mGlu-1) receptor, together with the rat mGlu-1 transmembrane region and C-terminal tail, retained amino acid sensing. Third, a CaR lacking residues 1–599 of the N-terminal extracellular head but retaining an intact CaR transmembrane region and a functional but truncated C terminus (headless-T903 CaR) failed to respond to l-amino acids but retained responsiveness to the type-II calcimimetic NPS R-467. Finally, a T903 CaR control that retained an intact N terminus also retained l-amino acid sensing. Taken together, the data indicate that the VFT domain of CaR is necessary for l-amino acid sensing and are consistent with the hypothesis that the VFT domain is the site of l-amino acid binding. The findings support the concept that the mGlu-1 amino acid binding site for l-glutamate is conserved as an l-amino acid binding site in its homolog, the CaR. We previously demonstrated that the human calcium-sensing receptor (CaR) is allosterically activated by l-amino acids (Conigrave, A. D., Quinn, S. J., and Brown, E. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4814–4819). However, the domain-based location of amino acid binding has been uncertain. We now show that the Venus Fly Trap (VFT) domain of CaR, but none of its other major domains, is required for amino acid sensing. Several constructs were informative when expressed in HEK293 cells. First, the wild-type CaR exhibited allosteric activation by l-amino acids as previously observed. Second, two CaR-mGlu chimeric receptor constructs that retained the VFT domain of CaR, one containing the extracellular Cys-rich region of CaR and the other containing the Cys-rich region of the rat metabotropic glutamate type-1 (mGlu-1) receptor, together with the rat mGlu-1 transmembrane region and C-terminal tail, retained amino acid sensing. Third, a CaR lacking residues 1–599 of the N-terminal extracellular head but retaining an intact CaR transmembrane region and a functional but truncated C terminus (headless-T903 CaR) failed to respond to l-amino acids but retained responsiveness to the type-II calcimimetic NPS R-467. Finally, a T903 CaR control that retained an intact N terminus also retained l-amino acid sensing. Taken together, the data indicate that the VFT domain of CaR is necessary for l-amino acid sensing and are consistent with the hypothesis that the VFT domain is the site of l-amino acid binding. The findings support the concept that the mGlu-1 amino acid binding site for l-glutamate is conserved as an l-amino acid binding site in its homolog, the CaR. The extracellular Ca2+-sensing receptor (CaR) 1The abbreviations used are: CaR, calcium-sensing receptor; mGlu-1, metabotropic glutamate type-1; VFT, Venus fly trap; T903 CaR, CaR truncated after residue 903. plays a key role in the regulation of whole body calcium metabolism. In keeping with this, the CaR-null mouse exhibits loss of feedback control of parathyroid hormone secretion, hyperparathyroidism, and a metabolic bone disease (2Ho C. Conner D.A. Pollak M.R. Ladd D.J. Kifor O. Warren H.B. Brown E.M. Seidman J.G. Seidman C.E. Nat. Genet. 1995; 11: 389-394Crossref PubMed Scopus (524) Google Scholar). Furthermore, inactivating and activating mutations of the receptor in humans have been shown to induce various disorders of calcium metabolism (for review, see Ref. 3Hofer A.M. Brown E.M. Nat. Rev. Mol. Cell Biol. 2003; 4: 530-538Crossref PubMed Scopus (532) Google Scholar). Although the CaR is a key molecular regulator of whole body calcium metabolism, it presents something of a conundrum. It is widely expressed in mammalian tissues, including tissues such as the brain, that are not clearly involved in calcium metabolism. Furthermore, its closest relatives in molecular terms are members of sub-group C of the G protein-coupled receptors, receptors for specific amino acids such as l-glutamate (mGlus) and glutamate analogs, e.g. GABA. The large extracellular heads of these receptors are related to nutrient-sensing, bacterial periplasmic-binding proteins (4Brauner-Osborne H. Jensen A.A. Sheppard P.O. O'Hara P. Krogsgaard-Larsen P. J. Biol. Chem. 1999; 274: 18382-18386Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). The finding that the CaR is allosterically activated by a broad spectrum of l-amino acids, including aromatics such as l-Phe and l-Trp and aliphatic and polar amino acids such as l-Ala and l-Ser, has the effect of drawing it closer functionally to other members of subgroup C (1Conigrave A.D. Quinn S.J. Brown E.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4814-4819Crossref PubMed Scopus (427) Google Scholar). However, the site of amino acid binding has been unclear. A site-directed mutagenesis study implicated a role for a triple serine motif (Ser-169/Ser-170/Ser-171) in the VFT domain (5Zhang 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 (100) Google Scholar). Furthermore, in an analysis of CaR mutants, differential effects of the type-II calcimimetic NPS R-467, whose binding site lies in the transmembrane region of the receptor, and the aromatic amino acid l-Phe indicated that their binding sites were distinct (6Zhang Z. Jiang Y. Quinn S.J. Krapcho K. Nemeth E.F. Bai M. J. Biol. Chem. 2002; 277: 33736-33741Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In the current study, we have evaluated the domain-based requirements for amino acid sensing using chimeric receptors devised by domain swapping between two receptor homologs, the human CaR, which responds to Ca2+ and aromatic and aliphatic amino acids, and the rat mGlu-1, which responds to l-glutamate. Functional analysis of three key chimeric receptor constructs (listed as VFT/Cys-rich region/transmembrane domain+C-tail), including CaR/CaR/mGlu-1, CaR/mGlu-1/mGlu-1, and –/–/T903 CaR constructs together with two controls, the wild-type CaR and T903 CaR, indicates that the VFT domain alone is required for amino acid sensing. Thus, the VFT domain of CaR is the likely site of l-amino acid binding. Materials—pcDNA3.1(+) (Invitrogen) containing the wild-type human calcium-sensing receptor (cassette version, Ref. 7Bai 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 (385) Google Scholar, was the kind gift of Dr. Mei Bai and Professor Edward Brown (Endocrine-Hypertension Division, Brigham and Womens Hospital, Boston, MA). pBluescript containing the wild-type rat mGluR1α receptor used in the synthesis of the hCaR/mGlu-1/mGlu-1 construct was generously provided by Professor S. Nakanishi (Kyoto University, Japan). DNA ligase and restriction enzymes BspE1, NotI, SpeI, XbaI, XhoI, and ApaI were obtained from New England Biolabs. PCR reagents were obtained from Invitrogen. Site-directed mutagenesis was performed using QuikChange™ kits (Stratagene, La Jolla, CA). pGEM T-Easy (Promega) was used to subclone the “headless” PCR product. Construction of hCaR, Rat mGlu-1 Chimeric Receptors—The two chimeric constructs studied (in the format VFT domain/Cys-rich region/transmembrane region+C-tail) were CaR/CaR/mGlu-1 and CaR/mGlu-1/mGlu-1. A CaR/CaR/mGlu-1 chimera encoded a protein containing the VFT and Cys-rich regions of hCaR (residues 1–598) fused to the transmembrane region plus cytoplasmic tail of the rat mGlu-1α (residues 579–1199). Its construction has previously been described in detail (8Hammerland L.G. Krapcho K.J. Garrett J.E. Alasti N. Hung B.C. Simin R.T. Levinthal C. Nemeth E.F. Fuller F.H. Mol. Pharmacol. 1999; 55: 642-648PubMed Google Scholar). CaR/mGlu-1/mGlu-1 was designed to encode a protein that contained the hCaR VFT domain (residues 1–540) with the rat mGlu-1 Cys-rich region, transmembrane region, and cytoplasmic tail (residues 524–1199). Site-directed mutagenesis was performed to introduce BspE1 restriction enzyme sites at residues 522 of rmGluR1α and 541 of CaR/CaR/mGlu-1. A second BspE1 site at amino acid 877 of the mGlu-1 sequence was removed by site-directed mutagenesis without disturbing the encoded peptide sequence prior to performing the above digestion and ligation reactions. The DNA fragments containing the bases encoding residues 1–540 of hCaR and 523–1199 of rmGluR1α, together with an interlinking single Gly residue, were then purified and ligated into pcDNA3.1(+) to create CaR/mGlu-1/mGlu-1. The junction was confirmed by double-stranded DNA sequencing (Australian Genome Research Facility, Brisbane, Qld, Australia). Headless CaR Construct—A headless CaR deletion mutant elsewhere referred to as Rho-C-CaR (9Zhao X.M. Hauache O. Goldsmith P.K. Collins R. Spiegel A.M. FEBS Lett. 1999; 448: 180-184Crossref PubMed Scopus (56) Google Scholar, 10Hauache O.M. Hu J. Ray K. Xie R. Jacobson K.A. Spiegel A.M. Endocrinology. 2000; 141: 4156-4163Crossref PubMed Google Scholar, 11Hu J. Reyes-Cruz G. Chen W. Jacobson K.A. Spiegel A.M. J. Biol. Chem. 2002; 277: 46622-46631Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) or T903-Rhoc (12Ray K. Northup J. J. Biol. Chem. 2002; 277: 18908-18913Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) containing 20 amino acids of the N terminus of rhodopsin and amino acids 600–903 of the hCaR was constructed in pcDNA3.1 using PCR as described previously (9Zhao X.M. Hauache O. Goldsmith P.K. Collins R. Spiegel A.M. FEBS Lett. 1999; 448: 180-184Crossref PubMed Scopus (56) Google Scholar, 11Hu J. Reyes-Cruz G. Chen W. Jacobson K.A. Spiegel A.M. J. Biol. Chem. 2002; 277: 46622-46631Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Briefly, the sense primer was 100 nucleotides long, corresponding to the nucleotide sequence of the N-terminal 20-amino acid signal peptide of bovine rhodopsin and nucleotide positions 1797–1832 of the hCaR (GenBank™ 20759). The antisense primer was a 46-base oligonucleotide that corresponded to CaR nucleotides 2667–2709. The PCR product encodes a protein in which the bovine rhodopsin signal peptide is fused to Ala-600 of the CaR sequence and the CaR is truncated after Gly-903. The construct lacks the VFT domain of CaR and retains just 12 residues at the C-terminal end of the Cys-rich region of CaR on the C-terminal side of the bovine rhodopsin signal peptide. The PCR product was subcloned into NotI- and XbaI-digested pcDNA3.1 via the pGEM T-Easy Vector System 1 (Promega). DNA sequencing confirmed successful construction of Rho-C-hCaR (Australian Genome Research Facility). Wild-type CaR Truncated after Residue 903—As a control for the headless construct described above, the wild-type receptor was also truncated after residue 903. This pcDNA 3.1 construct was made using XhoI and ApaI restriction enzymes to generate a 450-bp fragment from a XhoI restriction site at nucleotide 534 of the headless-T903 construct to an ApaI restriction site just beyond the 3′-end of the insert (nucleotide 997 of the empty vector, pcDNA3.1+). This fragment was then ligated into the wild-type CaR construct that had been predigested with XhoI and ApaI. Both sites occur uniquely in the human wild-type CaR construct in pcDNA3.1(+). The accuracy of the junctions was confirmed by DNA sequencing as described above. Site-directed Mutagenesis—Site-directed mutagenesis was performed using the Stratagene QuikChange™ kit according to the manufacturer's instructions. Briefly, a pair of complementary primers of 25–35 bases was designed for each mutagenesis with the mutation placed at the middle of the primers (sequences of primers available from authors on request). The template hCaR in pcDNA3.1(+) was amplified using Pfu DNA polymerase (Stratagene) with these primers for 12–16 cycles in a DNA thermal cycler (PerkinElmer). After digestion of the template DNA with DpnI (New England Biolabs), the amplified mutant DNA was transformed into Escherichia coli (DH5α or XL1-Blue). The incorporation of the desired mutations and absence of other mutations were confirmed by automated DNA sequencing (Australian Genome Research Facility). Transient Transfection of Wild-type, Mutants, and Chimeric Receptors in HEK293 Cells—Transfection-grade DNA was prepared using the High-speed™ midi kit (Qiagen). HEK293 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum. When the cells reached 90–95% confluence, they were with wild-type CaR, mutant CaR, or chimeric constructs using A of DNA was in Dulbecco's modified Eagle's with and to at for 20 to HEK293 cells in in the were performed in using of of each construct was with of the in as described previously J. N. Conigrave A.D. J. 2003; PubMed Scopus Google Scholar). of Wild-type, Mutants, and Chimeric Receptors in HEK293 cells were in and with of the wild-type CaR, mutant CaR, or chimeric constructs using according to the manufacturer's After cells were to and for a of was then in the of (Invitrogen) or were by for and studied by using of in used to HEK293 for analysis was performed as described previously J. N. Conigrave A.D. J. 2003; PubMed Scopus Google Scholar). analysis of amino and type-II activation of the CaR and CaR in Ca2+ were by after with HEK293 cells that had been with the wild-type CaR or one of the chimeric CaR constructs were cultured on in Dulbecco's modified Eagle's 10% fetal bovine in and with in containing 20 bovine 1 After the was and the cells were in for at cells were into a and placed in the of a as described previously Brown Conigrave A.D. J. 1996; PubMed Scopus Google Scholar). The control had the or 20 at and of and its using for was performed as described previously Brown Conigrave A.D. J. 1996; PubMed Scopus Google Scholar). for Ca2+ were expressed as or to Ca2+ using a Brown Conigrave A.D. J. 1996; PubMed Scopus Google Scholar). and Ca2+ data were expressed as and to the of the as shown in a C extracellular Ca2+ (in of Ca2+ that a and data are expressed as of of and NPS on HEK293 of HEK293 cells that expressed the wild-type human calcium-sensing receptor were After cells from exhibited extracellular Ca2+ that was allosterically activated by l-amino acids or the type-II calcimimetic NPS 1 and of HEK293 cells to l-Phe and at in a of the of the effects of various and acids was consistent with data (1Conigrave A.D. Quinn S.J. Brown E.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4814-4819Crossref PubMed Scopus (427) Google of amino acid sensing by the wild-type CaR or chimeric receptors that contained the VFT domain of the for in a of and NPS on Chimeric Receptors of mGlu-1 chimeric receptor constructs that were of an intact CaR VFT domain and mGlu-1 transmembrane domain were expressed in HEK293 cells and functionally in cells. The constructs (listed in the VFT domain/Cys-rich were CaR/CaR/mGlu-1 and CaR/mGlu-1/mGlu-1. In HEK293 cells that expressed these constructs exhibited extracellular of Ca2+ and In receptor constructs were activated by l-amino acids and but not by the type-II calcimimetic NPS for data not Thus, of the transmembrane domain of CaR with the mGlu-1 transmembrane domain in loss of the to type-II but not l-amino The for l-amino acids to for the wild-type and and chimeric receptor constructs of and NPS on a Headless (9Zhao X.M. Hauache O. Goldsmith P.K. Collins R. Spiegel A.M. FEBS Lett. 1999; 448: 180-184Crossref PubMed Scopus (56) Google Scholar) described a functionally headless CaR construct in which of the amino acid residues of the N-terminal extracellular domain VFT and Cys-rich were with a N terminus containing the bovine rhodopsin signal peptide In the C terminus of headless construct was truncated after residue to This construct and a corresponding wild-type receptor construct that was also truncated after residue were in the current study and used to the hypothesis that the VFT domain of CaR is required for l-amino acid sensing. In HEK293 cells with alone exhibited a to that was to l-Phe The in cells with alone and in cells was of that in cells with the wild-type CaR The wild-type CaR, the CaR truncated after residue T903 and the headless-T903 CaR exhibited in Ca2+ when with cells with alone The type-II R-467, the of three the to The finding that retains in the headless receptor is consistent with the that the transmembrane domain region of CaR binding sites for type-II O.M. Hu J. Ray K. Xie R. Jacobson K.A. Spiegel A.M. Endocrinology. 2000; 141: 4156-4163Crossref PubMed Google Scholar) as as (12Ray K. Northup J. J. Biol. Chem. 2002; 277: 18908-18913Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). However, exhibited differential on the control and headless 1 and activated the wild-type CaR and T903 CaR, activated the headless-T903 CaR loss of The headless-T903 CaR, T903 CaR or the wild-type CaR, was to l-Phe at and and to 100 In l-Phe failed to the of the headless-T903 CaR to 1 or Thus, the headless CaR retained to R-467, with but was to l-Phe at its (1Conigrave A.D. Quinn S.J. Brown E.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4814-4819Crossref PubMed Scopus (427) Google of l-Phe and NPS on and headless-T903 and for data are expressed as of the wild-type control The data were obtained in for constructs and for data are expressed as of the wild-type control The data were obtained in for constructs and for data are expressed as of the wild-type control The data were obtained in for constructs and l-Phe l-Phe The data are expressed as of the wild-type control The data were obtained in for constructs and in a The current has demonstrated that the VFT domain of CaR alone is required for l-amino acid sensing. the headless-T903 CaR exhibited to l-Phe at in (1Conigrave A.D. Quinn S.J. Brown E.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4814-4819Crossref PubMed Scopus (427) Google Scholar). Furthermore, the of CaR for aromatic and aliphatic but not or amino acids was by of the extracellular Cys-rich region of CaR or its transmembrane region and C-terminal tail with the corresponding from the rat mGlu-1 The data clearly the that are required for sensing by l-amino acids and type-II whose binding site has been previously to the transmembrane region (9Zhao X.M. Hauache O. Goldsmith P.K. Collins R. Spiegel A.M. FEBS Lett. 1999; 448: 180-184Crossref PubMed Scopus (56) Google Scholar, 10Hauache O.M. Hu J. Ray K. Xie R. Jacobson K.A. Spiegel A.M. Endocrinology. 2000; 141: 4156-4163Crossref PubMed Google Scholar, 11Hu J. Reyes-Cruz G. Chen W. Jacobson K.A. Spiegel A.M. J. Biol. Chem. 2002; 277: 46622-46631Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, K. Northup J. J. Biol. Chem. 2002; 277: 18908-18913Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The finding that the VFT and none of the other domains, is required for l-amino acid sensing in that amino acids the CaR functional M. Trivedi S. Kifor O. Quinn S.J. Brown E.M. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google it is likely that the l-amino acid binding site is in the VFT domain and not on a In amino acid binding in the VFT domain a for the that the l-amino acid and type-II calcimimetic binding sites are distinct and (6Zhang Z. Jiang Y. Quinn S.J. Krapcho K. Nemeth E.F. Bai M. J. Biol. Chem. 2002; 277: 33736-33741Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and that of the triple serine in the VFT domain amino acid sensing (5Zhang 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 (100) Google Scholar). analysis as for the rat mGlu-1 VFT domain N. Y. Y. M. Nakanishi S. H. K. 2000; PubMed Scopus Google Scholar, N. N. H. K. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar) or binding analysis as for amino acid binding to the VFT domain of the receptor Y. M. N. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The headless-T903 CaR used in the current study has been shown previously to to in the of the (9Zhao X.M. Hauache O. Goldsmith P.K. Collins R. Spiegel A.M. FEBS Lett. 1999; 448: 180-184Crossref PubMed Scopus (56) Google Scholar, 10Hauache O.M. Hu J. Ray K. Xie R. Jacobson K.A. Spiegel A.M. Endocrinology. 2000; 141: 4156-4163Crossref PubMed Google Scholar). In the current study, was with R-467. However, using to in Ca2+ the headless-T903 CaR was to in extracellular Ca2+ in the absence of This effect not to Ca2+ the in headless-T903 cells was that in cells or in cells with alone It is not in the absence of R-467, the headless-T903 CaR Ca2+ of Ca2+ from In of the that it Ca2+ of have shown that in the absence of the of the headless-T903 CaR on C is O.M. Hu J. Ray K. Xie R. Jacobson K.A. Spiegel A.M. Endocrinology. 2000; 141: 4156-4163Crossref PubMed Google Scholar, 11Hu J. Reyes-Cruz G. Chen W. Jacobson K.A. Spiegel A.M. J. Biol. Chem. 2002; 277: 46622-46631Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, K. Northup J. J. Biol. Chem. 2002; 277: 18908-18913Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). to was an in the of the type-II calcimimetic with to the headless-T903 CaR when with its wild-type and T903 but not 1 the of the headless is an allosteric that is in the absence of extracellular Ca2+ The in have from a to activation or from a of the transmembrane that the binding site K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of the binding sites for extracellular Ca2+ on headless receptors indicate that sensing of Ca2+ and by the transmembrane region alone J. Reyes-Cruz G. Chen W. Jacobson K.A. Spiegel A.M. J. Biol. Chem. 2002; 277: 46622-46631Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, K. Northup J. J. Biol. Chem. 2002; 277: 18908-18913Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). This is by the analysis of the headless receptor in the current study the other on chimeric receptors in which the transmembrane region of CaR is by the mGlu-1 transmembrane region (8Hammerland L.G. Krapcho K.J. Garrett J.E. Alasti N. Hung B.C. Simin R.T. Levinthal C. Nemeth E.F. Fuller F.H. Mol. Pharmacol. 1999; 55: 642-648PubMed Google Scholar, 10Hauache O.M. Hu J. Ray K. Xie R. Jacobson K.A. Spiegel A.M. Endocrinology. 2000; 141: 4156-4163Crossref PubMed Google Scholar) indicate that sensing is The that the CaR transmembrane region is not necessary for Ca2+ sensing is also by the current study and to one of these Ca2+ binding is by the VFT domain of CaR and its transmembrane Ca2+ binding to the transmembrane regions of the CaR and mGlu-1 This is consistent with for Ca2+ sensing by mGlu-1 Y. Y. PubMed Scopus Google Scholar). is required to HEK293 cells that expressed the CaR were to l-Phe cells that expressed it This was in the absence of but was in its and in the of R-467, l-Phe the for in HEK293 cells that expressed the wild-type CaR from to In the of R-467, l-Phe the for by in cells that expressed the wild-type or T903 CaR and and The of the in to l-Phe in and cells is not However, it in receptor or the e.g. from extracellular Ca2+ is a of the CaR, l-amino acids and the type-II calcimimetic NPS are allosteric Thus, the activating effects of l-amino acids and type-II the of a of which to the VFT is a an allosteric of Furthermore, the between glutamate and Ca2+ on mGlu-1 R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). on the of the activation for these receptors is clearly In the of the CaR, for it necessary to l-amino acids Ca2+ binding or the of the In study has indicated that the VFT but none of its other domains, is necessary for l-amino acid sensing by with other sub-group C receptors, the findings that the VFT domain is the likely site of amino acid binding. We Dr. Mei Bai and Professor Edward Brown of Brigham and Womens Hospital, Boston, and Professor S. University, for key DNA Dr. Bai provided a of the We Jacobson for