A Region in the Seven-transmembrane Domain of the Human Ca2+ Receptor Critical for Response to Ca2+

Abstract

Of 12 naturally occurring, activating mutations in the seven-transmembrane (7TM) domain of the human Ca2+ receptor (CaR) identified previously in subjects with autosomal dominant hypocalcemia (ADH), five appear at the junction of TM helices 6 and 7 between residue Ile819 and Glu837. After identifying a sixth activating mutation in this region, V836L, in an ADH patient, we studied the remaining residues in this region to determine whether they are potential sites for activating mutations. Alanine-scanning mutagenesis revealed five additional residues in this region that when substituted by alanine led to CaR activation. We also found that, whereas E837A did not activate the receptor, E837D and E837K mutations did. Thus, region Ile819–Glu837 of the 7TM domain represents a “hot spot” for naturally occurring, activating mutations of the receptor, and most of the residues in this region apparently maintain the 7TM domain in its inactive configuration. Unique among the residues in this region, Pro823, which is highly conserved in family 3 of the G protein-coupled receptors, when mutated to either alanine or glycine, despite good expression severely impaired CaR activation by Ca2+. Both the P823A mutation and NPS 2143, a negative allosteric modulator that acts on the 7TM through a critical interaction with Glu837, blocked activation of the CaR by various ADH mutations. These results suggest that the 7TM domain region Ile819–Glu837 plays a key role in CaR activation by Ca2+. The implications of our finding that NPS 2143 corrects the molecular defect of ADH mutations for treatment of this disease are also discussed. Of 12 naturally occurring, activating mutations in the seven-transmembrane (7TM) domain of the human Ca2+ receptor (CaR) identified previously in subjects with autosomal dominant hypocalcemia (ADH), five appear at the junction of TM helices 6 and 7 between residue Ile819 and Glu837. After identifying a sixth activating mutation in this region, V836L, in an ADH patient, we studied the remaining residues in this region to determine whether they are potential sites for activating mutations. Alanine-scanning mutagenesis revealed five additional residues in this region that when substituted by alanine led to CaR activation. We also found that, whereas E837A did not activate the receptor, E837D and E837K mutations did. Thus, region Ile819–Glu837 of the 7TM domain represents a “hot spot” for naturally occurring, activating mutations of the receptor, and most of the residues in this region apparently maintain the 7TM domain in its inactive configuration. Unique among the residues in this region, Pro823, which is highly conserved in family 3 of the G protein-coupled receptors, when mutated to either alanine or glycine, despite good expression severely impaired CaR activation by Ca2+. Both the P823A mutation and NPS 2143, a negative allosteric modulator that acts on the 7TM through a critical interaction with Glu837, blocked activation of the CaR by various ADH mutations. These results suggest that the 7TM domain region Ile819–Glu837 plays a key role in CaR activation by Ca2+. The implications of our finding that NPS 2143 corrects the molecular defect of ADH mutations for treatment of this disease are also discussed. The extracellular Ca2+ receptor (CaR) 1The abbreviations used are: CaR, extracellular Ca2+ receptor; GPCR, G protein-coupled receptor; VFT, Venus's flytrap; 7TM, seven-transmembrane domain; ADH, autosomal dominant hypocalcemia; hCaR, human extracellular Ca2+ receptor; DMEM, Dulbecco's modified Eagle's medium; PI, phosphoinositide; PIPES, 1,4-piperazinediethane-sulfonic acid; [Ca2+]o, extracellular calcium ion; WT, wild type. is a member of family 3 of the G protein-coupled receptor (GPCR) superfamily (1Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1230) Google Scholar, 2Hu J. Spiegel A.M. Trends Endocrinol. Metab. 2003; 14: 282-288Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Family 3 members are typically characterized by a large extracellular amino terminus comprised of Venus's flytrap (VFT)-like and cysteine-rich domains, in addition to the signature seven-transmembrane (7TM) domain of all GPCRs. The three-dimensional structure of the VFT domain of the rat metabotropic glutamate type 1 receptor showed that it is an intermolecular disulfide-linked dimer (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 (1110) Google Scholar). Agonist binding to the cleft of the VFT leads to VFT closure and a 70° rotation of one monomer relative to the other about an axis perpendicular to the dimer interface. How this agonist-induced conformational change in the VFT domain is transmitted to the 7TM domain to cause CaR activation is a key unanswered question. Naturally occurring CaR mutations identified in subjects with familial hypocalciuric hypercalcemia and autosomal dominant hypocalcemia (ADH), in addition to validating the physiologic importance of the CaR in extracellular Ca2+ homeostasis (4Brown E.M. MacLeod R.J. Physiol. Rev. 2001; 81: 239-297Crossref PubMed Scopus (1230) Google Scholar), provide unique insights into CaR structure and function (2Hu J. Spiegel A.M. Trends Endocrinol. Metab. 2003; 14: 282-288Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Missense, activating mutations identified in ADH could help identify regions of the CaR particularly important for receptor activation. More than 30 such activating mutations have been identified to date in subjects with ADH (5Pidasheva S. D'Souza-Li L. Canaff L. Cole D.E. Hendy G.N. Hum. Mutat. 2004; 24: 107-111Crossref PubMed Scopus (127) Google Scholar), and these are non-randomly distributed (Fig. 1). Ten ADH mutations cluster between residues 116–131 in a part of the VFT domain, loop 2, which is the site of intermolecular disulfide-linked dimerization (6Ray K. Hauschild B.C. Steinbach P.J. Goldsmith P.K. Hauache O. Spiegel A.M. J. Biol. Chem. 1999; 274: 27642-27650Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar) The corresponding loop in the glutamate type 1 receptor three-dimensional structure represents a “switch region” in that it is disordered in the active, agonist-bound form of the VFT, but in the inactive form residues equivalent to 117–123 of the CaR become α-helical. We suggested that ADH loop2 mutations cause activation of the CaR by facilitating the agonist-induced conformational change in the VFT domain (2Hu J. Spiegel A.M. Trends Endocrinol. Metab. 2003; 14: 282-288Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar).Fig. 1Schematic diagram showing amino acid sequence of the hCaR with boundaries of transmembrane helices predicted recently by Petrel et al. (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) based on alignment with rhodopsin rather than those predicted earlier by Garrett et al. (27Garrett 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 (459) Google Scholar) previously used by us. The location of signal peptide, N-linked glycosylation sites, and the sequence of synthetic polypeptide used to raise monoclonal antibody ADD is indicated. All cysteines are shown in black background. The beginning and end of the VFT domain and the four loops in lobe 1 of the VFT are indicated. Naturally occurring activating mutations identified previously in the hCaR and the V836L mutation reported herein as well as the inactivating V817I mutation (boxed) are indicated. Glu837, shown to be involved in binding of the allosteric modulators NPS R-568 and NPS 2143, and Pro823, reported herein to be critical for the function of the receptor, are shown in bold print. The two regions with clustering ADH mutations, residues 116–131 and residues 819–837, are shaded.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the present work, we identified a novel ADH mutation, V836L, in the 7TM domain and noted that it is located in another region of clustered ADH mutations, residues 819–837, at the junction of TM6 and TM7 proximal to the extracellular surface of the plasma membrane (Fig. 1). Moreover, we had previously identified Glu837 in TM7 as a residue critical for action of positive allosteric modulators of the CaR (7Hu 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 (85) Google Scholar). This suggested to us that the TM6/TM7 junction may also represent a switch region that is critically involved in the activation mechanism of the CaR. To begin to evaluate this possibility, we performed alanine-scanning mutagenesis of residues in this region. We identified multiple additional TM6/TM7 residues that lead to receptor activation upon substitution of alanine and a unique mutation in TM6, P823A, that blocks CaR activation, mimicking the effect of a negative allosteric modulator. Site-directed Mutagenesis of the hCaR—The full-length human CaR (hCaR) cDNA cloned in the pCR3.1 expression vector was described previously (8Ray K. Fan G.F. Goldsmith P.K. Spiegel A.M. J. Biol. Chem. 1997; 272: 31355-31361Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Site-directed mutagenesis was performed using the QuikChange™ site-directed mutagenesis kit (Stratagene, Inc., La Jolla, CA) according to the manufacturer's instructions. Parental hCaR cDNA in pCR3.1 vector was amplified using pfu Turbo DNA polymerase with mutagenic oligonucleotide primers (sequences available on request) for 16 cycles in a DNA thermal cycler (PerkinElmer Life Sciences). After digestion of the parental DNA with DpnI for 1 h, the amplified DNA with incorporated nucleotide substitution was transformed into Escherichia coli (DH-5α strain). The sequence of mutant receptors was confirmed by automated DNA sequencing using a dRho-damine Terminator cycle sequencing kit and ABI PRISM-373A DNA sequencer (PE Applied Biosystems, Foster City, CA). Transient Transfection of Wild Type and Mutant Receptors in HEK-293 Cells—Transfections were performed using 12 μg of plasmid DNA for each transfection in a 75-cm2 flask of HEK-293 cells. DNA was diluted in serum-free DMEM (BioFluids, Inc., Rockville, MD) mixed with diluted Lipofectamine (Invitrogen), and the mixture was incubated at room temperature for 30 min. The DNA-Lipofectamine complex was further diluted in 6 ml of serum-free DMEM and was added to 80% confluent HEK-293 cells plated in 75-cm2 flasks. After 5 h of incubation, 15 ml of complete DMEM containing 10% fetal bovine serum (BioFluids, Inc.) was added. 24 h after transfection, transfected cells were split and cultured in complete DMEM. Phosphoinositide (PI) Hydrolysis Assay—PI hydrolysis assay has been described previously (8Ray K. Fan G.F. Goldsmith P.K. Spiegel A.M. J. Biol. Chem. 1997; 272: 31355-31361Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Briefly, 24 h after transfection, transfected cells from a confluent 75-cm2 flask were split. Typically oneeighth of cells were plated in one well in a 6-well plate, and whole cell lysate was prepared 48 h post-transfection for Western blot assay. The remaining cells were plated in two 12-well plates in complete DMEM medium containing 3.0 μCi/ml of 3H-labeled myoinositol (PerkinElmer Life Sciences) and cultured for another 24 h. Culture medium was replaced by 1× PI buffer (60 mm NaCl, 2.5 mm KCl, 2.8 mm glucose, 0.2 mm MgCl2, 10 mm LiCl in 12.5 mm PIPES, pH 7.2) and incubated for 1 h at 37 °C. After removal of PI buffer, cells were incubated for an additional 1 h with different concentrations of Ca2+ in 1× PI buffer with or without 1 μm calcimimetic compound NPS R-568 or calcilytic compound NPS 2143. The reactions were terminated by the addition of 1 ml of acid-methanol (1:1,000 v/v) per well. Total inositol phosphates were purified by chromatography on Dowex 1-X8 columns, and radioactivity for each sample was counted with a liquid scintillation counter. Graphs of concentration dependence for stimulation of PI hydrolysis by [Ca2+]o for each transfection were drawn by using GraphPad Prism version 2.0 software. Each value on a curve is the mean of duplicate determinations unless otherwise indicated. Graphs shown in this paper were representative ones from at least three independent experiments. Synthetic Chemistry—The method for the synthesis of NPS R-568 was reported previously (9Hauache O.M. Hu J. Ray K. Xie R. Jacobson K.A. Spiegel A.M. 2000; PubMed Google Scholar). The synthesis of of NPS 2143 was the of and M. J. R. N. M. L. and M. S. Scholar). of the of in and treatment with the in than substitution with by the of the in was through a substitution of the and with in good the of and in NPS 2143, which was as the in a of acid T. T. T. K. K. T. S. N. K. K. Chem. PubMed Scopus Google Scholar). cells in 6-well plates were with and on in buffer containing mm pH mm NaCl, 1 mm and added mixture Applied of full-length receptors, μg of per with was on The on the were membrane and incubated with of monoclonal antibody ADD a synthetic corresponding to residues of hCaR the membrane was incubated with a antibody to at a of The hCaR was with an and of Naturally CaR in the TM6/TM7 identified a novel mutation V836L, in a with To the of this and five previously reported ADH mutations located in this region of the CaR T. T. T. R. N. H. N. H. S. Y. N. K. T. Yamamoto M. J. Endocrinol. Metab. 2002; PubMed Scopus Google Scholar, M. T. Brown E.M. T. T. J. 2004; PubMed Scopus Google Scholar, D. M. Y. T. Scholar, A. M. J.P. M. Y. Brown E.M. M. J. Endocrinol. Metab. 2001; PubMed Scopus Google Scholar, L. Canaff L. M. M. Brown E.M. Cole D.E. Hendy G.N. J. Endocrinol. Metab. 2002; PubMed Scopus (112) Google Scholar), we PI hydrolysis as a function of extracellular Ca2+ concentration and CaR expression by in HEK-293 cells transfected with and mutant CaR and all mutant receptors showed expression on (Fig. 2, of a shown previously (8Ray K. Fan G.F. Goldsmith P.K. Spiegel A.M. J. Biol. Chem. 1997; 272: 31355-31361Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) to to an form of the CaR at and a cell form at The V836L mutation led to of the CaR to Ca2+ activation with as did to the five other ADH mutations, as previously for other activating mutations identified in ADH, at concentrations of Ca2+ was than that of CaR (Fig. 2, and for [Ca2+]o stimulation of PI hydrolysis of wild type receptor and receptors containing activating mutations in the region of residues shown are of three to occurring activating mutation activating mutation in a and of Alanine-scanning of CaR in the TM6/TM7 evaluate the importance of other residues in the TM6/TM7 junction region in of CaR to activation by we mutated residues from from the identified as sites of ADH to alanine was mutated to and expression and of these residues when mutated to and CaR to Ca2+ (Fig. and substitution for other residues in this region and substitution for change in Ca2+ whereas alanine substitution for a in not substitution for Glu837, as we reported did not Ca2+ (7Hu 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 (85) Google Scholar). acid and substitution for Glu837 CaR to Ca2+ (Fig. and of P823A as a Unique CaR substitution for led to a in Ca2+ despite cell surface expression to the on the that is with that of CaR (Fig. substitution for also severely impaired CaR activation by the mutant was well at the cell surface not we studied three naturally occurring inactivating mutations identified in familial hypocalciuric V817I to in TM6, in and in The two mutations showed at 30 mm Ca2+ and on the cell surface expression not the V817I mutant showed expression on with CaR and showed a in to its that of the P823A mutant (Fig. NPS a positive allosteric modulator that Ca2+ of CaR, was to the of the P823A mutant but did not its to (Fig. the P823A mutant good CaR expression but severely Ca2+ we were in the effect of this mutation when on activating CaR mutations. of P823A with activating mutations in the VFT domain such as (Fig. or not showed severely Ca2+ with either or the activating P823A with activating mutations in the 7TM domain such as V836L (Fig. or not Ca2+ but not to the as for VFT domain mutations. In P823A with a activating mutation, and activation but did not activation (Fig. The in Ca2+ with these was not a function of receptor expression as by not concentration dependence for [Ca2+]o stimulation of PI hydrolysis of CaR in transfected HEK-293 cells hCaR and P823A mutant hCaR with or without treatment of 1 μm NPS concentration dependence for [Ca2+]o stimulation of PI hydrolysis of CaR in transfected HEK-293 cells hCaR, V836L, and and and and for of results are as in the to Each value on a curve is the mean of duplicate shown are representative of three Large Image Figure ViewerDownload Hi-res image Download (PPT) of a on Ca2+ of and Mutant 2143 is a negative allosteric modulator that CaR to Ca2+ (Fig. E.F. J. Endocrinol. 2002; PubMed Scopus Google Scholar). the positive allosteric modulator NPS NPS 2143 critically on Glu837 for its action the of to the glutamate (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, L. K. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). mutation of Glu837 to or to Ca2+ (Fig. but whereas E837D to activation by NPS R-568 and by NPS 2143, E837K to either allosteric modulator (Fig. We the effect of NPS 2143 on a of other activating mutations of the CaR to determine whether they were to by the negative allosteric modulator. VFT domain activating such as (Fig. (Fig. and as well as additional 7TM domain activating mutations such as V836L (Fig. all were by NPS 2143. In the activating mutant was not by NPS 2143 (Fig. dependence for [Ca2+]o stimulation of PI hydrolysis of CaR in transfected HEK-293 cells V836L and mutant with or without treatment of 1 μm NPS 2143. and for of results are as in the to that the is at 30 mm at mm V836L at mm and at 30 mm without NPS 2143. Each value on a curve is the mean of duplicate shown are representative of three Large Image Figure ViewerDownload Hi-res image Download (PPT) We J. S. G. Spiegel A.M. J. 2004; PubMed Scopus Google Scholar) and (5Pidasheva S. D'Souza-Li L. Canaff L. Cole D.E. Hendy G.N. Hum. Mutat. 2004; 24: 107-111Crossref PubMed Scopus (127) Google Scholar, in 2Hu J. Spiegel A.M. Trends Endocrinol. Metab. 2003; 14: 282-288Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) have identified activating CaR mutations in subjects with of function CaR mutations identified in subjects with familial hypocalciuric the of which cause either of expression or receptor to the cell mutations ADH may provide into the mechanism of CaR activation. the novel V836L mutation we the 7TM domain ADH mutations one mutation each in and in and two in and four each in TM6 and Of the cluster at the junction of TM6 and TM7 In addition to these naturally occurring activating mutations, we showed that five other residues in this region to activation by Ca2+ when mutated to Moreover, the remaining residues in this region could also be potential sites for activating mutation, as we found that whereas E837A was not E837D and E837K mutations did activate the our of not a role for other of the 7TM domain, these results suggest that a part of this domain, the extracellular surface proximal of TM6 and TM7 may a key role in CaR activation. for activation are based on the structure of bovine rhodopsin K. Kumasaka T. T. H. T. Yamamoto M. M. 2000; PubMed Scopus Google Scholar). is to in TM7 and is by residues in and and a of the 7TM domain in an inactive How the rhodopsin has not been but a that of TM6 is a key in the activation mechanism M. A. M. S. A. 2004; PubMed Scopus (127) Google Scholar). highly conserved in family 1 in TM6 in L. 2001; PubMed Scopus Google acts as a for rotation of the sequence between the 7TM of family 1 family 3 one be in from the rhodopsin three-dimensional structure to that of family 3 GPCRs. it is to that a rotation of TM6 in the CaR acts as part of a mechanism upon receptor activation. key role for in such a TM6 rotation is suggested by the of the P823A good expression of the mutant receptor, its to be by Ca2+ is positive allosteric modulator the Ca2+ of the mutant receptor but not to the P823A mutation blocked or the Ca2+ of activating VFT or 7TM mutations. is highly conserved in family 3 but is in a different location rather than than the in TM6 highly conserved in GPCRs. The of and TM in by et al. S. S. D. J.P. S. A. 2004; PubMed Scopus Google Scholar) suggested that to mutations not structure or function of in residues in other This not appear to be for in the CaR, and we suggest that mutation of this residue in other family 3 have The P823A mutation had not as the CaR negative allosteric NPS 2143. Both blocked the of and of activating CaR mutations but had effect on the mutation that among ADH mutations activation Hauache O. Goldsmith P.K. R. Spiegel A.M. 1999; PubMed Scopus Google Scholar). We and recently found by alanine substitution that residue Glu837 is critical for the action of allosteric modulators of the CaR with a such as NPS R-568 and NPS 2143 (7Hu 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 (85) Google Scholar, 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, L. K. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). was that a critical form between the glutamate and the in these We the of E837D and E837K that the negative at residue is for the of the CaR to NPS R-568 and NPS 2143. was when Glu837 was substituted by either a or How agonist-induced VFT closure and dimer rotation leads to activation of the 7TM domain of family 3 lead us to that the TM6/TM7 junction region plays a critical role in the CaR in its inactive of of the residues in this region, as well as the action of positive allosteric modulators binding to as NPS the in the 7TM domain, to Ca2+ activation. In negative allosteric modulators which also to Glu837 as NPS and the P823A mutation the inactive of the CaR by rotation of part of We suggest that the relative of the mutant to by NPS 2143 or by the P823A mutation the location of this mutation in The mutation may cause a change in the CaR domain that leads to G activation and is to by in the 7TM is the of NPS 2143 to the of all ADH mutations with the of ADH mutations, with the of the activating cause hypocalcemia and of and Ca2+ by the Ca2+ of the CaR in and treatment of ADH using and calcium is of the to allosteric modulators such as NPS 2143 the of of the molecular defect in ADH by the of the CaR to and Ca2+ allosteric CaR modulators are for in treatment of N. J. 2004; PubMed Scopus Google Scholar), but further in and in may be to evaluate such for a of hypocalcemia in subjects with We and for with of V836L mutation in the CaR of a with autosomal dominant