Dimerization of the Extracellular Calcium-sensing Receptor (CaR) on the Cell Surface of CaR-transfected HEK293 Cells

Abstract

The extracellular calcium (Ca2+o)-sensing receptor (CaR) is a G protein-coupled receptor that plays important roles in calcium homeostasis. In this study, we employed epitope tagging, cell-surface biotinylation, and immunoprecipitation techniques to demonstrate that the CaR is expressed mostly in the form of a dimer on the surface of transfected human embryonic kidney (HEK293) cells. Western analysis of cell-surface proteins under nonreducing conditions showed that the CaR exists in several forms with molecular masses greater than 200 kDa. Most of these high molecular mass forms of the receptor could be converted to a single monomeric species at 160 kDa under reducing conditions. This result suggests that the CaR forms dimers or even higher oligomers on the cell surface through intermolecular disulfide bonds that are sensitive to reducing agents. Consistent with this hypothesis, use of a cell-surface cross-linking agent substantially increases the proportion of the putative dimeric CaR at 280 kDa relative to the monomeric form of the receptor at 160 kDa under reducing conditions. Dimerization of the CaR in intact cells was further demonstrated when we co-transfected and co-immunoprecipitated the wild type, full-length receptor and a truncated form of the CaR lacking its cytoplasmic tail. Taken together, we conclude from these results that the functional CaR resides on the cell surface of transfected HEK293 cells in the form of a dimer. The extracellular calcium (Ca2+o)-sensing receptor (CaR) is a G protein-coupled receptor that plays important roles in calcium homeostasis. In this study, we employed epitope tagging, cell-surface biotinylation, and immunoprecipitation techniques to demonstrate that the CaR is expressed mostly in the form of a dimer on the surface of transfected human embryonic kidney (HEK293) cells. Western analysis of cell-surface proteins under nonreducing conditions showed that the CaR exists in several forms with molecular masses greater than 200 kDa. Most of these high molecular mass forms of the receptor could be converted to a single monomeric species at 160 kDa under reducing conditions. This result suggests that the CaR forms dimers or even higher oligomers on the cell surface through intermolecular disulfide bonds that are sensitive to reducing agents. Consistent with this hypothesis, use of a cell-surface cross-linking agent substantially increases the proportion of the putative dimeric CaR at 280 kDa relative to the monomeric form of the receptor at 160 kDa under reducing conditions. Dimerization of the CaR in intact cells was further demonstrated when we co-transfected and co-immunoprecipitated the wild type, full-length receptor and a truncated form of the CaR lacking its cytoplasmic tail. Taken together, we conclude from these results that the functional CaR resides on the cell surface of transfected HEK293 cells in the form of a dimer. calcium (Ca2+o)-sensing receptor G protein-coupled receptor polyacrylamide gel electrophoresis dithiothreitol bis(sulfosuccinimidyl) suberate. The extracellular calcium (Ca2+o)-sensing receptor (CaR)1 is a G protein-coupled receptor (GPCR) (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 (2366) Google Scholar). Activation of the CaR by elevated levels of Ca2+o stimulates phospholipase C and raises the cytosolic calcium concentration (Ca2+i). The physiological importance of the CaR in determining the level at which Ca2+o is set in vivo has been documented by the characterization of human syndromes resulting from activating or inactivating mutations of the CaR, which alter the function of parathyroid and kidney so as to produce hypo- or hypercalcemia, respectively (2Pollak 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 (908) Google Scholar, 3Pollak M.R. Brown E.M. Estep H.L. McLaine P.N. Kifor O. Park J. Hebert S.C. Seidman C.E. Seidman J.G. Nat. Genet. 1994; 8: 303-307Crossref PubMed Scopus (533) Google Scholar). The function and expression of the CaR have been assessed in transiently transfected human embryonic kidney cells (HEK293) (4Bai 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 (380) Google Scholar, 5Pearce S.H. Bai M. Quinn S.J. Kifor O. Brown E.M. Thakker R.V. J. Clin. Invest. 1996; 98: 1860-1866Crossref PubMed Scopus (168) Google Scholar, 6Pearce S.H. Williamson C. Kifor O. Bai M. Coulthard M.G. Davies M. Lewis-Barned N. McCredie D. Powell H. Kendall-Taylor P. Brown E.M. Thakker R.V. New Engl. J. Med. 1996; 335: 1115-1122Crossref PubMed Scopus (503) Google Scholar, 7Bai M. Pearce S.H. Kifor O. Trivedi S. Stauffer U.G. Thakker R.V. Brown E.M. Steinmann B. J. Clin. Invest. 1997; 99: 88-96Crossref PubMed Scopus (181) Google Scholar, 8De Luca F. Ray K. Mancilla E.E. Fan G.F. Winer K.K. Gore P. Spiegel A.M. Baron J. J. Clin. Endocrinol. Metab. 1997; 82: 2710-2715Crossref PubMed Scopus (95) Google Scholar, 9Bai M. Janicic N. Trivedi S. Quinn S.J. Cole D.E.C. Brown E.M. Hendy G.N. J. Clin. Invest. 1997; 99: 1917-1925Crossref PubMed Scopus (78) Google Scholar). CaR-transfected HEK293 cells respond to Ca2+o with an EC50 of 4.1 ± 0.1 mm (the effective concentration of Ca2+o producing one-half of the maximal Ca2+i response). The concentration-response curve for the Ca2+o-elicited Ca2+i responses in CaR-transfected HEK293 cells exhibits a relatively high Hill coefficient of about 3. This large Hill coefficient suggests a substantial degree of positive cooperativity in the binding of ligand and/or G protein by the CaR, which may involve intra- or intermolecular interactions (e.g. via formation of homodimers); namely, binding of the first ligand or G protein increases the affinity of the second ligand or G protein. Intermolecular interactions leading to cooperativity, for example, could result from dimerization of GPCRs (e.g. as for the muscarinic receptor and rhodopsin), which has been suggested as the molecular basis for cooperativity in the binding of their respective agonists (10Potter L.T. Ballesteros L.A. Bichajian L.H. Ferrendelli C.A. Fisher A. Hanchett H.E. Zhang R. Mol. Pharmacol. 1991; 39: 211-221PubMed Google Scholar, 11Hirschberg B.T. Schimerlik M.I. J. Biol. Chem. 1994; 269: 26127-26135Abstract Full Text PDF PubMed Google Scholar) or G proteins (12Wessling-Resnick M. Johnson G.L. J. Biol. Chem. 1987; 262: 3697-3705Abstract Full Text PDF PubMed Google Scholar, 13Willardson B.M. Pou B. Yoshida T. Bitensky M.W. J. Biol. Chem. 1993; 268: 6371-6382Abstract Full Text PDF PubMed Google Scholar). Our earlier biochemical studies had shown the presence of considerable amounts of CaR-specific, high molecular mass immunoreactivity by Western analysis, in addition to the putative monomeric form of the receptor, in membrane proteins prepared from both CaR-transfected HEK293 cells and native parathyroid cells (4Bai 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 (380) Google Scholar). These high molecular mass species likely correspond to dimeric and trimeric CaRs. The presence of dimeric forms of the CaR has also recently been described in detergent extracts prepared with the inner medulla of the rat kidney (14Ward D.T. Brown E.M. Harris H.W. J. Biol. Chem. 1998; 273: 14476-14483Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). A structurally related GPCR, mGluR5, forms homodimers via intermolecular disulfide linkages within the amino-terminal extracellular domain. The extracellular domains of mGluR5 and the CaR share 17 cysteines in equivalent positions, raising the possibility that the CaR could also dimerize in this manner (15Romano C. Yang W.L. O'Malley K.L. J. Biol. Chem. 1996; 271: 28612-28616Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). In addition, several less structurally related GPCRs have been demonstrated to form homodimers, including the muscarinic receptor (16Avissar S. Amitai G. Sokolovsky M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 156-159Crossref PubMed Scopus (88) Google Scholar), β2-adrenergic receptor (17Venter J.C. Fraser C.M. Fed. Proc. 1983; 42: 273-278PubMed Google Scholar, 18Hebert T.E. Moffett S. Morello J.P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. Chem. 1996; 271: 16384-16392Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar), glucagon receptor (19Herberg J.T. Codina J. Rich K.A. Rojas F.J. Iyengar R. J. Biol. Chem. 1984; 259: 9285-9294Abstract Full Text PDF PubMed Google Scholar), and δ-opioid receptor (20Cvejic S. Devi L.A. J. Biol. Chem. 1997; 272: 26959-26964Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar). A noncovalent hydrophobic dimerization motif has been suggested to mediate homodimerization of the β2-adrenergic receptor (18Hebert T.E. Moffett S. Morello J.P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. Chem. 1996; 271: 16384-16392Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar), which was also present in the CaR. Therefore, the CaR can potentially dimerize through two distinct types of intermolecular interactions, i.e.noncovalent hydrophobic interactions and formation of covalent intermolecular disulfide bonds. In this report, using molecular and biochemical approaches, we show that the functional CaR normally resides on the cell surface mostly as a homodimer. The unusually high degree of cooperativity in the activation of the CaR by its polycationic agonists suggests that dimerization of the CaR is likely to have functional implications. Site-directed mutagenesis was performed using the approach described by Kunkel (21Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4900) Google Scholar). The dut-1 ung-1 strain of Escherichia coli, CJ236, was transformed with cassette VI of a reconstructed CaR containing unique restriction sites introduced into the wild type human CaR as silent point mutations as described previously (4Bai 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 (380) Google Scholar). Uracil-containing, single-stranded DNA was produced by infecting the cells with the helper phage, VCSM13. The single-stranded DNA was then annealed to a mutagenesis primer, which contained a stop codon at the desired position and was flanked on both sides by a wild type sequence. The primer was then extended around the entire single-stranded DNA and ligated to generate closed circular heteroduplex DNA. DH5α-competent cells were transformed with the DNA heteroduplex, and incorporation of the desired mutation was confirmed in all cases by sequencing the entire cassette. Finally, the mutated cassette VIs were cloned into the XhoI and XbaI sites of the reconstructed CaR (4Bai 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 (380) Google Scholar). Cassette III in each of the mutant receptors was replaced with that in the Flag-tagged wild type receptor (9Bai M. Janicic N. Trivedi S. Quinn S.J. Cole D.E.C. Brown E.M. Hendy G.N. J. Clin. Invest. 1997; 99: 1917-1925Crossref PubMed Scopus (78) Google Scholar) by double restriction digestion with AflII and NheI followed by ligation of the larger restriction fragments containing point mutations and the smaller restriction fragment containing the Flag-tag. The DNA for transfection was prepared using the Midi Plasmid Kit (QIAGEN). LipofectAMINE (Life Technologies, Inc.) was employed as a DNA carrier for transfection (22Hawley-Nelson P. Ciccarone V. Gebeyehu G. Jessee J. Felgner P.L. Focus. 1993; 15: 73-79Google Scholar) according to the manufacturer's procedures. The HEK293 cells used for transient transfection were provided by NPS Pharmaceuticals, Inc. (Salt Lake City, UT) and cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with 10% fetal bovine serum The was prepared by DNA and LipofectAMINE in (Life Technologies, Inc.) and the at for The was then with and to HEK293 cells on mm using of DNA of or in using of DNA protein for immunoprecipitation and Western of at equivalent amounts of with fetal bovine serum were to the medium the transfected and the was replaced with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at The expressed receptor protein was the of of two each of the two were and used to HEK293 cells. to cell intact HEK293 cells transiently transfected with Flag-tagged CaR were with and with mm a at with for to the proteins on the cell The was then by the cells in for cross-linking we an of a into the with The and/or HEK293 cells were with and with mm mm mm mm including and immunoprecipitation at was by the cell at for at The were as cell The protein concentration was using the protein of of of immunoprecipitation and of containing of protein were to a The was at for of an was to the The was for an at of 10% (Life Technologies, Inc.) was to the for an at The was with immunoprecipitation and the species were in of electrophoresis at for The receptor of was by Western of proteins from CaR-transfected HEK293 cells was to polyacrylamide gel electrophoresis Nature. PubMed Scopus Google Scholar) using a of polyacrylamide The proteins on the gel were to a with the forms of the receptor present on the cell surface were using an followed by of the with an of the using the for the all forms of the CaR on the were using a a within the extracellular of the CaR, provided by and at followed by a and then an with HEK293 cells previously transfected with the CaR were for at with in containing mm mm mm bovine serum and and were then with a mm containing mm mm mm and bovine serum at for The were then in a containing the using a of the employed previously in this Quinn S.J. Brown E.M. J. 1991; Google Scholar). calcium was to the desired with of Ca2+o in of mm that were followed by mm a level of mm Ca2+o and mm a level of mm were at and with at ± through a The of was used to Ca2+i as described previously Quinn S.J. Brown E.M. J. 1991; Google Scholar). The EC50 for the wild type or truncated receptors in to of Ca2+o were from the EC50 for all of the and were expressed with the of the as the of of the EC50 was performed using analysis of or Google Scholar) of the described in the was performed at cell-surface expression of the CaR, we introduced the epitope into the extracellular of CaR. The function as as the and level of expression of the Flag-tagged CaR are to of the wild type receptor, as assessed by high and Western analysis, respectively (9Bai M. Janicic N. Trivedi S. Quinn S.J. Cole D.E.C. Brown E.M. Hendy G.N. J. Clin. Invest. 1997; 99: 1917-1925Crossref PubMed Scopus (78) Google Scholar). on the cell surface of HEK293 cells transiently transfected with the Flag-tagged CaR or were first with to the cells. The CaR was then with and with containing dithiothreitol or The of the native CaR were from disulfide bonds protein by including mm in the The CaR was first with an to the forms of the receptor expressed on the cell of the from the the CaR was with a (4Bai 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 (380) Google Scholar), to both cell-surface and forms of the using for we a at 160 kDa (the position of the monomeric in the with and with we with in two a were at the of the monomeric as as the putative dimeric and higher The surface forms of the monomeric receptor to the of the both of the were present in cells transfected with and Therefore, the are for the CaR. intermolecular disulfide we and the CaR, the receptor with lacking The monomeric CaR in was when conditions were employed we two 200 and kDa with as as higher molecular mass species and kDa for the wild type CaR In this the with in showed a of a high molecular mass species at 280 kDa (the position of the dimeric in addition to the monomeric This in with the of 200 and kDa in is likely that this in and a dimeric receptor than the at kDa in both are to the dimerization is we the intact cells with high mm in mm at for to surface nonreducing the of surface expression of the CaR were the in cells and with high Ca2+o that activation of the receptor alter its degree of was important in these studies to the formation of disulfide linkages protein by including mm in the is a with the on cysteines and the from disulfide bonds. shown in of mm in the the formation of a of CaR immunoreactivity that was in Western performed using the Therefore, the high molecular species in and are the of formation of disulfide bonds. show that the putative dimers of the CaR on the cell surface were of types of protein we forms of the receptor on the cell surface by with a we the cells with the The and cells were then in the presence of The Flag-tagged wild type receptor was and the CaR was with we the concentration of the of dimer to the is the CaR species on the cell surface with the dimer the species on the This result that the dimeric receptor is the species present on the cell The in of surface by the in the presence of both form covalent bonds with the of on the we employed to proteins to of the we that the in was less than on the CaR of the the at kDa in we that the CaR immunoreactivity at 160 kDa in we for the surface expression of the CaR in and all had of relative to dimers or oligomers Therefore, the of the receptor on the cell surface be that in surface the monomeric and forms as a result of cell-surface cross-linking produce in the of the of the when with the of the CaR cytoplasmic in several receptors with of of their were introduced stop at at the of the cytoplasmic and the respective receptors are to as and The truncated to a receptor at position that has been documented to be by Ray K. Fan G.F. Spiegel A.M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), and (9Bai M. Janicic N. Trivedi S. Quinn S.J. Cole D.E.C. Brown E.M. Hendy G.N. J. Clin. Invest. 1997; 99: 1917-1925Crossref PubMed Scopus (78) Google Scholar), were was also had a EC50 of ± 0.1 mm for high increases in Ca2+i than the full-length wild type receptor ± then transfected HEK293 cells with the truncated the and as Western analysis showed that the levels of expression of the truncated receptors were to or than and that of the wild type receptor, and all truncated receptors homodimers on the cell surface to the wild type receptor under nonreducing conditions. This result the possibility that the truncated receptors could form with the full-length receptor when the two receptors were high molecular species in nonreducing conditions were Therefore, we studies to that the high molecular species of the in and the wild type receptor and the truncated are the full-length and truncated receptors we co-transfected the truncated receptors with a Flag-tagged full-length receptor or Flag-tagged truncated receptors with a full-length receptor and the the and receptors we be to receptors with monomeric full-length and truncated receptors can be under reducing conditions on we be to the relative amounts of and which were to with containing on Western using as a we the co-transfected cells. we be to the of the dimers or higher on and that surface forms of receptors in co-transfected cells were co-immunoprecipitated using the which was from co-transfected receptors on a and with a the receptors from transfected cells could be by and oligomers were and monomeric forms of full-length or truncated receptors were on the surface of co-transfected cells shown in the of the Flag-tagged receptor was higher than that of the receptor, that the were substantial amounts of the homodimers of each of the co-transfected the expression of the full-length receptor was in cells co-transfected with with that in cells transfected with the full-length receptor The cross-linking and in the present study, with Western analysis of cell-surface CaR under nonreducing demonstrate that the CaR resides on the cell surface mostly as a dimer and as higher oligomers at all as a of homodimers cytoplasmic receptors suggests that the cytoplasmic is for and which dimerization activation Cell. 80: Full Text PDF PubMed Scopus Google Scholar), the dimerization of the CaR is that of the CaR in cell the on cysteines from disulfide bonds with cysteines in the the in the of were of of the cysteines that are present in the cytoplasmic of the CaR the of proteins for gel In the intact these cysteines likely in a form of the reducing by the of New New Scholar). was with a mutant CaR in which the entire was even in the of The of the surface dimeric form of the CaR and higher oligomers of the receptor under nonreducing conditions is converted to a single form of the monomeric receptors with a molecular mass of 160 kDa under reducing conditions Our earlier biochemical studies had shown that are monomeric forms of the CaR in membrane of CaR-transfected HEK293 cells with molecular masses of and 160 kDa on These forms of the receptor the CaR and receptors with types and of (4Bai 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 (380) Google Scholar), The species that we to as the the species high The of the surface CaR to a single form of monomeric receptor suggests that the CaR with the cell In as the forms of the with high to at the cell The of the dimeric receptor to reducing suggests that type of intermolecular dimerization is intermolecular disulfide bonds. is likely that the in the intermolecular disulfide of the CaR are in the in mGluR5 (15Romano C. Yang W.L. O'Malley K.L. J. Biol. Chem. 1996; 271: 28612-28616Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar), mGluR5 and the CaR share the relative of These cysteines may form disulfide bonds that are in the of both the CaR and or intermolecular disulfide bonds for dimerization of these two has been shown that the in the intermolecular disulfide of dimeric mGluR5 are within about 17 kDa of the (15Romano C. Yang W.L. O'Malley K.L. J. Biol. Chem. 1996; 271: 28612-28616Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). In this the CaR has including two within the putative that are likely by of the Therefore, or of the cysteines within 17 kDa of the and/or of the cysteines present in CaR may be in intermolecular disulfide bonds. we putative dimers even in the presence of reducing when we the CaR with to cell-surface expression and or an to the level of CaR protein expression and including both the and has been suggested that a noncovalent hydrophobic dimerization motif in the β2-adrenergic receptor (18Hebert T.E. Moffett S. Morello J.P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. Chem. 1996; 271: 16384-16392Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar) the formation of a important that is this dimerization motif for noncovalent hydrophobic interactions is also present in the CaR within that the the putative intermolecular are of GPCRs has suggested that is of the of all the likely this motif in receptor to the motif in receptor J. 1993; PubMed Scopus Google Scholar). Our studies showed that mutation in with a as as a in codon which normally resulting in a and a truncated CaR lacking and the of can form an dimer in the presence of mm S.H. Bai M. Quinn S.J. Kifor O. Brown E.M. Thakker R.V. J. Clin. Invest. 1996; 98: 1860-1866Crossref PubMed Scopus (168) Google Scholar). the putative dimerization motif in could to the formation of dimers of the receptor and/or of dimeric CaR In we have demonstrated that the CaR resides on the cell surface as homodimers that likely the form of the receptor and to the high degree of cooperativity for the CaR. is likely that the wild type and mutant form in a CaR on and an CaR on the present in that formation of wild type and mutant receptors may the function of the receptor in the example, a of the mutant receptor with wild type or with mutant receptors in the in could potentially the level of the wild type to of that present in O. Kifor for and J. V. A. F. R. T. and K. V. for with the HEK293 cells and the