Identification of Acidic Residues in the Extracellular Loops of the Seven-transmembrane Domain of the Human Ca2+ Receptor Critical for Response to Ca2+ and a Positive Allosteric Modulator
2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês
10.1074/jbc.m207100200
ISSN1083-351X
AutoresJianxin Hu, Guadalupe Reyes‐Cruz, Wangzhong Chen, Kenneth A. Jacobson, Allen M. Spiegel,
Tópico(s)Biochemical Analysis and Sensing Techniques
ResumoWe investigated the role of the eight acidic residues in the extracellular loops (exo-loops) of the seven-transmembrane domain of the human Ca2+ receptor (hCaR) in receptor activation by Ca2+ and in response to a positive allosteric modulator, NPS R-568. Both in the context of the full-length receptor and of a truncated receptor lacking the extracellular domain (Rho-C-hCaR), we mutated each acidic residue to alanine, singly and in combination, and tested the effect on expression of the receptor, on activation by Ca2+, and on NPS R-568 augmentation of sensitivity to Ca2+. Of the eight acidic residues, mutation of any of three in exo-loop 2, Asp758, Glu759, and Glu767, increased the sensitivity of both the full-length hCaR and of Rho-C-hCaR to activation by Ca2+. Mutation of all five acidic residues in exo-loop 2, whether in the full-length receptor or in Rho-C-hCaR, impaired cell surface expression of the mutant receptor and thereby largely abolished response to Ca2+. Mutation of Glu837 in exo-loop 3 to alanine did not alter Ca2+ sensitivity of the full-length receptor, but in both the latter context and in Rho-C-hCaR, alanine substitution of Glu837 drastically reduced sensitivity to NPS R-568. Our data point to a key role of three specific acidic residues in exo-loop 2 in hCaR activation and to Glu837 at the junction between exo-loop 3 and transmembrane helix seven in response to NPS R-568. We speculate on the basis of these results that the three acidic residues we identified in exo-loop 2 help maintain an inactive conformation of the seven-transmembrane domain of the hCaR. We investigated the role of the eight acidic residues in the extracellular loops (exo-loops) of the seven-transmembrane domain of the human Ca2+ receptor (hCaR) in receptor activation by Ca2+ and in response to a positive allosteric modulator, NPS R-568. Both in the context of the full-length receptor and of a truncated receptor lacking the extracellular domain (Rho-C-hCaR), we mutated each acidic residue to alanine, singly and in combination, and tested the effect on expression of the receptor, on activation by Ca2+, and on NPS R-568 augmentation of sensitivity to Ca2+. Of the eight acidic residues, mutation of any of three in exo-loop 2, Asp758, Glu759, and Glu767, increased the sensitivity of both the full-length hCaR and of Rho-C-hCaR to activation by Ca2+. Mutation of all five acidic residues in exo-loop 2, whether in the full-length receptor or in Rho-C-hCaR, impaired cell surface expression of the mutant receptor and thereby largely abolished response to Ca2+. Mutation of Glu837 in exo-loop 3 to alanine did not alter Ca2+ sensitivity of the full-length receptor, but in both the latter context and in Rho-C-hCaR, alanine substitution of Glu837 drastically reduced sensitivity to NPS R-568. Our data point to a key role of three specific acidic residues in exo-loop 2 in hCaR activation and to Glu837 at the junction between exo-loop 3 and transmembrane helix seven in response to NPS R-568. We speculate on the basis of these results that the three acidic residues we identified in exo-loop 2 help maintain an inactive conformation of the seven-transmembrane domain of the hCaR. The G protein-coupled [Ca2+]o receptor (CaR) 1The abbreviations used for: CaR, extracellular Ca2+ receptor; hCaR, human extracellular Ca2+receptor; GPCR, G protein-coupled receptor; mGluR1, metabotropic glutamate receptor type 1; PI, phosphoinositide; ECD, extracellular domain; 7TM, seven-transmembrane domain; VFT, Venus's-flytrap; HEK, human embryonic kidney; DMEM, Dulbecco's modified Eagle's medium; exo-loop, extracellular loop; PIPES, 1,4-piperazinediethanesulfonic acid; NPS R-568, (R)-N-(3-methoxy-α-phenylethyl)-3-(2′-chlorophenyl)-1-propylamine hydrochloride; NPS S-568, (S)-N-(3-methoxy-α-phenylethyl)-3-(2′-chlorophenyl)-1-propylamine hydrochloride. plays a central role in the regulation of [Ca2+]o homeostasis (1Brown E.M. MacLeod R.J. Physiol. Rev. 2001; 81: 239-297Crossref PubMed Scopus (1252) Google Scholar, 2Brown E.M. Cell Biochem. Biophys. 2000; 33: 63-95Crossref PubMed Scopus (12) Google Scholar). [Ca2+]o activates the CaR in the parathyroid, thereby inhibiting parathyroid hormone secretion, and in the kidney, causing increased urinary calcium excretion. The physiological importance of the CaR in determining the level at which [Ca2+]o is set in vivo has been documented by the identification of inactivating mutations in the CaR gene as the cause of familial hypocalciuric hypercalcemia and activating mutations as the cause of autosomal dominant hypocalcemia (3Bai 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, 4D'Souza-Li L. Yang B. Canaff L. Bai M. Hanley D.A. Bastepe M. Salisbury S.R. Brown E.M. Cole D.E.C. Hendy G.N. J. Clin. Endocrinol. Metab. 2002; 87: 1309-1318Crossref PubMed Scopus (112) Google Scholar). Naturally occurring CaR mutations identified in subjects with autosomal dominant hypocalcemia generally cause increased CaR sensitivity to [Ca2+]o rather than causing constitutive activation (5Zhao X.M. Hauache O. Goldsmith P.K. Collins R. Spiegel A.M. FEBS Lett. 1999; 448: 180-184Crossref PubMed Scopus (56) Google Scholar). The CaR belongs to a unique subfamily, family 3, of G protein-coupled receptors (GPCR) with an unusually large N-terminal, extracellular domain (ECD) comprised of Venus's-flytrap (VFT) and cysteine-rich domains, in addition to the seven-transmembrane domain (7TM) characteristic of all GPCR (6Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1241) Google Scholar). Studies with chimeric family 3 GPCR (7Hu J. Hauache O. Spiegel A.M. J. Biol. Chem. 2000; 275: 16382-16389Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 8Hammerland L.G. Krapcho K.J. Garrett J.E. Alasti N. Hung B.C.P. Simin R.T. Levinthal C. Nemeth E.F. Fuller F.H. Mol. Pharmacol. 1999; 55: 642-648PubMed Google Scholar, 9Brauner-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, 10Malitschek B. Schweizer C. Keir M. Heid J. Froestl W. Mosbacher J. Kuhn R. Henley J. Joly C. Pin J.P. Kaupmann K. Bettler B. Mol. Pharmacol. 1999; 56: 448-454Crossref PubMed Scopus (98) Google Scholar) and the three-dimensional structure of the metabotropic glutamate type 1 receptor (mGluR1) determined by x-ray crystallography (11Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1123) Google Scholar) show that the VFT is the site of agonist binding in family 3 GPCR. The precise site(s) for binding to the CaR, however, have not been identified. The activation of family 3 GPCR can also be positively modulated by compounds that bind to the 7TM domain and are presumed to act allosterically (12Knoflach F. Mutel V. Jolidon S. Kew J.N.C. Malherbe P. Vieira E. Wichmann J. Kemp J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13402-13407Crossref PubMed Scopus (219) Google Scholar). The phenylalkylamine, NPS R-568, a so-called calcimimetic, increases the sensitivity of the receptor to [Ca2+]o activation (13Nemeth E.F. Steffey M.E. Hammerland L.G. Hung B.C.P. Van Wagenen B.C. DelMar E.G. Balandrin M.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4040-4045Crossref PubMed Scopus (561) Google Scholar), acting as a positive allosteric modulator by binding to the CaR 7TM region (14Hauache O.M. Hu J. Ray K. Xie R. Jacobson K.A. Spiegel A.M. Endocrinology. 2000; 141: 4156-4163Crossref PubMed Google Scholar). A CaR mutant lacking the ECD that responds minimally to [Ca2+]o shows significant responsiveness when NPS R-568 is added (14Hauache O.M. Hu J. Ray K. Xie R. Jacobson K.A. Spiegel A.M. Endocrinology. 2000; 141: 4156-4163Crossref PubMed Google Scholar). This suggests that sites involved in [Ca2+]o activation are present within the 7TM domain and not only the ECD. Stretches of acidic residues within the VFT have been considered likely sites of Ca2+ binding to the CaR (15Brown 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 (2395) Google Scholar). Given that the 7TM domain also is capable of being activated by [Ca2+]o, we decided to investigate the role of acidic residues located in the extracellular loops of the 7TM domain in this activation. The exo-loops of the 7TM domain, like the ECD, are presumptively exposed at the cell surface and therefore could be involved in extracellular Ca2+ binding to and activation of the CaR. The full-length hCaR cDNA cloned in the pCR3.1 expression vector and a truncated receptor lacking the ECD, Rho-C-hCaR, were described previously (5Zhao X.M. Hauache O. Goldsmith P.K. Collins R. Spiegel A.M. FEBS Lett. 1999; 448: 180-184Crossref PubMed Scopus (56) Google Scholar, 16Ray 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). Rho-C-hCaR contains the first 20 amino acids of the N terminus of bovine rhodopsin fused to amino acid residues 600–903 2Rho-C-hCaR was incorrectly described by us previously (5Zhao X.M. Hauache O. Goldsmith P.K. Collins R. Spiegel A.M. FEBS Lett. 1999; 448: 180-184Crossref PubMed Scopus (56) Google Scholar) as fusing the N-terminal 20 residues of bovine rhodopsin to hCaR residue 599 rather than residue 600. of the wild type hCaR (see Fig. 1). Site-directed mutagenesis was performed using the QuikChangeTM site-directed mutagenesis kit (Stratagene Inc., La Jolla, CA), according to the manufacturer's instructions. Parental hCaR cDNA in pCR3.1 vector was amplified usingPfu 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 dRhodamine Terminator Cycle sequencing kit and ABI PRISM-373A DNA sequencer (PE Applied Biosystems, Foster City, CA). 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, the transfected cells were split and cultured in complete DMEM. PI hydrolysis assay has been described previously (16Ray 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 one-eighth 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 containing 3.0 μCi/ml of [3H]myo-inositol (PerkinElmer Life Sciences) and cultured for another 24 h. Culture medium was replaced by 1× PI buffer (120 mm NaCl, 5 mm KCl, 5.6 mm glucose, 0.4 mm MgCl2, 20 mm LiCl in 25 mm PIPES buffer, pH 7.2) and incubated for 1 h at 37 °C. After removal of PI buffer, the 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 NPS S-568. 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 the radioactivity for each sample was counted with 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. The graphs shown in this paper are representative ones from at least three independent experiments. The method for the synthesis of NPS R-568 was reported previously (14Hauache O.M. Hu J. Ray K. Xie R. Jacobson K.A. Spiegel A.M. Endocrinology. 2000; 141: 4156-4163Crossref PubMed Google Scholar). For the synthesis of NPS S-568, racemic 1-(3-methoxyphenyl)ethylamine was prepared by reductive amination of 3-methoxyacetophenone with ammonium acetate (17Borch R.F. Bernstein M.D. Durst H.D. J. Am. Chem. Soc. 1971; 93: 2897-2904Crossref Scopus (2095) Google Scholar), and the enantiomerically pure S-isomer was isolated through crystallization with S-mandelic acid (18Sakai K. Hashimoto Y. Kinbara K. Saigo K. Murakami H. Nohira H. Bull. Chem. Soc. Jpn. 1993; 66: 3414-3418Crossref Google Scholar). The final products were then obtained by reductive amination of 3-(2-chlorophenyl)propionaldehyde with the free base ofS-1-(3-methoxyphenyl)ethylamine using sodium cyanoborohydride in tetrahydrofuran containing a trace of acetic acid to provide S-568. The amine was isolated as the hydrochloric salt following treatment of the free base with anhydrous hydrochloric acid in dioxane. The specific rotations, proton NMR spectra and high resolution mass spectra were consistent with the assigned structure. Confluent cells in 6-well plates were rinsed with ice-cold phosphate-buffered saline and scraped on ice in lysis buffer containing 20 mm Tris-HCl (pH 6.8), 150 mm NaCl, 10 mm EDTA, 1 mm EGTA, 1% Triton X-100, and freshly added protease inhibitors mixture (Roche Molecular Biochemicals). For immunoblotting of full-length receptors, 50 μg of protein/lane reduced with β-mercaptoethanol (5%) was separated on 5% SDS-PAGE gel. The proteins on the gel were electrotransferred onto nitrocellulose membrane and incubated with 0.1 μg/ml of protein A-purified mouse monoclonal anti-hCaR antibody ADD (raised against a synthetic peptide corresponding to residues 214–235 of hCaR protein). Subsequently, the membrane was incubated with a secondary goat anti-mouse antibody conjugated to horseradish peroxidase (Amersham Biosciences) at a dilution of 1: 2,000. The hCaR protein was detected with an ECL system (Amersham Biosciences). For immunoblotting of Rho-C-hCaR constructs, 20 μg of protein/lane premixed with 5% β-mercaptoethanol for 1 h was separated on precast 4–20% gradient gels (Invitrogen), and mouse monoclonal anti-rhodopsin N terminus antibody B6–30, which was kindly provided by Paul Hargrave (University of Florida), was used as the primary antibody. For fluorescence immunocytochemistry, transfected cells grown on duplicate coverslips precoated with 20 μg/μl Fibronectin (Calbiochem, La Jolla, CA) from human plasma were fixed with paraformaldehyde (4%) in Dulbecco's phosphate-buffered saline (BioFluids Inc.) for 20 min with one set of the coverslips permeabilized with methanol (100%). After 1 h of incubation at 37 C with mouse anti-rhodopsin N terminus antibody B6–30, the cells were washed and incubated with fluorescein-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch Inc., West Grove, PA) for 45 min at room temperature. After washing, the cells on coverslips were mounted on glass slides using ProLong Antifade (Molecular Probes Inc., Eugene, OR) and examined by two independent observers, who assessed staining patterns without knowledge of the identity of cDNAs transfected, with a fluorescent microscope (Zeiss Axiophot, Zeiss, Germany). We previously described construction of a mutant hCaR truncated at residue 903 and with a deletion of most of the ECD (residues 1–599). The first 20 amino acids of bovine rhodopsin were fused to hCaR residue 600 to facilitate cell surface expression of this mutant construct designated Rho-C-hCaR (Fig.1) (5Zhao X.M. Hauache O. Goldsmith P.K. Collins R. Spiegel A.M. FEBS Lett. 1999; 448: 180-184Crossref PubMed Scopus (56) Google Scholar, 19Krautwurst D. Yau K.W. Reed R.R. Cell. 1998; 95: 917-926Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). We transfected wild type and Rho-C-hCaR cDNAs into HEK-293 cells and analyzed their function by [Ca2+]o-stimulated PI hydrolysis assay and their expression on immunoblots stained with either anti-hCaR monoclonal antibody ADD to detect full-length CaR or anti-rhodopsin N terminus antibody B6–30 to detect Rho-C-hCaR. Fig.2 shows that wild type hCaR responds to [Ca2+]o with an EC50 value of 3.08 ± 0.04 mm (mean ± S.E., n= 5). Under reducing conditions, ADD antibody detected two major bands of about 130 and 150 kDa (Fig. 2). Previous studies have shown that the monomeric ∼150-kDa band represents hCaR forms expressed at the cell surface and modified with N-linked, complex carbohydrates; the ∼130-kDa band represents high mannose-modified forms, trapped intracellularly and sensitive to endoglycosidase H digestion (3Bai 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, 16Ray 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,20Ray K. Clapp P. Goldsmith P.K. Spiegel A.M. J. Biol. Chem. 1998; 273: 34558-34567Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) Rho-C-hCaR showed minimal response to [Ca2+]o in the PI hydrolysis assay, suggesting that the ECD of the hCaR plays a critical role in ligand binding and activation of the receptor. Rho-C-hCaR, however, is capable of responding to [Ca2+]o upon addition of 1 μm NPS R-568. This calcimimetic compound did not directly activate the receptor but markedly potentiated Ca2+-stimulated responses not only of the wild type hCaR but also of Rho-C-hCaR (Fig. 2). The addition of 1 μm NPS S-568 has no effect on the response to [Ca2+]o of either WT hCaR or Rho-C-hCaR (data not shown). We found that imposing an activating mutation, F788C, identified in a subject with autosomal dominant hypocalcemia (21Watanabe T. Bai M. Lane C.R. Matsumoto S. Minamitani K. Minagawa M. Niimi H. Brown E.M. Yasuda T. J. Clin. Endocrinol. Metab. 1998; 83: 2497-2502Crossref PubMed Scopus (68) Google Scholar) onto Rho-C-hCaR slightly increased the basal activity of the mutant receptor and mimicked the effect of NPS R-568 in potentiating the [Ca2+]o response. Immunoblot of Rho-C-hCaR constructs shows a single band at ∼36 kDa for the monomeric form of the mutant receptor (Fig. 2, right panel). The immunoblot confirms the expression of Rho-C-hCaR constructs but does not distinguish between receptor proteins expressed at the cell surface and those retained intracellularly. The ability of Rho-C-hCaR to respond to extracellular calcium, however, indicates that at least some, if not all, of the receptor protein is expressed at the cell surface. Although there is strong evidence that the VFT domain of the ECD of family 3 GPCRs, including the CaR, is responsible for agonist binding (7Hu J. Hauache O. Spiegel A.M. J. Biol. Chem. 2000; 275: 16382-16389Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 8Hammerland L.G. Krapcho K.J. Garrett J.E. Alasti N. Hung B.C.P. Simin R.T. Levinthal C. Nemeth E.F. Fuller F.H. Mol. Pharmacol. 1999; 55: 642-648PubMed Google Scholar, 9Brauner-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, 10Malitschek B. Schweizer C. Keir M. Heid J. Froestl W. Mosbacher J. Kuhn R. Henley J. Joly C. Pin J.P. Kaupmann K. Bettler B. Mol. Pharmacol. 1999; 56: 448-454Crossref PubMed Scopus (98) Google Scholar), the ability of Rho-C-hCaR to respond to calcium upon addition of NPS R-568 or by imposing the F788C mutation indicates the existence of calcium-binding site(s) in the 7TM domain of the hCaR as well. Acidic residues in the VFT have been speculated to be involved in calcium binding to and activation of the CaR. This focused our attention on the eight acidic residues in the extracellular loops of the 7TM domain of the CaR (two in exo-loop 1, five in exo-loop 2, and one in exo-loop 3; Fig. 1) to assess their possible role in the activation of the receptor by calcium. We constructed by site-directed mutagenesis mutant full-length hCaRs with a single alanine mutation substituted for each of the respective acidic residues in exo-loops 1–3. Fig. 3 shows that the eight different mutants were expressed in the same pattern of two major bands as the wild type, indicating that the hCaR is tolerant of single alanine substitution in these sites without abolishing cell surface expression. The density of upper bands of these mutants varied slightly. Our recent study showed that different cell surface expression levels of hCaRs affect the maximal response of the receptor to [Ca2+]o but not the EC50 value of the receptor (22Hu J. Mora S. Colussi G. Proverbio M.C. Jones K.A. Bolzoni L. De Ferrara M.E. Civati G. Spiegel A.M. J. Bone Miner. Res. 2002; 17: 1461-1469Crossref PubMed Scopus (24) Google Scholar). Fig. 3 shows that three of the eight mutants, namely hCaR/D758A, hCaR/E759A, and hCaR/E767A, exhibited increased sensitivity to [Ca2+]o. The EC50 values for these activating mutants are 0.95 ± 0.05 mm (hCaR/D758A), 0.74 ± 0.02 mm (hCaR/E759A), and 1.83 ± 0.03 mm (hCaR/E767A) (mean ± S.E.; n = 3). The maximal activation by calcium for mutants D758A and E759A was 50–60% of that of the WT hCaR. In contrast, alanine substitution for each of the other five acidic residues did not change the sensitivity to calcium and in most cases minimally altered maximal response. E837A showed the lowest maximal response (∼70% of WT) of these five mutants. Because alanine substitution for several individual acidic residues within exo-loop 2 led to increased sensitivity of the CaR, we created additional mutants substituting alanine for multiple acidic residues in exo-loop 2 to determine whether this would lead to further increases in CaR sensitivity. As shown in Fig. 4, a receptor with the cluster of four adjacent acidic residues Glu755, Glu757, Asp758, and Glu759 mutated simultaneously to alanines (termed hCaR/4A) showed increased sensitivity to [Ca2+]o but only to a similar extent as the individual E767A mutant. However, changing all five acidic residues in exo-loop 2 to alanines simultaneously (termed hCaR/5A) not only did not lead to further increase in CaR sensitivity but instead resulted in significantly impaired function of the receptor with maximal activation <20% of wild type hCaR (Fig. 4). As shown by the faint upper band on immunoblot of the 5A mutant (Fig. 4, right panel), the loss of function of the 5A mutant is due to poor cell surface expression. We extended our study of exo-loop 2 alanine mutants by combining them with the naturally occurring F788C mutation (hCaR/F788C) identified in subjects with autosomal dominant hypocalcemia (21Watanabe T. Bai M. Lane C.R. Matsumoto S. Minamitani K. Minagawa M. Niimi H. Brown E.M. Yasuda T. J. Clin. Endocrinol. Metab. 1998; 83: 2497-2502Crossref PubMed Scopus (68) Google Scholar). In vitrostudy shows that this mutation causes a left shift in the response of the receptor to [Ca2+]o (Fig.5). The F788C mutant receptor retains increased sensitivity to [Ca2+]o when it is combined with the E767A mutation (hCaR/F788C/E767A) or 4A mutations (hCaR/F788C/4A), although maximal receptor activation is reduced. However, combining the hCaR/F788C mutant with the 5A mutations (hCaR/F788C/5A) resulted in complete loss of function in a [Ca2+]o-stimulated PI hydrolysis assay (Fig. 5). Loss of receptor function was due to a lack of cell surface expression as reflected in absence of the upper band on immunoblot (Fig. 5, right panel). We next tested the effects of alanine substitution of acidic residues in the exo-loops of Rho-C-hCaR that lacks the ECD. Mutant receptor function was tested by using the intact cell [Ca2+]o-stimulated PI hydrolysis assay with and without the addition of 1 μmNPS R-568. Fig. 6 A shows that without addition of NPS R-568, most receptor constructs showed minimal response to [Ca2+]o, even up to 30 mm. The E767A mutation, and to a much lesser extent, the 4A mutation revealed a significant [Ca2+]o response within the Rho-C-hCaR context. With the addition of 1 μmNPS R-568, as shown before, Rho-C-hCaR was significantly activated by [Ca2+]o. With NPS R-568, the E767A and 4A mutants showed greater activation by [Ca2+]o than Rho-C-hCaR, as did the E671A mutant (Fig. 6 B). The D674A mutant was also activated by [Ca2+]o in the presence of 1 μm NPS R-568 but to a lower extent than Rho-C-hCaR. Importantly, two mutant constructs, Rho-C-hCaR/5A and Rho-C-hCaR/E837A, exhibited no response to [Ca2+]o even in the presence of 1 μm NPS R-568. Studies with full-length hCaR/5A mutant showed that it is very poorly expressed at the cell surface, thus the lack of response of Rho-C-hCaR/5A could also reflect poor cell surface expression. Full-length hCaR/E837A mutant, however, is expressed at the cell surface and is clearly activated by [Ca2+]o. Thus the lack of [Ca2+]o activation of the Rho-C-hCaR/E837A mutant, even with addition of NPS R-568, was unexpected. Immunoblotting showed that all Rho-C-hCaR constructs are expressed, but unlike with full-length CaR, immunoblotting alone does not distinguish between cell surface-expressed and intracellular forms of receptor. Therefore additional approaches were needed to assess cell surface expression of the Rho-C-hCaR mutants, particularly Rho-C-hCaR/5A and Rho- C-hCaR/E837A. To document whether the Rho-C-hCaR/5A and Rho-C-hCaR/E837A mutants were expressed at the cell surface, immunocytochemistry was performed with the anti-rhodopsin N terminus antibody B6–30, the epitope of which is located in the extracellular N terminus. Fig. 7 shows that HEK-293 cells transfected with wild type Rho-C-hCaR or Rho-C-hCaR/E837A were stained by antibody B6–30 both under nonpermeabilized and permeabilized conditions, indicating successful cell surface expression of these receptors. In contrast, cells transfected with Rho-C-hCaR/5A were stained only under permeabilized conditions (Fig. 7), indicating that Rho-C-hCaR/5A was expressed intracellularly only. Given that the Rho-C-hCaR/E837A mutant is expressed at the cell surface and yet does not respond to [Ca2+]o in the presence of NPS R-568, we considered the possibility that Glu837 may be involved in the action of NPS R-568 on the hCaR and that mutating this residue to alanine impairs NPS R-568 modulation of hCaR activation. To test this possibility, we measured [Ca2+]o-stimulated PI hydrolysis of cells transfected with hCaR/E837A in the presence or absence of 1 μm NPS R-568, comparing it with two other alanine mutants, hCaR/E757A and hCaR/E767A. Fig.8 A shows that NPS R-568 failed to potentiate the response of hCaR/E837A to [Ca2+]o, whereas the calcimimetic compound significantly potentiated the response of hCaR/E757A to [Ca2+]o and even further potentiated the already left-shifted [Ca2+]o response of hCaR/E767A. To evaluate further the effect of the E837A mutation on responsiveness to NPS R-568, we tested a range of concentrations of NPS R-568 up to 100 μm in the PI hydrolysis assay at 2 mm[Ca2+]o. Fig. 8 B shows that concentrations as low as 0.1 μm NPS R-568 enhanced the WT hCaR response to [Ca2+]o and that the effect of NPS R-568 reached a maximum at 10 μm. In contrast, the hCaR/E837A mutant response to NPS R-568 was significantly right-shifted. No significant enhancement of [Ca2+]o response by the hCaR/E837A mutant was observed unless the concentration of NPS R-568 was 10 μmor greater. Even at maximally effective concentrations of NPS R-568, the enhancement of [Ca2+]o response of the hCaR/E837A mutant was less than 50% of that seen with WT hCaR. This difference in NPS R-568 effect was not a function of differences in receptor expression as seen on immunoblot (Fig. 8 B,right panel). We tested the hypothesis that acidic residues in the extracellular loops of the 7TM domain of the hCaR are involved in [Ca2+]o activation of the receptor by replacing each of these residues with alanine and measuring the ability of the respective mutant receptors to respond to [Ca2+]o. Alanine mutagenesis of extracellular loop acidic residues was done both in the context of the full-length receptor and in Rho-C-hCaR, a mutant receptor lacking most of the ECD. Of the eight acidic residues in exo-loops 1–3, we identified three, Asp758, Glu759, and Glu767, all in exo-loop 2, that based on the evidence summarized below appear to play important roles in [Ca2+]o activation of the receptor. We identified another acidic residue, Glu837 in exo-loop 3, as critical for receptor responsiveness to the positive allosteric modulator, NPS R-568. Interestingly, only these four of the eight acidic residues in exo-loops 1–3 are identically conserved in all CaR species sequenced to date. Glu755 is conservatively substituted by aspartate, and Glu671, Asp674, and Glu757 are nonconservatively substituted or deleted in some species (23Nearing J. Betka M. Quinn S. Hentschel H. Elger M. Baum M. Bai M. Chattopadyhay N. Brown E.M. Hebert S.C. Harris H.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9231-9236Crossref PubMed Scopus (128) Google Scholar, 24Naito T. Saito Y. Yamamoto J. Nozaki
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