Amino Acids in the Second and Third Intracellular Loops of the Parathyroid Ca2+-sensing Receptor Mediate Efficient Coupling to Phospholipase C
2000; Elsevier BV; Volume: 275; Issue: 26 Linguagem: Inglês
10.1074/jbc.m909613199
ISSN1083-351X
AutoresWenhan Chang, Tsui‐Hua Chen, Stacy Pratt, Dolores Shoback,
Tópico(s)Ion Transport and Channel Regulation
ResumoTo determine the role of amino acids in the second and third intracellular (IC) loops of the Ca2+-sensing receptor (CaR) in phospholipase C (PLC) activation, we mutated residues in these loops either singly or in tandem to Ala and assessed PLC activity by measuring high extracellular [Ca2+] ([Ca2+]o)-induced inositol phosphate accumulation and protein expression by immunoblotting and immunocytochemistry in human embryonic kidney 293 cells. Two CaR constructs in the second IC loop, F707A CaR and to a lesser extent L704A CaR, demonstrated reduced activation of PLC, despite levels of protein expression comparable with the wild-type (wt) CaR. Substitution of Tyr or His for Phe-707, but not Leu, Val, Glu, or Trp, partially restored the ability of high [Ca2+]o to activate PLC. Eight residues in the third IC loop were involved in PLC signaling. The responses to high [Ca2+]o in cells expressing CaRs with Ala substitutions at these sites were <35% of the wt CaR. The L798A, F802A, and E804A CaRs were dramatically impaired in their responses to [Ca2+]o even up to 30 mm. Substitutions of Leu-798 with other hydrophobic residues (Ile, Val, or Phe), but not with acidic, basic, or polar residues, produced reduced responses compared with wt. Phe-802 could be replaced with either Tyr or Trp with partial retention of the ability to activate PLC. Glu-804 could only be substituted with Asp or Gln and maintain its signaling capacity. Cell surface expression of the CaRs mutated at Leu-798 and Phe-802 appeared normal compared with wt CaR. Cell surface CaR expression was, however, reduced substantially in cells expressing several mutants at position Glu-804 by confocal microscopy. These studies strongly implicate specific hydrophobic and acidic residues in the second and third IC loops of the parathyroid CaR (and potentially larger stretches of the third loop) in mediating efficient high [Ca2+]o-induced PLC activation and or CaR expression. To determine the role of amino acids in the second and third intracellular (IC) loops of the Ca2+-sensing receptor (CaR) in phospholipase C (PLC) activation, we mutated residues in these loops either singly or in tandem to Ala and assessed PLC activity by measuring high extracellular [Ca2+] ([Ca2+]o)-induced inositol phosphate accumulation and protein expression by immunoblotting and immunocytochemistry in human embryonic kidney 293 cells. Two CaR constructs in the second IC loop, F707A CaR and to a lesser extent L704A CaR, demonstrated reduced activation of PLC, despite levels of protein expression comparable with the wild-type (wt) CaR. Substitution of Tyr or His for Phe-707, but not Leu, Val, Glu, or Trp, partially restored the ability of high [Ca2+]o to activate PLC. Eight residues in the third IC loop were involved in PLC signaling. The responses to high [Ca2+]o in cells expressing CaRs with Ala substitutions at these sites were <35% of the wt CaR. The L798A, F802A, and E804A CaRs were dramatically impaired in their responses to [Ca2+]o even up to 30 mm. Substitutions of Leu-798 with other hydrophobic residues (Ile, Val, or Phe), but not with acidic, basic, or polar residues, produced reduced responses compared with wt. Phe-802 could be replaced with either Tyr or Trp with partial retention of the ability to activate PLC. Glu-804 could only be substituted with Asp or Gln and maintain its signaling capacity. Cell surface expression of the CaRs mutated at Leu-798 and Phe-802 appeared normal compared with wt CaR. Cell surface CaR expression was, however, reduced substantially in cells expressing several mutants at position Glu-804 by confocal microscopy. These studies strongly implicate specific hydrophobic and acidic residues in the second and third IC loops of the parathyroid CaR (and potentially larger stretches of the third loop) in mediating efficient high [Ca2+]o-induced PLC activation and or CaR expression. Ca2+-sensing receptor G-protein-coupled receptor phospholipase C metabotropic glutamate receptor type B γ-aminobutyric acid receptor intracellular wild type base pair inositol phosphate human embryonic kidney cells tandem Ala CaRs1 are members of the G-protein-coupled receptor (GPCR) superfamily and couple to PLC activation, inhibition of cyclic AMP formation, and opening of nonselective cation channels (1.Brown 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 (2377) Google Scholar, 2.Ye C. Kanazirska M. Quinn S. Brown E.M. Vassilev P.M. Biochem. Biophys. Res. Commun. 1996; 224: 271-280Crossref PubMed Scopus (69) Google Scholar, 3.Chang W. Pratt S. Chen T.-H. Nemeth E. Huang Z. Shoback D. J. Bone Miner. Res. 1998; 13: 570-580Crossref PubMed Scopus (81) Google Scholar, 4.Chang W. Chen T.H. Pratt S. Shoback D. Am. J. Physiol. 1998; 275: E213-E221PubMed Google Scholar). CaRs share modest sequence homology with the metabotropic glutamate receptors (mGluRs) (5.Francesconi A. Duvoisin R.M. J. Biol. Chem. 1998; 273: 5615-5624Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 6.Gomeza J. Joly C. Kuhn R. Knopfel T. Bockaert J. Pin J.P. J. Biol. Chem. 1996; 271: 2199-2205Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), the type B γ-aminobutyric acid receptor (GABAB) (7.Kaupmann K. Huggel K. Heid J. Flor P.J. Bischoff S. Mickel S.J. McMaster G. Angst C. Bittiger H. Froestl W. Bettler B. Nature. 1997; 386: 239-246Crossref PubMed Scopus (878) Google Scholar), and a large group of pheromone receptors (8.Ryba N.J. Tirindelli R. Neuron. 1997; 19: 371-379Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 9.Cao Y. Oh B.C. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11987-11992Crossref PubMed Scopus (127) Google Scholar) and, thus, constitute the family 3 of GPCRs (10.Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1233) Google Scholar). Receptors in the CaR/mGluR subfamily share several structural features. These include a large extracellular amino-terminal domain, seven membrane-spanning regions, three IC loops, and a large cytoplasmic tail (see Fig. 1 a). The extracellular domains of CaRs and mGluRs are known to be critical for ligand recognition (11.Fan G.F. Ray K. Zhao X.M. Goldsmith P.K. Spiegel A.M. FEBS Lett. 1998; 436: 353-356Crossref PubMed Scopus (91) Google Scholar, 12.Brauner-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, 13.Parmentier M.L. Joly C. Restituito S. Bockaert J. Grau Y. Pin J.P. Mol Pharmacol. 1998; 53: 778-786Crossref PubMed Scopus (70) Google Scholar). Naturally occurring mutants of the CaR, implicated in the pathogenesis of abnormal Ca2+-sensing in vivo, occur predominantly in the large amino-terminal domain of this receptor (14.Bai M. Int. J. Mol. Med. 1999; 4: 115-125PubMed Google Scholar,15.Pearce S.H. Trump D. Wooding C. Besser G.M. Chew S.L. Grant D.B. Heath D.A. Hughes I.A. Paterson C.R. Whyte M.P. Thakker R.V. J. Clin. Invest. 1995; 96: 2683-2692Crossref PubMed Scopus (330) Google Scholar). Point mutations in this domain, responsible for either gain-of-function or loss-of-function, indicate its key role in the Ca2+-sensing function of the receptor. IC domains of receptors in the CaR/mGluR subfamily are likely, by analogy to other GPCRs, to be responsible for coupling to G-protein-mediated signal transduction (10.Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1233) Google Scholar, 16.Pin J.P. Joly C. Heinemann S.F. Bockaert J. EMBO J. 1994; 13: 342-348Crossref PubMed Scopus (165) Google Scholar, 17.Wess J. FASEB J. 1997; 11: 346-354Crossref PubMed Scopus (511) Google Scholar). A comparison of CaRs with the mGluR 1–8 indicates limited sequence conservation in their second IC loops (<10%) but striking conservation (67 to 85%) in their third IC loops (see Fig. 1 b). This observation suggests these latter regions likely share similar function. Mutagenesis of mGluR1 and R5 previously demonstrated that specific residues in IC loops 2 and 3 contribute to PLC activation, whereas other residues were involved in the regulation of cyclic AMP formation (5.Francesconi A. Duvoisin R.M. J. Biol. Chem. 1998; 273: 5615-5624Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Domains of comparable functional significance in the CaR have, to date, not been identified. Studies of kindred with familial benign hypercalcemia and neonatal severe hyperparathyroidism indicated that a CaR with a mutation at residue 795 (R795W) in the amino-terminal portion of the third IC loop had a reduced ability to mobilize intracellular Ca2+ (18.Bai 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 (382) Google Scholar). The remaining residues within the second and third IC loops have not been carefully examined. In these studies, we mutated amino acids in IC loops 2 and 3 of the bovine CaR to identify the positions of key signaling residues and structural requirements at those sites. Phe-707 in the second IC loop and 2 residues in the third IC loop, Leu-798 and Phe-802, proved critical to the activation of PLC. Glu-804 proved essential for efficient cell surface expression of CaRs. This work supports the presence of several functional determinants in IC loops 2 and 3 in the stimulation of PLC by and expression of CaRs in mammalian cells. The full-length bovine parathyroid CaR cDNA (1.Brown 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 (2377) Google Scholar) was provided by Dr. Edward Brown (Harvard Medical School, Boston, MA). The Chameleon double-stranded, site-directed mutagenesis kit and pBluescript II SK- (pBS) were obtained from Stratagene (La Jolla, CA). pcDNA1/Amp and pCEP4 were purchased from Invitrogen (Carlsbad, CA). Restriction enzymes were from Stratagene, Life Technologies, Inc., and Promega (Madison, WI). Fluorescein-conjugated goat-anti rabbit IgG for immunocytochemistry was obtained from Molecular Probes, Inc. (Eugene, OR). Other supplies were from previously noted sources (3.Chang W. Pratt S. Chen T.-H. Nemeth E. Huang Z. Shoback D. J. Bone Miner. Res. 1998; 13: 570-580Crossref PubMed Scopus (81) Google Scholar,19.Chang W. Tu C. Bajra R. Komuves L. Miller S. Strewler G. Shoback D. Endocrinology. 1999; 140: 1911-1919Crossref PubMed Google Scholar). The SmaI fragment (nucleotides 248–3819) of the wt bovine parathyroid CaR was ligated into pBS (wt CaR/pBS) as described previously (3.Chang W. Pratt S. Chen T.-H. Nemeth E. Huang Z. Shoback D. J. Bone Miner. Res. 1998; 13: 570-580Crossref PubMed Scopus (81) Google Scholar) and used as the template for mutagenesis. Mutagenesis was performed using the Chameleon kit according to the manufacturer's instructions. Briefly, in each reaction a selection primer was used to convert a unique KpnI site in wt CaR/pBS to an SrfI site, and a mutagenic primer was used to introduce the desired mutation and a new restriction site (i.e. NotI, NspV, SpeI,HindIII, SmaI, Nar I, orDraI). Primers were mixed and annealed with heat-denatured plasmid cDNA template. Synthesis of the (−) strand of plasmid cDNA was achieved by DNA polymerase and ligase. The reaction mixture was then treated with KpnI to linearize any double-stranded wt CaR cDNA. Uncut circular hybrid cDNA was then transformed into XL-mutS blue Escherichia coli. cDNA amplified from this transformation containing both wt and mutant cDNA was again treated with KpnI to linearize the remaining wt CaR cDNA. This DNA mixture was then re-transformed into XL-1 blue E. coli. Transformants were selected on Luria-Bertani broth agar plates containing ampicillin. After DNA amplification, mutations were confirmed by automated DNA sequencing (Biomolecular Resource Center, University of California, San Francisco). Subcloning of mutant CaR constructs into pcDNA1/Amp for transient transfections were done by gel-purifying the 2213-bpPinAI-XbaI fragment, which contained the mutations in the second IC loop, and ligating this insert into the 6308-bp PinAI-XbaI fragment of wt CaR/pcDNA1/Amp. The latter construct was made by ligating the 3619-bp fragment of wt CaR/pBS into pcDNA1/Amp cut withNotI and HindIII. For constructs with mutations in IC loop 3, a 1073-bp XhoI-XbaI fragment from the relevant pBS mutant CaR construct was gel-purified and ligated into the 7448-bp XhoI-XbaI fragment of wt CaR/pcDNA1/Amp. To generate mutant CaR constructs for stable transfections, the Srf1 site in mutant CaR/pBS constructs was first converted back to KpnI site by mutagenesis. TheKpnI- NotI fragment comprising the mutant CaR cDNA of interest was then subcloned into pCEP4 as described (3.Chang W. Pratt S. Chen T.-H. Nemeth E. Huang Z. Shoback D. J. Bone Miner. Res. 1998; 13: 570-580Crossref PubMed Scopus (81) Google Scholar). HEK293 cells were grown in Dulbecco's modified Eagle's medium with fetal bovine serum (10% v/v) and transfected as described previously using CaCl2 precipitation (3.Chang W. Pratt S. Chen T.-H. Nemeth E. Huang Z. Shoback D. J. Bone Miner. Res. 1998; 13: 570-580Crossref PubMed Scopus (81) Google Scholar). For transient expression, cells were washed twice with phosphate-buffered saline after a 24-h incubation and then plated in 6-well culture plates. After allowing 48 to 72 h for attachment and growth, InsP production and receptor expression were assessed. For stable expression of CaR constructs, transfected cells were selected in medium containing hygromycin B (200 μg/ml) for at least 4 weeks before experiments. Total InsP accumulation was measured in triplicate after labeling transfected HEK293 cells with [3H]myoinositol (2 μCi/ml) for 18 to 24 h in Dulbecco's modified Eagle's medium, 10% fetal bovine serum as described (3.Chang W. Pratt S. Chen T.-H. Nemeth E. Huang Z. Shoback D. J. Bone Miner. Res. 1998; 13: 570-580Crossref PubMed Scopus (81) Google Scholar). [3H]InsP accumulation in the presence of LiCl (10 mm) was measured after a 60-min incubation with different [Ca2+]o at 37 °C. Total [3H]InsPs were extracted from cells and isolated by anion-exchange chromatography (3.Chang W. Pratt S. Chen T.-H. Nemeth E. Huang Z. Shoback D. J. Bone Miner. Res. 1998; 13: 570-580Crossref PubMed Scopus (81) Google Scholar, 20.Shoback D.M. McGhee J.M. Endocrinology. 1988; 122: 2833-2839Crossref PubMed Scopus (24) Google Scholar). Results in all experiments are reported as the average fold-increase in total [3H]InsP and calculated as total [3H]InsPs produced by increasing [Ca2+]o/basal [3H]InsPs at 0.5 mm Ca2+. Experiments in both transiently and stably transfected cells were repeated at least three times unless otherwise indicated. Crude membrane protein fractions were prepared from HEK293 cells, electrophoresed on SDS-polyacrylamide electrophoresis gels, and transferred to nitrocellulose membranes as described (3.Chang W. Pratt S. Chen T.-H. Nemeth E. Huang Z. Shoback D. J. Bone Miner. Res. 1998; 13: 570-580Crossref PubMed Scopus (81) Google Scholar, 19.Chang W. Tu C. Bajra R. Komuves L. Miller S. Strewler G. Shoback D. Endocrinology. 1999; 140: 1911-1919Crossref PubMed Google Scholar). Membranes were blotted with one of two affinity-purified rabbit antisera (21825B, 50 nm or 321113A, 10 nm) (3.Chang W. Pratt S. Chen T.-H. Nemeth E. Huang Z. Shoback D. J. Bone Miner. Res. 1998; 13: 570-580Crossref PubMed Scopus (81) Google Scholar, 19.Chang W. Tu C. Bajra R. Komuves L. Miller S. Strewler G. Shoback D. Endocrinology. 1999; 140: 1911-1919Crossref PubMed Google Scholar), and signals were detected with an enhanced chemiluminescence (ECL™) assay kit (Amersham Pharmacia Biotech). Protein expression of all mutant CaR constructs was tested by immunoblotting at least twice along with wt controls. Immunocytochemistry with 3,3′-diaminobenzidene- and fluorescein-conjugated antibodies was performed on cells grown on chamber slides, fixed for 30 min, incubated with primary and secondary antibodies, and then stained as previously detailed (3.Chang W. Pratt S. Chen T.-H. Nemeth E. Huang Z. Shoback D. J. Bone Miner. Res. 1998; 13: 570-580Crossref PubMed Scopus (81) Google Scholar, 19.Chang W. Tu C. Bajra R. Komuves L. Miller S. Strewler G. Shoback D. Endocrinology. 1999; 140: 1911-1919Crossref PubMed Google Scholar). Primary antibody was either a CaR antiserum (21825A; 500 nm), this antiserum preincubated with 100-fold excess peptide, or non-immune rabbit serum. The antiserum was raised against a peptide derived from the extracellular domain of the bovine parathyroid CaR (3.Chang W. Pratt S. Chen T.-H. Nemeth E. Huang Z. Shoback D. J. Bone Miner. Res. 1998; 13: 570-580Crossref PubMed Scopus (81) Google Scholar, 19.Chang W. Tu C. Bajra R. Komuves L. Miller S. Strewler G. Shoback D. Endocrinology. 1999; 140: 1911-1919Crossref PubMed Google Scholar). For 3,3′-diaminobenzidine-staining, slides were counterstained with aqueous hematoxylin. For fluorescein staining, coverslips with cells were mounted on glass slides using Gel-Mount (Biomeda, Forster City, CA) without counterstaining and examined with a Leica TCS NT/SP confocal microscope (Leica Microsystems, Heidelberg, Germany). All mutants were analyzed from at least two separate transfections along with wt CaR-expressing cells. Coverslips were coded and then examined by two blinded observers, who assessed staining patterns without knowledge of the mutation status. Differences between wt and mutant CaR responses were tested by analysis of variance with the F-test using Excel 98 (MicroSoft, Seattle, WA). To assess the importance of residues in the second IC loop of the bovine parathyroid CaR to signal transduction, we constructed seven mutants in which four sequential amino acids were converted to Ala (tandem Ala (TA) constructs) (see Fig. 1 a). The signaling properties of these mutants and the wt CaR were initially assessed in HEK293 cells by their ability to produce InsPs in response to raising [Ca2+]o from 0.5 to 5.0 mm. In cells transiently expressing wt CaRs, this increment in [Ca2+]o produced a 6.0 ± 0.8-fold increase in total InsPs (Fig. 2 a). High [Ca2+]o had no effect on cells transfected with vector only (Fig. 2 a). The ability of the TA mutants of the second IC loop of CaR to increase InsPs varied. InsP responses in cells expressing Ala (708–711) and Ala (712–715) CaR mutants were comparable with the wt CaR (Fig. 2 a). InsP responses to high [Ca2+]o in cells expressing Ala(716–719), Ala(720–723), and Ala(724–727) CaRs were reduced by 30–40% compared with wt CaR (p < 0.01) (Fig. 2 a). The most striking result, however, was seen in cells expressing Ala(700–703) and Ala(704–707) CaR mutants. Their ability to generate InsPs with a 4.5 mm increment in [Ca2+]o was only 1.0 ± 0.2- and 0.1 ± 0.1-fold (p < 0.001versus control), respectively. This was only 17 and 2% of wt CaR responses for the Ala(700–703) CaR and Ala(704–707) CaR, respectively (Fig. 2 a). Immunoblotting and immunocytochemistry with anti-CaR antiserum confirmed that the sizes, intensity, and patterns of the protein bands detected in cells expressing all seven TA mutant CaR constructs were comparable with those of wt CaR-expressing HEK293 cells (Fig.2 b and data not shown). Protein bands in membranes from both wt and mutant CaR-expressing cells were ≈140 and 160 kDa and of equivalent intensity. Thus, differences in the relative quantity or forms of receptor proteins expressed do not explain the reduced signaling properties of these two mutants. To identify specific signaling determinants within the amino-terminal part of the second IC loop, we mutated residues 700–707 individually to Ala and assessed the ability of these point mutants to support InsP production. Cells transiently transfected with L705A and V706A CaR cDNAs responded to raising [Ca2+]o from 0.5 to 5.0 mm with 9.5–10-fold increases in InsPs, comparable with the responses of the wt CaR (10.7 ± 0.3-fold) in these experiments. Cells transiently expressing the mutants T700A, N701A, R702A, and V703A CaRs had mildly reduced InsP responses to the same increment in [Ca2+]o of 6–9-fold. These were ≈15 to 40% lower than the wt CaR (Fig.3 a). The most dramatic defects, however, were observed in cells expressing L704A and F707A CaR mutants. A 4.5 mm increment in [Ca2+]o produced increases in InsPs of only 2.9 ± 0.4 (L704A CaR; p < 0.01 versuswt) and 0.2 ± 0.1-fold (F707A CaR; p < 0.001versus wt), which is only 27 and 2% of wt CaR responses, respectively (Fig. 3 a). Dose-response studies showed that the L704A CaR mutant had an ED50 for Ca2+ that was right-shifted to 7.3 ± 0.09 mm Ca2+ (p < 0.01, compared with ≈5.5 ± 0.2 mm Ca2+for wt CaR; see Fig. 3 b). This mutant and the wt CaR produced comparable maximal InsP responses (R max) of 6.8 ± 1.1 and 7.3 ± 1.1-fold increases at 30 mm Ca2+, respectively. In contrast, the F707A CaR mutant generated anR max of only a 0.91 ± 0.3-fold increase over basal (p < 0.001 versus wt) (Fig.3 b). Immunoblotting and immunocytochemistry confirmed comparable levels of receptor expression for the eight single-site mutants including L704A and F707A CaRs in HEK293 cells (Fig.3 c and data not shown). To confirm that the signaling defects of the IC loop 2 mutants were not related to transient expression, we stably transfected wt and mutant L704A and F707A CaRs into HEK293 cells. Responses of the wt CaR-expressing cells to [Ca2+]o were much greater in stablyversus transiently transfected cells, as previously reported (3.Chang W. Pratt S. Chen T.-H. Nemeth E. Huang Z. Shoback D. J. Bone Miner. Res. 1998; 13: 570-580Crossref PubMed Scopus (81) Google Scholar), due to higher levels of receptor expression. In cells stably expressing wt CaRs, increasing [Ca2+]o from 0.5 to 5.0, 10, or 30 mm increased InsPs by 36.5 ± 2.7-, 47.0 ± 4.6-, or 36.7 ± 5.4-fold, respectively (Fig.3 d). Experiments with cells stably expressing L704A and F707A CaRs confirmed the severe reduction in InsP responses to high [Ca2+]o (see Fig. 3 d), similar to the results from transient transfections. The ED50 was significantly right-shifted from 3.3 ± 0.1 (wt CaR) to 5.1 ± 0.2 mm Ca2+ (L704A CaR) (p< 0.03) (Fig. 3 d), similar to the shift observed in transient transfections (Fig. 3 b). TheR max at 30 mm Ca2+ of the L704A CaR was also significantly reduced to a 21.2 ± 5.6-fold-increase compared with that of the wt CaR, which yielded 47.0 ± 4.5-fold increases in InsPs in stably transfected cells (p < 0.01) (Fig. 3 d). In contrast, we had observed no substantial difference between maximal signaling (at 30 mm Ca2+) of the L704A CaR and the wt CaR in transient transfections (Fig. 3 b). The F707A CaR, however, remained severely impaired in its ability to activate PLC in stably transfected cells. Its ED50 was 6.6 ± 0.2 mm Ca2+, which was significantly greater (p < 0.01) than that of the wt CaR (3.3 ± 0.1 mm Ca2+). The R max of this mutant averaged a 7.3 ± 0.6-fold increase at 30 mm Ca2+, which was only 16% of wt CaR responses (p < 0.001) (Fig. 3 d). Both L704A and F707A CaRs were expressed at levels comparable with wt CaR by immunoblotting and immunocytochemistry (Fig. 3 e and data not shown). Overall, these findings suggest that the signaling defects seen with L704A or F707A CaR mutants were not due to substantial reductions in receptor expression or alterations in receptor processing. These results support the idea that Leu-704 plays a secondary role, whereas Phe-707 is absolutely essential in PLC-mediated signal transduction by the CaR. To test whether the phenyl side chain of Phe-707 is essential for activation of PLC, we mutated this residue to others with functional groups of different sizes and charges (e.g. Val, Leu, Glu, His, Tyr, and Trp). Substitution of Phe-707 with the hydrophobic residues Val or Leu produced CaR mutants that did not respond to raising [Ca2+]o from 0.5 to 5.0 mm(Fig. 3 f). HEK293 cells transiently expressing F707E CaRs, in which Phe was converted to a negatively charged amino acid, were also unresponsive to raising [Ca2+]o to 5.0 mm (Fig. 3 f). Substitution of Phe-707 with positively charged His yielded a CaR mutant whose response to 5 mm Ca2+ was reduced by ≈75% compared with the wt receptor (p < 0.01) (Fig. 3 f). Substitution of Phe-707 with Trp, an even bulkier side group than Phe, generated a CaR mutant that was markedly impaired in its ability to activate PLC (Fig. 3 f). Substitution of Phe-707 with Tyr produced a receptor whose ability to increase InsPs was ≈50% that of wt responses (p < 0.05) (Fig. 3 f). The CaRs mutated at position 707 that we studied were expressed at levels comparable with the wt CaR by immunoblotting (data not shown). These results suggested that there was relatively little tolerance for changes of the nonpolar aromatic side chain of Phe-707 in mediating CaR signaling through PLC. Failure of the Trp substitution to maintain CaR function further suggested that an aromatic side chain larger than Phe also disrupted the function that this residue serves in the CaR. To assess contributions of the third IC loop of the CaR in signal transduction, we next individually mutated amino acids 794–807 to Ala (except 805, which is an Ala in the wt sequence) and assessed the ability of these mutants to activate PLC in HEK293 cells (Fig. 1). Mutations of 11 residues in this region produced CaRs with altered signaling responses, which we divided into two groups (Fig.4 a). Group 1 mutants, including K794A, N801A, and F807A CaRs, were able to increase InsPs with raising [Ca2+]o from 0.5 to 5.0 mm by ≈6.8–7.7-fold. Their responses, however, were ≈55–63% that of the wt CaR (12.2 ± 1.1-fold) and were statistically significantly reduced (p < 0.01versus wt; Fig. 4 a). Group 2 mutants, comprising the eight remaining CaR constructs, were more impaired than group 1 mutants; their InsP responses to the same increment in [Ca2+]o were <35% that of the wt CaR (p < 0.003; see asterisks in Fig.4 a). Dose-response studies were performed on the eight group 2 mutants. Three distinct patterns of responses emerged (Fig. 4, b andc). TheR max of the N803A CaR was a 14.4 ± 0.6-fold increase in InsPs at 30 mm Ca2+, which was comparable with the wt CaR (15.2 ± 1.2-fold). The ED50 of this mutant (≈7.3 mmCa2+) was, however, modestly shifted to the right compared with wt CaR (≈3.5 mm Ca2+) (Fig.4 b). Four mutants exhibited both reducedR max and increased ED50 values (i.e. R796A, K797A, P799A, and K806A CaRs). Their ED50 values ranged from 6 to 8 mmCa2+, and their responses to 30 mmCa2+ were only ≈36–60% that of the wt CaR responses (p < 0.01; Fig. 4 c). Three mutant receptors (L798A, F802A, and E804A CaRs) were unable to activate PLC even at 30 mm Ca2+. Their responses to high [Ca2+]o were only 1.2–1.8-fold above basal (at 0.5 mm Ca2+) and were equivalent to the increases in the vector controls at 30 mm Ca2+(1.4 ± 0.2-fold; Fig. 4 b). To address whether any of the above signaling abnormalities could be attributed to defective receptor expression, we analyzed CaR expression by Western blotting and immunocytochemistry. The levels and patterns of CaR protein bands for both wt and mutant CaRs were similar (Fig.4 d). Immunocytochemistry of cells expressing the Ala mutants was performed and showed that cell surface expression in all but one mutant (E804A CaR) was comparable with the wt CaR (see Fig.5). Cells expressing this mutant had less receptor staining on the membrane and increased staining in intracellular organelles (Fig. 5: wt versus E804A). These observations supported the idea that the marked decreases in PLC activation observed with the Ala mutants of the third IC loop of CaR (except for E804A) were likely due to defective receptor-effector signaling and not due to reduced expression of the receptor. Since E804A CaRs were so aberrant in their expression pattern, their ability/inability to mediate PLC activation could not be tested. The three sites identified above (798, 802, and 804) were then further mutagenized to address the amino acid requirements at these positions to support PLC activation and efficient receptor expression. The observation that a CaR mutant with Ala substituted for Leu-798 did not activate PLC equivalently to the wt CaR led us to hypothesize that the size of the hydrophobic residue at this position was critical for signal transduction. To test this hypothesis, we substituted Leu-798 with amino acids with different types of side chains. As shown in Fig. 6 a, only the substitution of Leu-798 with very closely related Ile produced a receptor that could increase InsPs at 30 mmCa2+ to levels comparable with the wt CaR. The ED50 for the L798I CaR was modestly shifted to the right from 5 to 7.5 mm Ca2+. All other substitutions for Leu-798 (namely Val, Phe, Glu, Pro, and Lys) produced CaRs that were marked defective (i.e. reduced by > 75%) in their ability to activate PLC, even with [Ca2+]o as high as 30 mm (Fig. 6,a and b). The substitution of a basic (Lys) or acidic (Glu) residue for Leu-798 was poorly tolerated, as was the nonpolar rigid side chain of Pro. High [Ca2+]o-induced InsP responses of cells expressing these three mutant CaRs were equivalent to vector controls (Fig. 6 b). Taken together, these findings underscored the importance of a specific, nonpolar hydrocarbon side chain at position 798 in the ability of CaRs to mediate PLC activation. To examine the role of the side chain at position 802 in the CaR in PLC activation, we mutated this Phe to Val, Leu, Glu, His, Tyr, and Trp. As expected, closely related Tyr was essentially interchangeable with Phe. The F802Y CaR mutant increased InsPs similarly to the wt CaR (Fig.7 a). Substitution of Trp, a bulkier residue than Phe, produced a mutant with anR max ≈ 60
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