Homology Modeling of the Transmembrane Domain of the Human Calcium Sensing Receptor and Localization of an Allosteric Binding Site
2004; Elsevier BV; Volume: 279; Issue: 8 Linguagem: Inglês
10.1074/jbc.m307191200
ISSN1083-351X
AutoresSusanne U. Miedlich, Lúcio Gama, Klaus Seuwen, Romain M. Wolf, Gerda E. Breitwieser,
Tópico(s)Biochemical Analysis and Sensing Techniques
ResumoA homology model for the human calcium sensing receptor (hCaR) transmembrane domain utilizing bovine rhodopsin (bRho) structural information was derived and tested by docking the allosteric antagonist, NPS 2143, followed by mutagenesis of predicted contact sites. Mutation of residues Phe-668 (helix II), Arg-680, or Phe-684 (helix III) to Ala (or Val or Leu) and Glu-837 (helix VII) to Ile (or Gln) reduced the inhibitory effects of NPS 2143 on [Ca2+]i responses. The calcimimetic NPS R-568 increases the potency of Ca2+ in functional assays of CaR. Mutations at Phe-668, Phe-684, or Glu-837 attenuated the effects of this compound, but mutations at Arg-680 had no effect. In all cases, mutant CaRs responded normally to Ca2+ or phenylalanine, which act at distinct site(s). Discrimination by the Arg-680 mutant is consistent with the structural differences between NPS 2143, which contains an alkyl bridge hydroxyl group, and NPS R-568, which does not. The homology model of the CaR transmembrane domain robustly accounts for binding of both an allosteric antagonist and agonist, which share a common site, and provides a basis for the development of more specific and/or potent allosteric modulators of CaR. These studies suggest that the bRho backbone can be used as a starting point for homology modeling of even distantly related G protein-coupled receptors and provide a rational framework for investigation of the contributions of the transmembrane domain to CaR function. A homology model for the human calcium sensing receptor (hCaR) transmembrane domain utilizing bovine rhodopsin (bRho) structural information was derived and tested by docking the allosteric antagonist, NPS 2143, followed by mutagenesis of predicted contact sites. Mutation of residues Phe-668 (helix II), Arg-680, or Phe-684 (helix III) to Ala (or Val or Leu) and Glu-837 (helix VII) to Ile (or Gln) reduced the inhibitory effects of NPS 2143 on [Ca2+]i responses. The calcimimetic NPS R-568 increases the potency of Ca2+ in functional assays of CaR. Mutations at Phe-668, Phe-684, or Glu-837 attenuated the effects of this compound, but mutations at Arg-680 had no effect. In all cases, mutant CaRs responded normally to Ca2+ or phenylalanine, which act at distinct site(s). Discrimination by the Arg-680 mutant is consistent with the structural differences between NPS 2143, which contains an alkyl bridge hydroxyl group, and NPS R-568, which does not. The homology model of the CaR transmembrane domain robustly accounts for binding of both an allosteric antagonist and agonist, which share a common site, and provides a basis for the development of more specific and/or potent allosteric modulators of CaR. These studies suggest that the bRho backbone can be used as a starting point for homology modeling of even distantly related G protein-coupled receptors and provide a rational framework for investigation of the contributions of the transmembrane domain to CaR function. The calcium sensing receptor (CaR) 1The abbreviations used are: CaR, calcium sensing receptor; hCaR, human CaR; GPCR, G protein-coupled receptor; mGluR, metabotropic glutamate receptor; GABABR, γ-aminobutyric acid (type B) receptor; bRho, bovine rhodopsin; TM, transmembrane; GFP, green fluorescent protein; EGFP, enhanced GFP; IP, immunoprecipitation; ELISA, enzyme-linked immunosorbent assay; wt, wild type. 1The abbreviations used are: CaR, calcium sensing receptor; hCaR, human CaR; GPCR, G protein-coupled receptor; mGluR, metabotropic glutamate receptor; GABABR, γ-aminobutyric acid (type B) receptor; bRho, bovine rhodopsin; TM, transmembrane; GFP, green fluorescent protein; EGFP, enhanced GFP; IP, immunoprecipitation; ELISA, enzyme-linked immunosorbent assay; wt, wild type. is a member of family C of the G protein-coupled receptor (GPCR) superfamily, which includes metabotropic glutamate receptors (mGluRs), γ-aminobutyric acid receptors (GABABRs), and a large family of putative pheromone and taste receptors. In addition to the seven transmembrane helices, which are the signature characteristic of all GPCRs, members of this family have large extracellular domains (with structural homology to bacterial periplasmic binding proteins) that contain the agonist binding site(s) (1Kunishima N. Shimada Y. Tsuji Y. Sato T. Tamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1096) Google Scholar). CaR signaling includes Gq-mediated activation of phosphatidylinositol phospholipase C, production of inositol 1,4,5-trisphosphate and diacylglycerol, followed by increases in intracellular Ca2+ in all cell types examined, activation of Gi-mediated pathways in some cell types (reviewed in Ref. 2Brown E.M. MacLeod R.J. Physiol. Rev. 2001; 81: 239-297Crossref PubMed Scopus (1216) Google Scholar), and, through an interaction of the CaR carboxyl terminus with filamin A, activation of the mitogen-activated protein kinase cascade (3Hjälm G. MacLeod R.J. Kifor O. Chattopadhyay N. Brown E.M. J. Biol. Chem. 2001; 276: 34880-34887Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 4Awata H. Huang C. Handlogten M.E. Miller R.T. J. Biol. Chem. 2001; 276: 34871-34879Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar).CaR not only binds and is activated by Ca2+ at its agonist binding site (localized to the amino-terminal 500 amino acids) (5Bräuner-Osborne H. Jensen A.A. Sheppard P.O. O'Hara P. Krogsgaard-Larsen P. J. Biol. Chem. 1999; 274: 1882-18386Abstract Full Text Full Text PDF Scopus (155) Google Scholar), but also interacts with allosteric modulators via several sites that have been shown to be distinct from the agonist binding site. CaR activity (in the presence of Ca2+) is allosterically modulated by amino acids (6Conigrave A.D. Quinn S.J. Brown E.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4814-4819Crossref PubMed Scopus (412) Google Scholar), small peptides (7Brown E.M. Katz C. Butters R. Kifor O. J. Bone Miner. Res. 1991; 6: 1217-1225Crossref PubMed Scopus (68) Google Scholar, 8Quinn S.J. Ye C.-P. Diaz R. Kifor O. Bai M. Vassilev P. Brown E. Am. J. Physiol. 1997; 42: C1315-C1323Crossref Google Scholar), as well as a family of structurally related phenylalkylamines (9Nemeth 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 (547) Google Scholar, 10Cohen A. Silverberg S.J. Curr. Opin. Pharmacol. 2002; 2: 734-739Crossref PubMed Scopus (31) Google Scholar). The phenylalkylamines are of particular interest, because both allosteric agonists (calcimimetics) and antagonists (calcilytics) have been identified, typified by NPS R-467 or R-568 (calcimimetics) (9Nemeth 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 (547) Google Scholar) and NPS 2143 (calcilytic) (11Nemeth E.F. Delmar E.G. Heaton W.L. Miller M.A. Lambert L.D. Conklin R.L. Gowen M. Gleason J.G. Bhatnagar P.K. Fox J. J. Pharmacol. Exp. Ther. 2001; 299: 323-331PubMed Google Scholar). The putative binding site(s) for amino acids and potentially for peptides (including poly-l-arginine, protamine, β-amyloid) have been identified within the extracellular agonist binding domain by homology with metabotropic glutamate receptors, and have been localized to a triple serine motif, Ser-169-171 (12Zhang Z. Qiu W. Quinn S.J. Conigrave A.D. Brown E.M. Bai M. J. Biol. Chem. 2002; 277: 33727-33735Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Mutant CaRs, which are no longer modulated by amino acids, still exhibit altered potency of Ca2+ in the presence of phenylalkylamines (13Zhang Z. Jiang Y. Quinn S.J. Krapcho K. Nemeth E.F. Bai M. J. Biol. Chem. 2002; 277: 33736-33741Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), confirming that the phenylalkylamines interact at a distinct site. Chimeras between CaR and mGluRs have shown that the phenylalkylamine binding site is localized to the transmembrane domain of CaR (13Zhang Z. Jiang Y. Quinn S.J. Krapcho K. Nemeth E.F. Bai M. J. Biol. Chem. 2002; 277: 33736-33741Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 14Hauache O.M. Hu J. Ray K. Xie R. Jacobson K.A. Spiegel A.M. Endocrinology. 2000; 141: 4156-4163Crossref PubMed Google Scholar, 15Ray K. Northup J. J. Biol. Chem. 2002; 277: 18908-18913Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and recent studies have suggested that negatively charged residues within the e2 and e3 loops may contribute to binding of NPS R-568 (15Ray K. Northup J. J. Biol. Chem. 2002; 277: 18908-18913Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 16Hu 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 (83) Google Scholar). Identification and characterization of the phenylalkylamine binding site on CaR might provide a basis for development of more specific compounds capable of modulating CaR activity on the background of the relatively constant extracellular Ca2+ concentrations observed in vivo.With one high resolution crystal structure of a GPCR available (bovine rhodopsin bRho (17Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (4992) Google Scholar)), any homology modeling of GPCRs based on experimental structural information remains speculative and is only useful in combination with reliable experimental data. Implications of the structure of bRho on generating models for other GPCRs are addressed by Ballesteros and Palczweski (Ref. 18Ballesteros J. Palczewski K. Curr. Opin. Drug Discov. Dev. 2001; 4: 561-574PubMed Google Scholar). Various attempts to build and refine atomic-level GPCR models from structural information (for example, see reviews in Refs. 19Gershengorn M.C. Osman R. Endocrinology. 2001; 141: 2-10Crossref Scopus (114) Google Scholar, 20Ballesteros J. Weinstein H. Methods Neurosci. 1995; 25: 366-428Crossref Scopus (2419) Google Scholar, 21Bikker J.A. Trumpp-Kallmeyer S. Humblet C. J. Med. Chem. 1998; 41: 2911-2927Crossref PubMed Scopus (128) Google Scholar, 22Flower D.R. Biochim. Biophys. Acta. 1999; 1422: 207-234Crossref PubMed Scopus (219) Google Scholar) and/or first principles (e.g. 23Pogozheva I.D. Lomize A.L. Mosberg H.I. Biophys. J. 1997; 70: 1963-1985Abstract Full Text PDF Scopus (143) Google Scholar, 24Vaidehi N. Floriano W.B. Trabanino R. Hall S.E. Freddolino P. Jung Choi E. Zamakos G. Goddard III, W.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12622-12627Crossref PubMed Scopus (252) Google Scholar) have been reported. In this work, we have opted for the pragmatic assumption that the backbone of bRho is a reasonable starting point for building a model of the 7TM region of human CaR, despite the fact that CaR is not a member of the rhodopsin family of GPCRs. Furthermore, distinctions regarding active or inactive morphologies in the structure were not incorporated into the model. We have assumed that the ligands of interest interact primarily with the helical transmembrane (7TM) parts of the receptor and refrained from building the extra- and intracellular loops (except for extracellular loop 1, connecting helices II and III, see below and in "Experimental Procedures"). Here we report the general features of the model for the transmembrane domain of human CaR, as well as the identification of residues predicted to contact the phenylalkylamines NPS 2143 and NPS R-568. Mutations at the predicted residues eliminate or severely attenuate the efficacy of the allosteric compounds, whereas the abilities of Ca2+ or the amino acid phenylalanine to activate the receptor are largely unaffected. In addition to providing a basis for the development of more specific and/or potent allosteric modulators of CaR, these studies provide an example of the generalizability of the bRho backbone as a starting point for homology modeling of even distantly related GPCRs.EXPERIMENTAL PROCEDURESGeneration of the Homology Model—The helical parts of bRho were isolated from the x-ray coordinates (protein data base file 1F88.pdb) and used as a template. The selection was moderately extended on both ends of the helices. The final sequence fragments of bRho chosen to serve as a template for model building are those listed in Table I. A specific routine was used to perform the sequence alignment of the 7TM domain of human CaR to the selected residues of the corresponding regions in bRho, taking into account that no insertions or deletions can be applied for the superposition of helices. In addition to the bRho sequence, the consensus sequence of the entire rhodopsin family was used in this procedure. The alignment was optimized with regard to various physical properties of the amino acids such as their formal charge, hydrophobicity, and size of side chains. Once a given sequence alignment was established, the side chains in bRho were replaced by the corresponding ones of CaR, using version 2.9 of the SCWRL program (25Bower M.J. Cohen F.E. Dunbrack Jr., R.L. J. Mol. Biol. 1997; 267: 1268-1282Crossref PubMed Scopus (482) Google Scholar, 26Dunbrack Jr., R.L. Proteins: Struct. Funct. Genet. Suppl. 1999; 3: 81-87Crossref PubMed Scopus (66) Google Scholar) with the side-chain conformation library bbdep01. Jul.sortlib. The resulting structure was refined by molecular mechanics, using the parm94 parameter set of AMBER (27Cornell W.D. Cieplak P. Bayly C.I. Gould I.R. Merz Jr., K.M. Ferguson D.M. Spellmeyer D.C. Fox T. Caldwell J.W. Kollman P.A. J. Am. Chem. Soc. 1995; 117: 5179-5197Crossref Scopus (11452) Google Scholar) with the conjugate gradient minimizer as implemented in our in-house software Wit!P. The energy minimization was carried out with all Cα atoms being fixed at this stage. Electrostatics were evaluated with the original force field charges, using a distance-dependent dielectric function with ϵ = 1 × r. Helices II and III (presumed shorter than in bRho, according to the sequence alignment in Table I) were then connected to form the extracellular loop 1, and the final structure was again refined, this time allowing all the residues connecting helices II and III to move freely without any constraints.Table IAlignment of the human CaR sequence to the 7TM regions of bRho and the rhodopsin family consensus sequence Open table in a new tab The ligand NPS 2143 was assigned standard AMBER atom types resulting in acceptable force field parameters for this type of structure. Partial charges were computed with the MPEOE method in the Wit!P software. These charges are close to the RESP charges to be expected for such connectivities and were considered as suitable for the current work. Docking of the ligand into the receptor was carried out manually. The starting point for docking was the presumed close interaction between Glu-837 and the charged nitrogen on the ligand. The rest of the ligand was then positioned by avoiding severe steric overlap with the receptor, trying to embed the aromatic (i.e. hydrophobic) groups as deeply as possible into the receptor while respecting the internal strain of the ligand. The eventual refinement of the entire complex was again carried out by conjugated gradient minimization with the same settings as for the receptor alone, but this time allowing all Cα in the receptor to move freely within 0.5 Å, applying a harmonic force of 10 kcal/mol/Å2 beyond that distance ("tethering"). The resulting geometry is essentially free of strain in the framework of the applied force field. The docking of NPS R-568 into CaR was performed in the same way as for NPS 2143, however, the docking mode for the latter was used as the starting point.Mutagenesis and Cell Transfections—Point mutations were produced in the CaR-EGFP background (fusion protein previously described in Ref. 28Gama L. Breitwieser G.E. J. Biol. Chem. 1998; 273: 29712-29718Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) by site-directed PCR mutagenesis and confirmed by restriction endonuclease digestion and direct sequencing. Sequences of primer pairs used for mutagenesis are available upon request. HEK-293 cells (American Type Culture Collection, Rockville, MD) were grown in high glucose Dulbecco's modified Eagle's medium (Invitrogen, Grand Island, NY), supplemented with 10% fetal bovine serum (Invitrogen), penicillin (50 units/ml), streptomycin (50 μg/ml), and amphotericin B (2 μg/ml) at 37 °C, 5% CO2. For transient transfections, 1 μg of each CaR construct, 2 μl of NovaFECTOR (Venn Nova LLC, Pompano, FL), and 100 μl of medium were premixed, then added to HEK-293 cells in a 24-well plate, supplemented with regular medium as previously described (28Gama L. Breitwieser G.E. J. Biol. Chem. 1998; 273: 29712-29718Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Experiments were carried out 68-72 h after transfection.IP Formation Assay—Transiently transfected HEK-293 cells were seeded to confluence in 24-well plates and maintained for 1 day in full medium before labeling with myo[3H]inositol (100 MBq/ml; ART/Anawa Trading, Wangen, Switzerland) for 24 h in serum-free Dulbecco's modified Eagle's medium. Cells were then washed and incubated at 37 °C in a phosphate-free physiologic salt solution, and experiments were carried out exactly as previously described (45Mailland M. Waelchli R. Ruat M. Boddeke H.G. Seuwen K. Endocrinology. 1997; 138: 3601-3605Crossref PubMed Scopus (87) Google Scholar).Enzyme-linked Immunoassay to Quantitate CaR Surface Localization—Transiently transfected cells were replated onto 96-well poly-l-lysine-precoated plates (BD Biosciences, Bedford, MA) after 24 h. Cells were fixed 68-72 h after transfection in 4% paraformaldehyde (30 min) and blocked with 5% fetal bovine serum in phosphate-buffered saline for 60 min (at room temperature). Subsequently, cells were incubated with 100 μl of a 1:500 dilution of the primary antibody (polyclonal antibody produced against an amino-terminal epitope (LRG, residues 374-391, first described by Goldsmith et al. (29Goldsmith P.K. Fan G. Miller J.L. Rogers K.V. Spiegel A.M. J. Bone Miner. Res. 1997; 12: 1780-1788Crossref PubMed Scopus (45) Google Scholar), Genemed Synthesis, Inc., San Francisco, CA) for 60 min, then with 100 μl of a 1:1000 dilution of the secondary antibody (horseradish peroxidase-conjugated anti-rabbit IgG, Amersham Biosciences, Piscataway, NJ). The 3,3′,5,5′-tetramethylbenzidine liquid substrate system (Sigma, St. Louis, MO) was used to develop the plate; reactions were halted with 1 m H2SO4. The plate was analyzed on a PowerWaveX™ microplate scanning spectrophotometer at 450 nm (BIOTEK Instruments, Inc., Winooski, VT).Measurement of Intracellular Ca2+—Single cell fluorescence measurements of [Ca2+]i were made as previously described (28Gama L. Breitwieser G.E. J. Biol. Chem. 1998; 273: 29712-29718Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 32Miedlich S.U. Gama L. Breitwieser G.E. J. Biol. Chem. 2002; 277: 49691-49699Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 33Breitwieser G.E. Gama L. Am. J. Physiol. 2001; 280: C1412-C1421Crossref PubMed Google Scholar). Solutions containing the calcilytic NPS 2143 N-[(R)-2-hydroxy-3-(2-cyano-3-chlorophenoxy)propyl]-1,1-dimethyl-3-(2-naphthyl)ethylamine (Fig. 1A) or the calcimimetic NPS R-568 (R)-N-(3-methoxy-α-phenylethyl)-3-(2′-chlorophenyl)-1-propylamine hydrochloride (Fig. 1B) were prepared from stock solutions in Me2SO and added to the extracellular solutions immediately prior to the experiment. NPS R-568 was synthesized as described by Nemeth et al. (9Nemeth 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 (547) Google Scholar); NPS 2143 was prepared as in a previous study (46Doggrell S.A. Del Fresno M. Castaner J. Drugs Future. 2002; 27: 140-142Crossref Scopus (4) Google Scholar). All other chemicals were obtained from Sigma Chemical Co. Me2SO had no effect on the parameters measured when tested under identical experimental conditions. All experiments were carried out at room temperature (22-24 °C). 10-20 cells were analyzed during each experiment; experiments were repeated in at least three independent transfections unless otherwise noted. Selected regions of cells were excited at 340/380 nm and emitted light collected at 510 nm at intervals of 10 s using an imaging system (Universal Imaging Corp., West Chester, PA) based on the MetaFluor software package. Background images were obtained at the beginning of each experiment from an area devoid of cells. Calibration of [Ca2+]i was done with a series of buffered calcium standards (Molecular Probes Inc., Eugene, OR) assuming a Kd of 145 nm determined in vitro at 22 °C (30Haugland R.P. Handbook of Fluorescent Probes and Research Products. 9th Ed. Molecular Probes, Eugene, OR2002: 775Google Scholar).Fig. 1Compound structures. A, calcilytic NPS 2143, N-[(R)-2-hydroxy-3-(2-cyano-3-chlorophenoxy)propyl]-1,1-dimethyl-3-(2-naphthyl)ethylamine. B, calcimimetic NPS R-568, (R)-N-(3-methoxy-α-phenylethyl)-3-(2′-chlorophenyl)-1-propylamine hydrochloride.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Data Analysis—Average [Ca2+]i in a given condition was calculated as the average of at least 10 consecutive time points for each cell analyzed. For studies of intracellular Ca2+ oscillations, a cell was characterized as exhibiting oscillations if three or more consecutive peaks could be clearly differentiated, and the amplitude of the peaks was ≥ 10 nm [Ca2+]i. Data were fitted to the Hill equation by least squares minimization using the Marquardt-Levenberg algorithm (NFIT, Island Products, Galveston, TX). All statistical analyses were done with SPSS 10.0 (SPSS Inc., Chicago, IL) and SigmaPlot 2000 for Windows version 6.0 (SPSS Inc.). To compare the fractions of cells having intracellular Ca2+ oscillations under distinct conditions, Chi-squared tests or Fisher's exact tests, for group frequencies of less than 5 cells, were applied. A p value of <0.05 was considered significant.RESULTSSequence Alignment of hCaR with Bovine Rhodopsin—The alignment shown in Table I is the result of an iterative process following several rounds of model building and experimental verification or falsification. For all helices, the global location in the sequence is consistent with earlier estimates based on hydrophobicity analysis. Possible alignments with a good overall score (see "Experimental Procedures") were taken as a starting point; the final alignment emanates from additional information provided by the mutational studies. The very low homology of the 7TM domain of human CaR to that of bRho (the template) or to the rhodopsin family consensus, and the absence of various key residues usually conserved in the rhodopsin family, did not allow the straightforward use of standard sequence alignment tools. Nevertheless, the alignment algorithm that was used worked well for helices I, III, V, VI, and VII. Difficulties were encountered particularly for helix IV, and the alignment in Table I was ultimately chosen based on the proline doublet toward the C-terminal of the helix. Note that helix IV is not involved in any direct interactions with the ligands considered here, and experimental data challenging this alignment are therefore not available. Similar problems were encountered for helix II. In this case, however, the mutational studies demonstrating the importance of Phe-668 over Phe-667 in ligand binding provided critical evidence in favor of the alignment shown.Building the Model and Docking the Ligands—Selection of residues to be mutated was originally based on a model for human CaR, which was built using the Baldwin Cα trace of rhodopsin (31Baldwin J.M. Schertler G.F.X. Unger V.M. J. Mol. Biol. 1997; 272: 144-164Crossref PubMed Scopus (632) Google Scholar). Glu-837 at helix VII was selected as the main anchor point for binding of NPS 2143, and the ligand was embedded as described under "Experimental Procedures"; mutational studies concentrated on amino acids that were within a distance of 6 Å from the ligand in the preliminary 3-dimensional model, preferentially considering residues within the helices rather than within the loops. The primary results from the mutations were then incorporated into the refinement of the model of the CaR·NPS 2143 complex, which also resulted in refinement of the sequence alignment. Fig. 2A illustrates the predicted location of the allosteric modulator site at the extracellular transmembrane interface. Fig. 2B shows the results of docking trials using the calcilytic NPS 2143 and indicates the residues predicted to participate in bonding interactions with the ligand. The final model indicates that if the carboxylic group of Glu-837 forms a salt bridge with the protonated amino group in NPS 2143, then Phe-668 and Phe-684 may form hydrophobic interactions with the planar rings of the ligand. Additional interactions are suggested with Arg-680. As mentioned above, in earlier models based on different possible sequence alignments, it was also possible that Phe-667 might interact with the ligand. The final results from mutation studies (see below) indicate that this residue is not involved in the binding of the ligand, supporting the model presented here.Fig. 2Model for NPS 2143 binding to human CaR. A, location of NPS 2143 with respect to the seven transmembrane helices of CaR. Model was generated as discussed under "Experimental Procedures." B, close up view of hCaR residues (shown in ball and stick representation, with enclosing transparent molecular surface), which contribute to the binding of NPS 2143 (shown as a Cory-Pauling-Koltun space-filling model). The predominant interaction is the salt bridge to Glu-837 via the charged nitrogen in the ligand. Arg-680 can interact with the -OH group in the ligand, and Phe-668 and Phe-684 make close hydrophobic contacts and π-stacking with ligand rings. For both panels A and B, roman numerals indicate helix number.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Initial Characterization of Predicted Ligand Interaction Sites—The initial model predicted potential interaction(s) of NPS 2143 with Phe-667 or Phe-668 in helix II, Arg-680 and Phe-684 in helix III, and Glu-837 in helix VII at the extracellular interface. Point mutations at each site were generated, with substitutions of Ala, Leu, and Val for each phenylalanine, and Ile and Gln for Glu-837. To screen out mutants that were inefficiently trafficked to the plasma membrane, we performed ELISA assays utilizing paraformaldehyde-fixed, non-permeabilized cells. The data, presented in Fig. 3, indicate that most mutants were well expressed at the plasma membrane; exceptions were the F668A, F668L, F668V and R680L, R680V mutants, which were present at ≤40% of wt CaR levels. As a preliminary screen of mutants for functional activity, HEK-293 cells were transiently transfected with wild type CaR-EGFP or a mutated CaR and IP formation was measured. A measure of the general functionality of the mutants was obtained from the responses to a range of extracellular Ca2+ concentrations (2, 5, and 8 mm) (Fig. 4A), the relative potency of the allosteric antagonist NPS 2143 (at 0.1, 1, and 10 μm) was determined in the presence of 8 mm Ca2+ (Fig. 4B), and the potency of the allosteric agonist NPS R-568 (at 1 and 10 μm) was determined in the presence of 2 mm Ca2+ (Fig. 4C). Wild type CaR is activated in a concentration-dependent manner by extracellular Ca2+; NPS 2143 causes a progressive decrease in IP formation at 8 mm Ca2+, whereas NPS R-568 causes a dramatic increase in IP formation at 2 mm Ca2+. Mutations at position Phe-667 had activation characteristics comparable to wild type CaR (Table II), whereas the adjacent position Phe-668 exhibits normal activation by Ca2+ but attenuated responses to either NPS 2143 or NPS R-568 (Fig. 4, A-C). Mutations at positions Phe-684 and Glu-837 also exhibit attenuated responses to both allosteric ligands. Finally, mutations at position Arg-680 attenuate responses to NPS 2143 but have no effect on activation by extracellular Ca2+ and NPS R-568 relative to wt CaR (Fig. 4).Fig. 3Plasma membrane localization of wild type CaR and various point mutants. ELISA assay of wt CaR-EGFP and CaR-EGFP bearing individual point mutations at the locations indicated. Cells were treated as described under "Experimental Procedures"; the horizontal dashed line denotes the signal derived from untransfected HEK-293 cells (nonspecific signal). Bars represent means ± S.E. obtained from at least three independent transfections. Plasma membrane localization is characterized in arbitrary absorbance units (A.U.).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4IP formation of wt CaR and receptors with point mutations at predicted NPS 2143 contact sites. Experiments were performed as described under "Experimental Procedures." For all panels: closed circles, wt CaR; open circles, F668A
Referência(s)