Insights into Interactions between the α-Helical Region of the Salmon Calcitonin Antagonists and the Human Calcitonin Receptor using Photoaffinity Labeling
2005; Elsevier BV; Volume: 280; Issue: 31 Linguagem: Inglês
10.1074/jbc.m503272200
ISSN1083-351X
AutoresVi Pham, Maoqing Dong, John D. Wade, Laurence J. Miller, Craig J. Morton, Hooi‐Ling Ng, Michael W. Parker, Patrick M. Sexton,
Tópico(s)Receptor Mechanisms and Signaling
ResumoFish-like calcitonins (CTs), such as salmon CT (sCT), are widely used clinically in the treatment of bone-related disorders; however, the molecular basis for CT binding to its receptor, a class II G protein-coupled receptor, is not well defined. In this study we have used photoaffinity labeling to identify proximity sites between CT and its receptor. Two analogues of the antagonist sCT(8-32) containing a single photolabile p-benzoyl-l-phenylalanine (Bpa) residue in position 8 or 19 were used. Both analogues retained high affinity for the CT receptor and potently inhibited agonist-induced cAMP production. The [Bpa19]sCT(8-32) analogue cross-linked to the receptor at or near the equivalent cross-linking site of the full-length peptide, within the fragment Cys134-Lys141 (within the amino terminus of the receptor, adjacent to transmembrane 1) (Pham, V., Wade, J. D., Purdue, B. W., and Sexton, P. M. (2004) J. Biol. Chem. 279, 6720-6729). In contrast, proteolytic mapping and mutational analysis identified Met49 as the cross-linking site for [Bpa8]sCT(8-32). This site differed from the previously identified cross-linking site of the agonist [Bpa8]human CT (Dong, M., Pinon, D. I., Cox, R. F., and Miller, L. J. (2004) J. Biol. Chem. 279, 31177-31182) and may provide evidence for conformational differences between interaction with active and inactive state receptors. Molecular modeling suggests that the difference in cross-linking between the two Bpa8 analogues can be accounted for by a relatively small change in peptide orientation. The model was also consistent with cooperative interaction between the receptor amino terminus and the receptor core. Fish-like calcitonins (CTs), such as salmon CT (sCT), are widely used clinically in the treatment of bone-related disorders; however, the molecular basis for CT binding to its receptor, a class II G protein-coupled receptor, is not well defined. In this study we have used photoaffinity labeling to identify proximity sites between CT and its receptor. Two analogues of the antagonist sCT(8-32) containing a single photolabile p-benzoyl-l-phenylalanine (Bpa) residue in position 8 or 19 were used. Both analogues retained high affinity for the CT receptor and potently inhibited agonist-induced cAMP production. The [Bpa19]sCT(8-32) analogue cross-linked to the receptor at or near the equivalent cross-linking site of the full-length peptide, within the fragment Cys134-Lys141 (within the amino terminus of the receptor, adjacent to transmembrane 1) (Pham, V., Wade, J. D., Purdue, B. W., and Sexton, P. M. (2004) J. Biol. Chem. 279, 6720-6729). In contrast, proteolytic mapping and mutational analysis identified Met49 as the cross-linking site for [Bpa8]sCT(8-32). This site differed from the previously identified cross-linking site of the agonist [Bpa8]human CT (Dong, M., Pinon, D. I., Cox, R. F., and Miller, L. J. (2004) J. Biol. Chem. 279, 31177-31182) and may provide evidence for conformational differences between interaction with active and inactive state receptors. Molecular modeling suggests that the difference in cross-linking between the two Bpa8 analogues can be accounted for by a relatively small change in peptide orientation. The model was also consistent with cooperative interaction between the receptor amino terminus and the receptor core. Calcitonins (CTs) 1The abbreviations used are: CT, calcitonin; CTR, calcitonin receptor; hCT, human calcitonin; hCTR, human calcitonin receptor; Bpa, p-benzoyl-l-phenylalanine; [Bpa8]sCT(8-32), [Arg11,18, Bpa8]sCT(8-32); [Bpa19]sCT(8-32), [Arg11,18, Bpa19]sCT(8-32); BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; HA, hemagglutinin; HPLC, high performance liquid chromatography; PTH, parathyroid hormone; sCT, salmon calcitonin; TM, transmembrane; TMH, transmembrane helix; WT, wild-type; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. 1The abbreviations used are: CT, calcitonin; CTR, calcitonin receptor; hCT, human calcitonin; hCTR, human calcitonin receptor; Bpa, p-benzoyl-l-phenylalanine; [Bpa8]sCT(8-32), [Arg11,18, Bpa8]sCT(8-32); [Bpa19]sCT(8-32), [Arg11,18, Bpa19]sCT(8-32); BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; HA, hemagglutinin; HPLC, high performance liquid chromatography; PTH, parathyroid hormone; sCT, salmon calcitonin; TM, transmembrane; TMH, transmembrane helix; WT, wild-type; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. are 32-amino acid peptide hormones with a wide spectrum of biological activity. The most recognized action is the inhibition of osteoclast-mediated bone resorption, which forms the basis for its primary clinical use in the treatment of bone-related disorders such as Paget disease, osteoporosis, and hypercalcemia of malignancy (1.Sexton P.M. Findlay D.M. Martin T.J. Curr. Med. Chem. 1999; 6: 1067-1093PubMed Google Scholar, 2.Pondel M. Int. J. Exp. Pathol. 2000; 81: 405-422Crossref PubMed Scopus (125) Google Scholar, 3.Purdue B.W. Tilakaratne N. Sexton P.M. Receptors Channels. 2002; 8: 243-255Crossref PubMed Scopus (89) Google Scholar). CT, however, also has activity that includes modulation of renal ion excretion (4.Hosking D.J. Gilson D. Q. J. Med. 1984; 53: 359-368PubMed Google Scholar, 5.Carney S.L. Clin. Exp. Pharmacol. Physiol. 1992; 19: 433-438Crossref PubMed Scopus (11) Google Scholar, 6.De Rouffignac C. Elalouf J.M. Roinel N. Pflugers Arch. 1991; 419: 472-477Crossref PubMed Scopus (18) Google Scholar, 7.Muff R. Kaufmann M. Born W. Fischer J.A. Endocrinology. 1994; 134: 1593-1596Crossref PubMed Scopus (10) Google Scholar), analgesia (8.Gennari C. Bone (N.Y.). 2002; 30: 67S-70SCrossref PubMed Scopus (61) Google Scholar), inhibition of appetite (9.Chait A. Suaudeau C. De Beaurepaire R. Brain Res. Bull. 1995; 36: 467-472Crossref PubMed Scopus (30) Google Scholar), and gastric acid secretion (10.Demol P. Hotz J. Muller M.K. Trompeter R. Muttathil J. Goebell H. Arch. Int. Physiol. Biochim. 1986; 94: 331-338PubMed Google Scholar, 11.Lenz H.J. Klapdor R. Hester S.E. Webb V.J. Galyean R.F. Rivier J.E. Brown M.R. Gastroenterology. 1986; 91: 905-912Abstract Full Text PDF PubMed Scopus (39) Google Scholar, 12.Guidobono F. Netti C. Pagani F. Bettica P. Sibilia V. Pecile A. Zanelli J. Farmaco. 1991; 46: 555-563PubMed Google Scholar), as well as effects on reproduction via effects on embryological implantation and sperm function (13.Fraser L.R. Adeoya-Osiguwa S.A. Vitam. Horm. 2001; 63: 1-28Crossref PubMed Scopus (24) Google Scholar, 14.Adeoya-Osiguwa S.A. Fraser L.R. Mol. Reprod. Dev. 2003; 65: 228-236Crossref PubMed Scopus (23) Google Scholar, 15.Kumar S. Brudney A. Cheon Y.P. Fazleabas A.T. Bagchi I.C. Biol. Reprod. 2003; 68: 1318-1323Crossref PubMed Scopus (13) Google Scholar). Calcitonin receptors (CTRs) belong to the class II subfamily of G protein-coupled receptors, which also includes receptors for other peptides such as parathyroid hormone (PTH) and PTH-related peptide, secretin, vasoactive intestinal peptide, glucagon, glucagon-like peptide-1, growth hormone-releasing hormone, calcitonin gene-related peptide, and corticotropin-releasing factor. These peptide hormone class II G protein-coupled receptors share 30-50% amino acid identity as well as a number of conserved structural motifs and are thought to interact with their ligands in a similar manner (16.Gether U. Endocr. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1002) Google Scholar, 17.Gether U. Asmar F. Meinild A.K. Rasmussen S.G. Pharmacol. Toxicol. 2002; 91: 304-312Crossref PubMed Scopus (73) Google Scholar, 18.Pham V. Sexton P.M. J. Pept. Sci. 2004; 10: 179-203Crossref PubMed Scopus (20) Google Scholar). Alternative RNA splicing yields multiple CTR mRNA isoforms. In man, at least six potential variants exist (19.Gorn A.H. Lin H.Y. Yamin M. Auron P.E. Flannery M.R. Tapp D.R. Manning C.A. Lodish H.F. Krane S.M. Goldring S.R. J. Clin. Investig. 1992; 90: 1726-1735Crossref PubMed Scopus (198) Google Scholar, 20.Frendo J.L. Pichaud F. Mourroux R.D. Bouizar Z. Segond N. Moukhtar M.S. Jullienne A. FEBS Lett. 1994; 342: 214-216Crossref PubMed Scopus (38) Google Scholar, 21.Nakamura M. Hashimoto T. Nakajima T. Ichii S. Furuyama J. Ishihara Y. Kakudo K. Biochem. Biophys. Res. Commun. 1995; 209: 744-751Crossref PubMed Scopus (24) Google Scholar, 22.Albrandt K. Brady E.M. Moore C.X. Mull E. Sierzega M.E. Beaumont K. Endocrinology. 1995; 136: 5377-5384Crossref PubMed Google Scholar, 23.Gorn A.H. Rudolph S.M. Flannery M.R. Morton C.C. Weremowicz S. Wang T.Z. Krane S.M. Goldring S.R. J. Clin. Investig. 1995; 95: 2680-2691Crossref PubMed Scopus (97) Google Scholar, 24.Moore E.E. Kuestner R.E. Stroop S.D. Grant F.J. Matthewes S.L. Brady C.L. Sexton P.M. Findlay D.M. Mol. Endocrinol. 1995; 9: 959-968Crossref PubMed Google Scholar, 25.Nussenzveig D.R. Mathew S. Gershengorn M.C. Endocrinology. 1995; 136: 2047-2051Crossref PubMed Google Scholar, 26.Chen W.J. Armour S. Way J. Chen G. Watson C. Irving P. Cobb J. Kadwell S. Beaumont K. Rimele T. Kenakin T. Mol. Pharmacol. 1997; 52: 1164-1175Crossref PubMed Scopus (70) Google Scholar); however, the most common hCTR isoforms differ by the presence (hCTRb) or absence (hCTRa) of a 16-amino acid insert between amino acids 174 and 175, within the first intracellular loop of the receptor (23.Gorn A.H. Rudolph S.M. Flannery M.R. Morton C.C. Weremowicz S. Wang T.Z. Krane S.M. Goldring S.R. J. Clin. Investig. 1995; 95: 2680-2691Crossref PubMed Scopus (97) Google Scholar). Of these, the hCTRa is the major human receptor isoform and is expressed in essentially all tissues known to express the CTR. CTs from different species can be subdivided into three major classes: human/rodent, artiodactyl, and teleost/avian. Of these, the members of the teleost/avian group are generally the most potent, although relative potency varies in a species- and isoform-specific manner (19.Gorn A.H. Lin H.Y. Yamin M. Auron P.E. Flannery M.R. Tapp D.R. Manning C.A. Lodish H.F. Krane S.M. Goldring S.R. J. Clin. Investig. 1992; 90: 1726-1735Crossref PubMed Scopus (198) Google Scholar, 27.Niall H.D. Keutmann H.T. Copp D.H. Potts Jr., J.T. Proc. Natl. Acad. Sci. U. S. A. 1969; 64: 771-778Crossref PubMed Scopus (130) Google Scholar, 28.Findlay D.M. de Luise M. Michelangeli V.P. Ellison M. Martin T.J. Cancer Res. 1980; 40: 1311-1317PubMed Google Scholar, 29.Nicholson G.C. Moseley J.M. Sexton P.M. Mendelsohn F.A. Martin T.J. J. Clin. Investig. 1986; 78: 355-360Crossref PubMed Scopus (412) Google Scholar, 30.Nicholson G.C. D'Santos C.S. Evans T. Moseley J.M. Kemp B.E. Martin T.J. Biochem. J. 1988; 253: 505-510Crossref PubMed Scopus (8) Google Scholar, 31.Nicholson G.C. D'Santos C.S. Evans T. Moseley J.M. Kemp B.E. Michelangeli V.P. Martin T.J. Biochem. J. 1988; 250: 877-882Crossref PubMed Scopus (30) Google Scholar). The higher potency combined with a longer in vivo half-life has led to fish-like CTs, exemplified by salmon CT (sCT), being the principle form of CT used for the clinical treatment of bone disorders (32.Gonzalez D. Ghiringhelli G. Mautalen C. Calcif. Tissue Int. 1986; 38: 71-75Crossref PubMed Scopus (22) Google Scholar, 33.Zaidi M. Inzerillo A.M. Troen B. Moonga B. Abe E. Burckhardt P. Bilezikian J.P. Raisz L.G. Rodan G.A. Principles of Bone Biology. 2. Academic Press, San Diego, CA2002: 1423-1440Google Scholar). However, the usefulness of CT is limited by the development of clinical resistance. This can be due to the development of circulating antibodies against non-human CT (34.Singer F.R. Aldred J.P. Neer R.M. Krane S.M. Potts Jr., J.T. Bloch K.J. J. Clin. Investig. 1972; 51: 2331-2338Crossref PubMed Scopus (109) Google Scholar, 35.Plehwe W.E. Hudson J. Clifton-Bligh P. Posen S. Med. J. Aust. 1977; 1: 577-581Crossref PubMed Scopus (14) Google Scholar, 36.Singer F.R. Fredericks R.S. Minkin C. Arthritis Rheum. 1980; 23: 1148-1154Crossref PubMed Scopus (93) Google Scholar, 37.Tagliaro F. Dorizzi R. Luisetto G. Horm. Metab. Res. 1995; 27: 31-34Crossref PubMed Scopus (6) Google Scholar, 38.Grauer A. Ziegler R. Raue F. Exp. Clin. Endocrinol. Diabetes. 1995; 103: 345-351Crossref PubMed Scopus (37) Google Scholar), but it also occurs from loss of responsiveness to CT, presumably via receptor down-regulation and inhibition of new receptor synthesis (39.Ikegame M. Ejiri S. Ozawa H. J. Bone Miner. Res. 1994; 9: 25-37Crossref PubMed Scopus (24) Google Scholar, 40.Takahashi S. Goldring S. Katz M. Hilsenbeck S. Williams R. Roodman G.D. J. Clin. Investig. 1995; 95: 167-171Crossref PubMed Google Scholar, 41.Wada S. Martin T.J. Findlay D.M. Endocrinology. 1995; 136: 2611-2621Crossref PubMed Google Scholar). The optimal use of CTs remains unresolved, a situation that stems in part from lack of understanding of the bimolecular interaction between CT and its receptor and how this leads to receptor activation. Like other class II receptor ligands, CT has a diffuse pharmacophore with residues throughout the peptide sequence contributing to binding affinity and/or agonist potency. Whereas it is clear that much of the binding energy for high affinity interaction of the peptide and its receptor derives from interaction of the peptide with the amino-terminal extracellular domain of the receptor (42.Stroop S.D. Kuestner R.E. Serwold T.F. Chen L. Moore E.E. Biochemistry. 1995; 34: 1050-1057Crossref PubMed Scopus (92) Google Scholar, 43.Bergwitz C. Gardella T.J. Flannery M.R. Potts Jr., J.T. Kronenberg H.M. Goldring S.R. Juppner H. J. Biol. Chem. 1996; 271: 26469-26472Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 44.Stroop S.D. Nakamuta H. Kuestner R.E. Moore E.E. Epand R.M. Endocrinology. 1996; 137: 4752-4756Crossref PubMed Scopus (62) Google Scholar), detailed information on how receptor and ligand interact is scant. Photoaffinity labeling, which relies on the spatially restricted (within 3.1 Å) cross-linking of photolabile amino acids within peptide ligands and their receptors, presents a mechanism for establishing proximity between individual amino acids of a peptide ligand and small fragments (or even individual amino acids) of the receptor. This provides essential information about nature of the interaction. We have previously shown that position 19 of sCT is in close contact with the receptor region Cys134-Lys141 (45.Pham V. Wade J.D. Purdue B.W. Sexton P.M. J. Biol. Chem. 2004; 279: 6720-6729Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In human CT (hCT), amino acid 16, located one helical turn apart from residue 19, cross-linked to Phe137, consistent with orientation of the α-helix of agonist peptides with the membrane-proximal region of the receptor amino terminus (46.Dong M. Pinon D.I. Cox R.F. Miller L.J. J. Biol. Chem. 2004; 279: 1167-1175Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). We have also demonstrated that hCT amino acid 26 interacts with Thr30 in the distal amino terminus (46.Dong M. Pinon D.I. Cox R.F. Miller L.J. J. Biol. Chem. 2004; 279: 1167-1175Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), whereas hCT amino acid 8 interacts with Leu368 in the third extracellular loop (47.Dong M. Pinon D.I. Cox R.F. Miller L.J. J. Biol. Chem. 2004; 279: 31177-31182Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Amino-terminal truncation of the disulfide-bridged loop (amino acids 1-7) of sCT leads to the generation of potent antagonists (48.Feyen J.H. Cardinaux F. Gamse R. Bruns C. Azria M. Trechsel U. Biochem. Biophys. Res. Commun. 1992; 187: 8-13Crossref PubMed Scopus (42) Google Scholar, 49.Pozvek G. Hilton J.M. Quiza M. Houssami S. Sexton P.M. Mol. Pharmacol. 1997; 51: 658-665Crossref PubMed Scopus (36) Google Scholar) and allows for comparative analysis of the site of cross-linking of equivalent amino acids of agonist and antagonist peptides. In this study we have generated antagonist peptides substituted with the photolabile amino acid p-benzoyl-l-phenylalanine (Bpa) at residue 8 ([Bpa8]sCT(8-32)) and 19 ([Bpa19]sCT(8-32)) and compared their interaction with the published sites of interaction for full-length agonist peptides. The data show that the [Bpa19]sCT(8-32) analogue cross-links to the equivalent domain of the receptor labeled by the full-length peptide. In contrast, [Bpa8]sCT(8-32) cross-linked to Met49 in the distal amino terminus, a region quite distinct from the site labeled by [Bpa8]hCT. Materials—125I-Na (specific activity, 2200 Ci/mmol) was purchased from Amersham Biosciences. sCT, sCT(8-32), hCT, and all amino acid derivatives were synthesized by Auspep (Parkville, Australia). CNBr, protein G-agarose beads, 3-isobutyl-1-methylxanthine, and iodoacetamide were obtained from Sigma. Sequencing grade endoproteinases Lys-C and Asp-N, N-glycosidase F, and Complete protease inhibitor mixture tablets were purchased from Roche Applied Science. Endoglycosidase F was prepared as previously described (50.Pearson R.K. Miller L.J. Hadac E.M. Powers S.P. J. Biol. Chem. 1987; 262: 13850-13856Abstract Full Text PDF PubMed Google Scholar). Bovine serum albumin (BSA) was from ICN. Penicillin G/streptomycin was from Multicell Technologies (Warwick, RI). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, HEPES, fungizone, Lipofectamine transfection reagent, and protein molecular mass markers were obtained from Invitrogen. Tissue culture disposables and plastic ware were purchased from Falcon (Bedford, MA). Synthesis of the sCT(8-32) Photoactive Analogues—The two sCT(8-32) antagonist analogues were synthesized with a Bpa moiety incorporated into either position 8 (designated as [Bpa8]sCT(8-32)) or position 19 (designated as [Bpa19]sCT(8-32)), where 8 is the most amino-terminal amino acid (Fig. 1). In addition, for each of the photoactive analogues, lysine residues at positions 11 and 18 in the native sCT sequence were replaced with arginines to render the ligand resistant to enzymatic cleavage by endoproteinase Lys-C; this substitution does not alter activity of the peptide (51.D'Santos C.S. Nicholson G.C. Moseley J.M. Evans T. Martin T.J. Kemp B.E. Endocrinology. 1988; 123: 1483-1488Crossref PubMed Scopus (19) Google Scholar). Both photoactive peptide analogues were prepared by solid phase peptide synthesis as described previously (45.Pham V. Wade J.D. Purdue B.W. Sexton P.M. J. Biol. Chem. 2004; 279: 6720-6729Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Analysis of the synthetic sCT analogues, [Bpa19]sCT(8-32) and [Bpa8]sCT(8-32), by matrix-assisted laser desorption ionization time-of-flight mass spectrometry showed the principal products with the expected molecular masses (Bpa8 = 2937.6 Da and Bpa19 = 2925 Da), and amino acid analysis further validated the authenticity of the analogues. The synthetic peptides were purified to homogeneity by analytical reversed-phase high performance liquid chromatography (HPLC) in good overall yield. Iodination of Peptides—sCT, [Bpa8]sCT(8-32), and [Bpa19]sCT(8-32) were iodinated using chloramine-T method, as described previously, with specific activity of about 700 Ci/mmol (29.Nicholson G.C. Moseley J.M. Sexton P.M. Mendelsohn F.A. Martin T.J. J. Clin. Investig. 1986; 78: 355-360Crossref PubMed Scopus (412) Google Scholar, 45.Pham V. Wade J.D. Purdue B.W. Sexton P.M. J. Biol. Chem. 2004; 279: 6720-6729Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In some experiments, the [Bpa8]sCT(8-32) peptide analogue was also radiolabeled using IODO-BEAD (Pierce) as a solid-phase oxidant of a chloramine-T analogue followed by reversed-phase HPLC (52.Powers S.P. Pinon D.I. Miller L.J. Int. J. Pept. Protein Res. 1988; 31: 429-434Crossref PubMed Scopus (97) Google Scholar). Briefly, 15 μg of the [Bpa8]sCT(8-32) peptide was solubilized in 100 μl of 40% acetonitrile followed by 100 μl of 0.2 m borate buffer, pH 9.0, and 10 μl of Na125I (1 mCi). This reaction mixture was exposed to IODO-BEAD for 15 s and subsequently diluted with 0.5 ml of 0.1% trifluoroacetic acid before purification by reversed-phase HPLC on a C18 column. Fractions containing the radioactive peaks (specific activity, ∼ 2000 Ci/mmol) from the HPLC were collected and stored in 50-μl aliquots at -20 °C until use. Receptor Mutagenesis—The WT HA-hCTRa (Leu447 polymorphic variant) was generated as described previously (45.Pham V. Wade J.D. Purdue B.W. Sexton P.M. J. Biol. Chem. 2004; 279: 6720-6729Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The CTR mutants M48I, M59L, and M376L were generated using QuikChange site-directed mutagenesis (Stratagene), using the WT HA-hCTRa as template. All other receptor mutants were generated as previously described (45.Pham V. Wade J.D. Purdue B.W. Sexton P.M. J. Biol. Chem. 2004; 279: 6720-6729Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Oligonucleotide primer pairs (sense and antisense) were synthesized by GeneWorks (Hindmarsh, Australia). Selected clones were chosen for plasmid isolation (Qiagen), and the fidelity of mutations was confirmed by nucleotide sequencing (Australian Genome Research Facility, Parkville, Australia). Once the authenticity of mutagenesis was confirmed by sequencing, large-scale plasmid preparations were purified using a Qiagen kit (Qiagen). Cell Culture and DNA Transfection—Monkey kidney epithelium (COS-7 and COS-1) or human embryonic kidney HEK-293 cells stably expressing the hCTRa were maintained in complete DMEM supplemented with either 5% heat-inactivated fetal bovine serum or fetal clone-2 (Hyclone Laboratories, Logan, UT), 100 units/ml penicillin G, 100 μg/ml streptomycin, 16 mm HEPES, and 50 μg/ml fungizone at 37 °C in a humidified atmosphere of 95% air/5% CO2. For radioligand receptor binding assay, COS-7 cells were seeded into 24-well plates. For cAMP assay and cross-linking studies, COS-7 cells were grown in 60-cm2 dishes and 140-cm2 dishes, respectively. Once the cell monolayers were at 95% confluence, cells were transfected in serum- and antibiotic-free DMEM using 0.1, 3, or 7 μg of plasmid DNA for 24-well plates, 60-cm2 dishes, and 140-cm2 dishes, respectively, as instructed by the manufacturer. Before addition to cells, DNA was complexed with the transfection lipid reagent Lipofectamine at a ratio of 1 μl/0.1 μg DNA. Following transfection, cells were incubated for at least 4 h at 37 °C in a CO2 incubator, and the culture medium was replaced with complete DMEM as described above. All transient transfections were performed with plasmids encoding either the WT HA-hCTRa or the required mutant constructs. Transfected cells were grown for at least 48 h before radioligand binding studies, cAMP assay, or photoaffinity labeling. Receptor Binding Assays—The binding of the radiolabeled analogues to HA-hCTRa was assessed in 24-well plates as previously described (45.Pham V. Wade J.D. Purdue B.W. Sexton P.M. J. Biol. Chem. 2004; 279: 6720-6729Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Briefly, the cells were incubated with ∼90 pm 125I-sCT, 125I-sCT(8-32), or [125I-Bpa]sCT(8-32) analogue in binding buffer (DMEM and 0.1% BSA) in the absence (total binding) or presence of varying concentrations of unlabeled peptides. Cells were incubated for 1 h at 37 °C and subsequently washed with phosphate-buffered saline and lysed with 0.5 m NaOH. Nonspecific binding was determined in wells containing 1 μm unlabeled sCT(8-32), and maximal specific binding at each competing ligand concentration was calculated as a percentile of the total specific binding observed in the absence of competitor. The entire cell lysate was counted in a PerkinElmer Life Sciences γ-irradiation counter to determine the bound radioactivity. cAMP Assays—Intracellular cAMP assay was performed in 384-well plates using an Alpha Screen cAMP kit (PerkinElmer Life Sciences). In brief, transiently transfected COS-7 cells in 60-cm2 dishes were harvested, counted, and resuspended in stimulation buffer (phenol red-free DMEM, 0.1% BSA, and 1 mm 3-isobutyl-1-methylxanthine) and preincubated at 37 °C for ∼20 min. Required concentrations of peptides and cAMP were prepared in stimulation buffer. To generate a cAMP standard curve, increasing concentrations of cAMP (10-11 to 10-6 m) were added to wells. To generate agonist dose-response curves, cells (10,000 cells/well) were then incubated at 37 °C for 30 min in the presence of increasing concentrations of agonists (10-13 to 10-8 m), either sCT or sCT analogues. To generate antagonist dose-response curves, cells (10,000 cells/well) were incubated at 37 °C for 30 min with increasing concentrations of hCT (10-12 to 10-5 m) in the presence of 0, 10-6, 10-7, or 10-8 m of the antagonist sCT(8-32) or its analogues. Following incubation, cells were lysed with lysis buffer (5 mm HEPES, 0.3% Tween 20, and 0.1% BSA). Anti-cAMP acceptor beads (7.5 μg/ml), which were prepared in lysis buffer, were added to all wells and incubated at room temperature for 30 min in the dark. The detection mix of biotinylated cAMP (5 mm)/streptavidin donor beads (10 μg/ml) in lysis buffer, which was preincubated in the dark for 30 min at room temperature, was then added to all wells. The assay plate was incubated overnight at room temperature before reading on a Fusion plate reader (PerkinElmer Life Sciences). For each experiment, forskolin and cAMP dose-response curves were performed in parallel to allow translation of the α-screen signal to either cAMP or a percentage of the maximum forskolin response. Data were analyzed using Graphpad Prism 4.02. (San Diego, CA). In each assay, the quantity of cAMP generated was back-calculated from the raw data using a cAMP standard curve. For agonist responses, concentration-effect curves were fitted to a four-parameter logistic equation (53.Motulsky H. Christopoulos A. Fitting Models to Biological Data using Linear and Nonlinear Regression: A Practical Guide to Curve Fitting. GraphPad Software Inc., San Diego, CA2003Google Scholar). For calculation of antagonist potency, agonist concentration-response curves in the absence and presence of antagonist were globally fitted to the following equation using Prism (53.Motulsky H. Christopoulos A. Fitting Models to Biological Data using Linear and Nonlinear Regression: A Practical Guide to Curve Fitting. GraphPad Software Inc., San Diego, CA2003Google Scholar): Response = Emin+(Emax−Emin)[A]nH[A]nH+(10−pEC50[1+([B]/10−pA2)s])nH(Eq. 1) where Emax represents the maximal asymptote of the concentration-response curves, Emin represents the lowest asymptote of the concentration-response curves, pEC50 represents the negative logarithm of the agonist EC50 in the absence of antagonist, [A] represents the concentration of the agonist, [B] represents the concentration of the antagonist, nH represents the Hill slope of the agonist curve, s represents the Schild slope for the antagonist, and pA2 represents the negative logarithm of the concentration of antagonist that shifts the agonist EC50 by a factor of 2. Photoaffinity Labeling of Analogues to the Receptors and Mapping of the Binding Domain—The radiolabeled photoactive peptide [Bpa19]sCT(8-32) or [Bpa8]sCT(8-32) was cross-linked to hCTRs expressed in intact COS-7 cells followed by multiple enzymatic/chemical cleavage as previously described (45.Pham V. Wade J.D. Purdue B.W. Sexton P.M. J. Biol. Chem. 2004; 279: 6720-6729Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Briefly, WT or mutated receptor HA-hCTRa transiently transfected into COS-7 cells was incubated for 1 h in darkness with binding buffer (DMEM and 0.1% BSA) containing ∼900 pm 125I-Bpa-substituted sCT(8-32). Labeled cells were washed with phosphate-buffered saline and immediately irradiated for 30 min with a 365-nm UV lamp on ice. Cells were collected in 0.1 m Tris and 10 mm EDTA, pH 7.3, and subsequently subjected to immunoprecipitation according to the methodology of Sengstag et al. (54.Sengstag C. Stirling C. Schekman R. Rine J. Mol. Cell. Biol. 1990; 10: 672-680Crossref PubMed Scopus (52) Google Scholar). The precipitated receptors were then subjected to enzymatic or chemical cleavage using methods described previously (45.Pham V. Wade J.D. Purdue B.W. Sexton P.M. J. Biol. Chem. 2004; 279: 6720-6729Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). For the [Bpa8]sCT(8-32) probe, a modified approach was also used because it yielded more complete digestion of the receptor. Briefly, plasma membranes from COS-1 cells transiently expressing WT or mutant receptor (M59L, M376L, or M48I) containing 50 μg of protein were incubated with 0.1 nm 125I-[Bpa8]sCT(8-32) in Krebs-Ringer-HEPES buffer (0.25 m HEPES, pH 7.4, 1.04 m NaCl, 50 mm KCl, 10 mm KH2PO4, and 12 mm MgSO4)in the dark for 1 h. The reaction was immediately photolysed in a Rayonet photochemical reactor (Southern New England Ultraviolet, Hamden, CT) at 4 °C for 30 min and then washed twice with Krebs-Ringer-HEPES buffer. Photolabeled membranes were then subjected to reduction/alkylation (45.Pham V. Wade J.D. Purdue B.W. Sexton P.M. J. Biol. Chem. 2004; 279: 6720-6729Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) or solubilized in 1× Tris-glycine SDS sample buffer for SDS-PAGE followed by elution, lyophilization, ethanol/acetone precipitation, and endoglycosidase F/CNBr cleavage (55.Hadac E.M. Pinon D.I. Ji Z. Holicky E.L. Henne R.M. Lybrand T.P. Miller L.J. J. Biol. Chem. 1998; 273: 12988-12993Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Polyacrylamide Electrophoresis and Autoradiography—Radioactive protein samples were warmed to 70 °C for 10 min and then analyzed using a combination of 10% SDS-glycine, 16.5% SDS-Tricine, or 10% NuPAGE bis-Tris pre-cast gels (Invitrogen), depending on the molecular weight of the protein of interest. Following electrophoresis, if necessary, gels were stained with Coomassie Blue G,
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