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• W2009274925 abstract "The principal receptor-binding domain (Ser17–Val31) of parathyroid hormone (PTH) is predicted to form an amphiphilic α-helix and to interact primarily with the N-terminal extracellular domain (N domain) of the PTH receptor (PTHR). We explored these hypotheses by introducing a variety of substitutions in region 17–31 of PTH-(1–31) and assessing, via competition assays, their effects on binding to the wild-type PTHR and to PTHR-delNt, which lacks most of the N domain. Substitutions at Arg20 reduced affinity for the intact PTHR by 200-fold or more, but altered affinity for PTHR-delNt by 4-fold or less. Similar effects were observed for Glu substitutions at Trp23, Leu24, and Leu28, which together form the hydrophobic face of the predicted amphiphilic α-helix. Glu substitutions at Arg25, Lys26, and Lys27 (which forms the hydrophilic face of the helix) caused 4–10-fold reductions in affinity for both receptors. Thus, the side chains of Arg20, together with those composing the hydrophobic face of the ligand's putative amphiphilic α-helix, contribute strongly to PTHR-binding affinity by interacting specifically with the N domain of the receptor. The side chains projecting from the opposite helical face contribute weakly to binding affinity by different mechanisms, possibly involving interactions with the extracellular loop/transmembrane domain region of the receptor. The data help define the roles that side chains in the binding domain of PTH play in the PTH-PTHR interaction process and provide new clues for understanding the overall topology of the bimolecular complex. The principal receptor-binding domain (Ser17–Val31) of parathyroid hormone (PTH) is predicted to form an amphiphilic α-helix and to interact primarily with the N-terminal extracellular domain (N domain) of the PTH receptor (PTHR). We explored these hypotheses by introducing a variety of substitutions in region 17–31 of PTH-(1–31) and assessing, via competition assays, their effects on binding to the wild-type PTHR and to PTHR-delNt, which lacks most of the N domain. Substitutions at Arg20 reduced affinity for the intact PTHR by 200-fold or more, but altered affinity for PTHR-delNt by 4-fold or less. Similar effects were observed for Glu substitutions at Trp23, Leu24, and Leu28, which together form the hydrophobic face of the predicted amphiphilic α-helix. Glu substitutions at Arg25, Lys26, and Lys27 (which forms the hydrophilic face of the helix) caused 4–10-fold reductions in affinity for both receptors. Thus, the side chains of Arg20, together with those composing the hydrophobic face of the ligand's putative amphiphilic α-helix, contribute strongly to PTHR-binding affinity by interacting specifically with the N domain of the receptor. The side chains projecting from the opposite helical face contribute weakly to binding affinity by different mechanisms, possibly involving interactions with the extracellular loop/transmembrane domain region of the receptor. The data help define the roles that side chains in the binding domain of PTH play in the PTH-PTHR interaction process and provide new clues for understanding the overall topology of the bimolecular complex. Parathyroid hormone (PTH) 2The abbreviations used are: PTH, parathyroid hormone; PTHR, parathyroid hormone receptor; PTHrP, parathyroid hormone-related protein; N domain, N-terminal extracellular domain; J domain, juxtamembrane domain; IP, inositol phosphate; Aib, α-aminoisobutyric acid; Har, homoarginine; Bpa, para-benzoyl-l-phenylalanine; Bp, benzophenone; Cha, cyclohexylalanine; HPLC, high pressure liquid chromatography; Nle, norleucine; rPTH, rat parathyroid hormone; PipGly, (S)-4-piperidyl-N-amidino)glycine; Apa, (S)-2-amino-4-((2-amino)pyrimidinyl)butanoic acid; Gph, l-(4-guanidino)phenylalanine. 2The abbreviations used are: PTH, parathyroid hormone; PTHR, parathyroid hormone receptor; PTHrP, parathyroid hormone-related protein; N domain, N-terminal extracellular domain; J domain, juxtamembrane domain; IP, inositol phosphate; Aib, α-aminoisobutyric acid; Har, homoarginine; Bpa, para-benzoyl-l-phenylalanine; Bp, benzophenone; Cha, cyclohexylalanine; HPLC, high pressure liquid chromatography; Nle, norleucine; rPTH, rat parathyroid hormone; PipGly, (S)-4-piperidyl-N-amidino)glycine; Apa, (S)-2-amino-4-((2-amino)pyrimidinyl)butanoic acid; Gph, l-(4-guanidino)phenylalanine. plays a key role in calcium and phosphate homeostasis and has potent effects on the bone-remodeling process. PTH interacts with a Class 2 G protein-coupled receptor that is prominently expressed in bone osteoblasts and in cells located in the proximal and distal portions of the renal convoluted tubules. The PTH receptor (PTHR) is also expressed in the primordia of developing long bones, heart, mammary glands, and other tissues, where it mediates the morphogenic actions of PTH-related protein (PTHrP) (1Strewler G.J. N. Engl. J. Med. 2000; 342: 177-185Crossref PubMed Scopus (357) Google Scholar). For both PTH and PTHrP, the bioactive portions of the molecule reside within the first 34 amino acids of the processed polypeptides. Within region 1–34, the principal determinants of receptor-binding affinity and receptor-signaling activity map to the C- and N-terminal domains, respectively (2Tregear G.W. Van Rietschoten J. Greene E. Keutmann H.T. Niall H.D. Reit B. Parsons J.A. Potts Jr., J.T. Endocrinology. 1973; 93: 1349-1353Crossref PubMed Scopus (274) Google Scholar, 3Nussbaum S.R. Rosenblatt M. Potts Jr., J.T. J. Biol. Chem. 1980; 255: 10183-10187Abstract Full Text PDF PubMed Google Scholar). Solution-phase NMR studies of PTH-(1–34)-based ligands typically show a well formed α-helix in the region of the C-terminal binding domain (4Pellegrini M. Royo M. Rosenblatt M. Chorev M. Mierke D.F. J. Biol. Chem. 1998; 273: 10420-10427Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 5Chen Z. Xu P. Barbier J.-R. Willick G. Ni F. Biochemistry. 2000; 39: 12766-12777Crossref PubMed Scopus (43) Google Scholar). This C-terminal α-helix, extending approximately from Ser17 to Val31 (5Chen Z. Xu P. Barbier J.-R. Willick G. Ni F. Biochemistry. 2000; 39: 12766-12777Crossref PubMed Scopus (43) Google Scholar), exhibits strong amphiphilic character (6Neugebauer W. Surewicz W.K. Gordon H.L. Somorjai R.L. Sung W. Willick G.E. Biochemistry. 1992; 31: 2056-2063Crossref PubMed Scopus (57) Google Scholar, 7Epand R.E. Mol. Cell. Biol. 1983; 57: 41-47Google Scholar, 8Barbier J.-R. Gardella T.J. Dean T. MacLean S. Potetinova Z. Whitfield J.F. Willick G.E. J. Biol. Chem. 2005; 280: 23771-23777Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). PTH analog substitution studies have shown that the side chains of Trp23, Leu24, and Leu28, which form the hydrophobic face of this α-helix, are particularly important for efficient interaction with the receptor (9Gardella T.J. Wilson A.K. Keutmann H.T. Oberstein R. Potts Jr., J.T. Kronenberg H.M. Nussbaum S.R. Endocrinology. 1993; 132: 2024-2030Crossref PubMed Scopus (70) Google Scholar, 10Oldenburg K.R. Epand R.F. D'Orfani A. Vo K. Selick H. Epand R.M. J. Biol. Chem. 1996; 271: 17582-17591Abstract Full Text Full Text PDF PubMed Google Scholar, 11Reidhaar-Olson J. Davis R. De Souza-Hart J. Selick H. Mol. Cell. Endocrinol. 2000; 160: 135-147Crossref PubMed Scopus (10) Google Scholar). The mechanism by which PTH interacts with its receptor has been investigated via the approaches of ligand analog design, receptor mutagenesis, and photochemical cross-linking (reviewed in Ref. 12Gensure R.C. Gardella T.J. Jüppner H. Biochem. Biophys. Res. Commun. 2005; 328: 666-678Crossref PubMed Scopus (252) Google Scholar). The view that has emerged from these studies is that the overall mechanism consists of two principal and, to some extent, autonomous components: 1) an interaction between the C-terminal helical domain of the ligand and the N-terminal extracellular domain (N domain; spanning Tyr23 to approximately Ile190) of the mature receptor and 2) an interaction between the N-terminal portion of the ligand and the juxtamembrane domain (J domain) of the receptor containing the extracellular loops and seven transmembrane helices. The N domain component of the interaction is thought to provide the major portion of binding energy and stability to the complex, and the J domain component is thought to mediate the conformational changes involved in receptor activation (13Hoare S.R.J. Gardella T.J. Usdin T.B. J. Biol. Chem. 2001; 276: 7741-7753Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). It now seems likely that most, if not all, of the 15 or so other Class 2 G protein-coupled receptors utilize a similar two-site binding mechanism for interacting with their cognate peptide ligands (14Tan Y.V. Couvineau A. Murail S. Ceraudo E. Neumann J.M. Lacapere J.J. Laburthe M. J. Biol. Chem. 2006; 281: 12792-12798Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 15Grace C.R. Perrin M.H. DiGruccio M.R. Miller C.L. Rivier J.E. Vale W.W. Riek R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12836-12841Crossref PubMed Scopus (183) Google Scholar, 16Dong M. Pinon D.I. Miller L.J. Mol. Endocrinol. 2005; 19: 1821-1836Crossref PubMed Scopus (13) Google Scholar). Consistent with such a two-site binding mechanism for PTH and the PTHR, we have shown that N-terminal PTH peptide fragments, such as PTH-(1–14), bind only extremely weakly to the receptor, but can nevertheless induce at least measurable increases in cAMP levels in PTHR-expressing cells (17Luck M.D. Carter P.H. Gardella T.J. Mol. Endocrinol. 1999; 13: 670-680Crossref PubMed Google Scholar). The potency of such N-terminal PTH fragments can be significantly enhanced by introducing substitutions that improve the affinity of the ligand for the PTHR J domain (18Dean T. Linglart A. Mahon M.J. Bastepe M. Jüppner H. Potts Jr., J.T. Gardella T.J. Mol. Endocrinol. 2006; 20: 931-942Crossref PubMed Scopus (66) Google Scholar, 19Shimizu M. Potts Jr., J.T. Gardella T.J. J. Biol. Chem. 2000; 275: 21836-21843Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 20Shimizu N. Guo J. Gardella T.J. J. Biol. Chem. 2001; 276: 49003-49012Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Thus, the analog [Aib1,3,Gln10,Har11,Trp14]PTH-(1–14)-NH2 is equipotent to PTH-(1–34) in stimulating cAMP as well as inositol phosphate (IP) production in PTHR-expressing cells. Moreover, such optimized N-terminal PTH analogs retain full potency in cells expressing PTHR-delNt, a PTHR construct that lacks most (Ala24–Arg181) of the N domain, whereas unmodified PTH-(1–34) exhibits at least 100-fold reductions in potency and affinity for PTHR-delNt compared with its actions on the intact PTHR (18Dean T. Linglart A. Mahon M.J. Bastepe M. Jüppner H. Potts Jr., J.T. Gardella T.J. Mol. Endocrinol. 2006; 20: 931-942Crossref PubMed Scopus (66) Google Scholar, 19Shimizu M. Potts Jr., J.T. Gardella T.J. J. Biol. Chem. 2000; 275: 21836-21843Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 20Shimizu N. Guo J. Gardella T.J. J. Biol. Chem. 2001; 276: 49003-49012Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The large reduction in affinity/potency that unmodified PTH-(1–34) exhibits for PTHR-delNt can be attributed, according to the two-site interaction model, to the loss of the binding interactions that normally occur between the C-terminal helical domain of the ligand and the N domain of the receptor. The cross-linking approach has indeed established spatial proximities between PTH residues 23, 27, and 28, when substituted with para-benzoyl-l-phenylalanine (Bpa), and the N domain of the receptor (21Mannstadt M. Luck M.D. Gardella T.J. Jüppner H. J. Biol. Chem. 1998; 273: 16890-16896Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 22Gensure R.C. Gardella T.J. Jüppner H. J. Biol. Chem. 2001; 276: 28650-28658Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). On the other hand, [Lys27(Bp)2]PTH-(1–34)-NH2, which contains the photoreactive Bp moiety attached to the Lys27 side chain amino groups, was shown to cross-link to the first extracellular loop of the PTHR (23Greenberg Z. Bisello A. Mierke D.F. Rosenblatt M. Chorev M. Biochemistry. 2000; 39: 8142-8152Crossref PubMed Scopus (69) Google Scholar). This observation raised the possibility that the C-terminal binding domain of PTH can functionally interact with the receptor J domain. Consistent with this possibility, we recently showed that methylation of several backbone nitrogen atoms in region 17–31 of PTH-(1–31) impairs, albeit modestly, the capacity of the ligand to stimulate cAMP formation in cells expressing PTHR-delNt (8Barbier J.-R. Gardella T.J. Dean T. MacLean S. Potetinova Z. Whitfield J.F. Willick G.E. J. Biol. Chem. 2005; 280: 23771-23777Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Taken together, these observations point to the uncertainty that exists in our understanding of the specific mechanisms by which the C-terminal domain of PTH contributes to the PTHR-binding process. This study was undertaken to examine further the mode of action used by the C-terminal binding domain of PTH. We sought to address the roles that the side chains in this domain play in the PTHR-binding process, the general functional importance of amphiphilicity, and the potential for binding interactions with the receptor J domain. Our strategy was to introduce a variety of conservative and non-conservative substitutions in region 17–31 of PTH-(1–31)-NH2 and to assess their effects on binding to the intact PTHR and to PTHR-delNt. The overall results indicate a dominant role for specific interactions between side chains on the hydrophobic face of the C-terminal helix and the receptor N-terminal domain. They also provide evidence for weak interactions between side chains projecting from the hydrophilic face of the helix and the PTHR J domain. The data also shed new light on the overall topology of the bimolecular complex, as they support folding of the bound ligand and proximity of the binding sites in the N and J domains of the receptor. Peptide Synthesis—Peptides were based on the human PTH-(1–31)-NH2 sequence (SVSEIQLMHNLGKHLNSMERVEWLRKKLQDV-NH2). The alanine substitutions were incorporated into this otherwise unmodified PTH-(1–31)-NH2 scaffold. Subsequently, cyclohexylalanine (Cha) and Glu substitutions were incorporated into the [Ala1,Arg19]PTH-(1–31)-NH2 scaffold, which, because of the Ser1 → Ala and Glu19 → Arg substitutions, exhibits improved affinity for PTHR-delNt (19Shimizu M. Potts Jr., J.T. Gardella T.J. J. Biol. Chem. 2000; 275: 21836-21843Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 24Shimizu M. Shimizu N. Tsang J.C. Petroni B.D. Khatri A. Potts Jr., J.T. Gardella T.J. Biochemistry. 2002; 41: 13224-13233Crossref PubMed Scopus (24) Google Scholar). These Ala-, Cha-, and Glu-substituted PTH-(1–31)-NH2 and [Ala1,Arg19]PTH-(1–31)-NH2 peptides and their corresponding parental controls were synthesized by the Massachusetts General Hospital Biopolymer Core facility using conventional methodologies as we described previously (20Shimizu N. Guo J. Gardella T.J. J. Biol. Chem. 2001; 276: 49003-49012Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Additional analogs of PTH-(1–31)-NH2 with substitutions at position 20 were prepared as part of a previous study (25Barbier J.-R. MacLean S. Whitfield J.F. Morley P. Willick G.E. Biochemistry. 2001; 40: 8955-8961Crossref PubMed Scopus (9) Google Scholar). All peptides were verified by analytical HPLC, matrix-assisted laser desorption ionization mass spectrometry, and amino acid analysis, and peptide concentrations of stock solutions were established by amino acid analysis. The radioligands 125I-[Nle8,21,Tyr34]rPTH-(1–34)-NH2 and 125I-[Aib1,3,Nle8,Gln10,Har11,Ala12,Trp14,Tyr15]rPTH-(1–15)-NH2 (henceforth referred to as 125I-[Aib1,3,M]rPTH-(1–15)-NH2) were prepared by the oxidative chloramine-T procedure using Na125I (specific activity of 2200 Ci/mmol; PerkinElmer Life Sciences) and purified by reversed-phase HPLC. Circular Dichroism—CD spectra were obtained on a Jasco J-600 spectropolarimeter at 20 °C. Four spectra were averaged, and the data were smoothed by the Jasco software. The instrument was calibrated with ammonium (+)-10-camphorsulfonate. Data are expressed as the number of helical residues/peptide chain as calculated from –[θ]222 × 30/28,000, where [θ]222 is the mean residue ellipticity at 222 nm, as we described previously (8Barbier J.-R. Gardella T.J. Dean T. MacLean S. Potetinova Z. Whitfield J.F. Willick G.E. J. Biol. Chem. 2005; 280: 23771-23777Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Cell Culture—Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (HyClone, Logan UT), 100 units/ml penicillin G, and 100 μg/ml streptomycin sulfate (Invitrogen). For binding and cAMP experiments performed with the intact PTHR, the HKRK-B7 and ROS 17/2.8 cell lines were used. HKRK-B7 cells are derived from the porcine kidney cell line LLC-PK1 and express, via stable DNA transfection, the wild-type human PTHR at an approximate surface density of 950,000 PTH-binding sites/cell (26Takasu H. Guo J. Bringhurst F. J. Bone Miner. Res. 1999; 14: 11-20Crossref PubMed Scopus (84) Google Scholar). ROS 17/2.8 cells are rat osteosarcoma cells and express the endogenous PTHR at an approximate surface density of 70,000 PTH-binding sites/cell (27Yamamoto I. Shigeno C. Potts Jr., J.T. Segre G.V. Endocrinology. 1988; 122: 1208-1217Crossref PubMed Scopus (100) Google Scholar). The cells were plated and assayed in 24-well plates. PTHR-delNt was derived from the human PTHR by site-directed mutagenesis and lacks most (Ala24–Arg181) of the N domain (28Shimizu M. Carter P.H. Khatri A. Potts Jr., J.T. Gardella T.J. Endocrinology. 2001; 142: 3068-3074Crossref PubMed Scopus (62) Google Scholar). PTHR-delNt was expressed in COS-7 cells via transient DNA transfection. For binding assays, cell membranes were prepared from the transfected COS-7 cells. To increase the maximum binding of 125I-[Aib1,3,M]rPTH-(1–15)-NH2 to PTHR-delNt in these membranes, the cells were cotransfected with a negative-dominant mutant Gαs protein (Gαs(α3β5/Gly226 → Ala/Ala366 → Ser); hereafter referred to as GαsND). This mutant Gαs subunit is thought to couple to cognate receptors, and thus stabilize high affinity receptor conformations more efficiently than does wild-type Gαs without increasing basal cAMP levels (29Berlot C.H. J. Biol. Chem. 2002; 277: 21080-21085Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). We recently used a precursor of this Gαs mutant, Gαs(α3β5), to increase the binding of 125I-[Aib1,3,M]rPTH-(1–15)-NH2 to PTHR-delNt in COS-7 cell membranes (18Dean T. Linglart A. Mahon M.J. Bastepe M. Jüppner H. Potts Jr., J.T. Gardella T.J. Mol. Endocrinol. 2006; 20: 931-942Crossref PubMed Scopus (66) Google Scholar). We subsequently found that GαsND, which contains the same five-amino acid replacement of the corresponding Gαi residues in the α3β5 loop as does Gαs(α3β5) plus the point mutations Gly226 → Ala, which increases affinity for Gβ/γ, and Ala366 → Ser, which decreases affinity for GDP (29Berlot C.H. J. Biol. Chem. 2002; 277: 21080-21085Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), yielded ∼2-fold higher levels of specific binding of 125I-[Aib1,3,M]rPTH-(1–15)-NH2 than did Gαs(α3β5) (data not shown). The COS-7 cells were cotransfected in 6-well plates using plasmid DNA encoding PTHR-delNt (1 μg/well), plasmid DNA encoding GαsND (1 μg/well), and FuGENE 6 reagent (6 μl/well; Roche Diagnostics). Control experiments were performed with COS-7 cells similarly cotransfected with GαsND and the wild-type PTHR. Cells were harvested 3 days after transfection, and membranes were prepared as described (18Dean T. Linglart A. Mahon M.J. Bastepe M. Jüppner H. Potts Jr., J.T. Gardella T.J. Mol. Endocrinol. 2006; 20: 931-942Crossref PubMed Scopus (66) Google Scholar). Receptor Binding—Binding to the intact PTHR in HKRK-B7 and ROS 17/2.8 cells was assessed using 125I-[Nle8,21, Tyr34]rPTH-(1–34)-NH2 as a tracer radioligand as described (28Shimizu M. Carter P.H. Khatri A. Potts Jr., J.T. Gardella T.J. Endocrinology. 2001; 142: 3068-3074Crossref PubMed Scopus (62) Google Scholar). In brief, confluent cells in 24-well plates (∼500,000 cells/well) were incubated in binding buffer (50 mm Tris-HCl, 100 mm NaCl, 5 mm KCl, 2 mm CaCl2, 5% heat-inactivated horse serum, 0.5% fetal bovine serum, adjusted to pH 7.7 with HCl) containing radioligand (∼100,000 cpm/well) with or without unlabeled peptide ligand (3 × 10–9 to 1 × 10–5 m) for 4 h at 15 °C. The binding mixture was then removed by aspiration' the cells were rinsed three times with binding buffer and lysed in 1 m NaOH; and the entire lysate was counted for γ-irradiation in a γ-counter. Binding to PTHR-delNt in COS-7 cell membranes was assessed in 96-well vacuum filtration plates (Multi-Screen-HV Durapore, 0.65-μm membranes; Millipore Corp.) using 125I-[Aib1,3,M]rPTH-(1–15)-NH2 as a tracer radioligand as described (18Dean T. Linglart A. Mahon M.J. Bastepe M. Jüppner H. Potts Jr., J.T. Gardella T.J. Mol. Endocrinol. 2006; 20: 931-942Crossref PubMed Scopus (66) Google Scholar). In brief, cell membranes (20 μg/well) were incubated in membrane binding buffer containing radioligand (∼30,000 cpm/well) with or without unlabeled peptide ligand (3 × 10–9 to 1 × 10–5 m) for 90 min at 21 °C (reaction volume of 200 μl). The plates were then subjected to rapid vacuum filtration, and the filters were washed once with buffer, airdried, detached from the plate, and counted for γ-irradiation in a γ-counter. Nonspecific binding was defined as the binding observed in the presence of 1 × 10–6 m PTH-(1–31)-NH2 for HKRK-B7 and ROS 17/2.8 cells and of 1 × 10–6 m [Aib1,3,M]rPTH-(1–15)-NH2 for PTHR-delNt. Specifically bound radioactivity was calculated as a percentage of the radioactivity specifically bound in the absence of competing ligand. Stimulation of Intracellular cAMP and IP—The capacities of the ligands to stimulate formation of cAMP were assessed in intact ROS 17/2.8 cells, as described (28Shimizu M. Carter P.H. Khatri A. Potts Jr., J.T. Gardella T.J. Endocrinology. 2001; 142: 3068-3074Crossref PubMed Scopus (62) Google Scholar). In brief, cells in 24-well plates were incubated in binding buffer containing the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (2 mm) with or without a peptide ligand (3 × 10–11 to 1 × 10–6 m) for 30 min at room temperature. The medium was then removed, and the cells were lysed by adding 50 mm HCl and freezing the plate on dry ice. The cAMP in the thawed lysate was quantified by radioimmunoassay. The stimulation of production of inositol phosphates (IP1 + IP2 + IP3) was assessed in COS-7 cells transfected with the intact human PTHR as we described previously (28Shimizu M. Carter P.H. Khatri A. Potts Jr., J.T. Gardella T.J. Endocrinology. 2001; 142: 3068-3074Crossref PubMed Scopus (62) Google Scholar). In brief, intact transfected COS-7 cells in 24-well plates were labeled with myo-[3H]inositol (specific activity of 25 Ci/mmol; PerkinElmer Life Sciences) for 16 h. The labeled cells were then treated for 30 min with ligand in the presence of LiCl2 (30 mm), and the medium was removed and replaced with ice-cold trichloroacetic acid (5%). After 2 h on ice, the acid lysates were extracted with ether and processed by ion-exchange chromatography (0.5-ml resin bed), and the ammonium formate-eluted [3H]inositol phosphates were quantified by liquid scintillation counting. Data and Statistical Calculations—Binding and cAMP data were processed for curve fitting and derivation of IC50 and EC50 values using least-squares nonlinear regression analysis and the following equation: y = ymin + (ymax–ymin/1 + (IC50/x)n, where y, ymin, and ymax are the observed, minimum, and maximum response values, respectively; x is the ligand concentration; and n is the slope factor. In cases in which incomplete inhibition of binding occurred, e.g. with certain PTH-(1–31)-NH2 analogs binding to PTHR-delNt, the curves were extrapolated to nonspecific binding. Paired data sets were statistically compared using a two-tailed Student's t test, assuming unequal variance for the two sets. Alanine Scan of Region 17–31 of PTH—We first individually replaced each residue in region 17–31 of PTH-(1–31)-NH2 with alanine and assessed the effects of the substitutions on binding to the intact human PTHR stably expressed in HKRK-B7 cells. Binding was assessed by competition methods using 125I-[Nle8,21,Tyr34]rPTH-(1–34)-NH2 as a tracer radioligand. The parental PTH-(1–31)-NH2 peptide fully inhibited the binding of this tracer with an IC50 of 68 ± 10 nm (Fig. 1A and Table 1). The various alanine substitutions had a range of effects on this binding. Most dramatic was that of the Arg20 → Ala substitution, which abolished detectable binding (Fig. 1A). The alanine substitutions at Trp23 and Leu24 reduced the apparent binding affinity by 19- and 12-fold, respectively, relative to the parental peptide (p < 0.05). The alanine substitutions at Val21, Arg25, Lys27, Leu28, and Val31 reduced affinity by ∼3-fold, and the remaining alanine substitutions altered affinity by 2-fold or less (Fig. 1, A and B; and Table 1). The alanine substitutions at Glu19, Glu22, and Gln29 each produced a small (≤2-fold) enhancement of the apparent binding affinity, as did the Glu19 → Arg substitution, which we have shown previously to enhance cAMP-stimulating potency in PTH-(1–34) and PTH-(1–20) peptides (24Shimizu M. Shimizu N. Tsang J.C. Petroni B.D. Khatri A. Potts Jr., J.T. Gardella T.J. Biochemistry. 2002; 41: 13224-13233Crossref PubMed Scopus (24) Google Scholar). The Ala22 substitution was thus paired with Ala19 as well as with Arg19, but neither pairing improved affinity further relative to the single substitutions alone (Table 1).TABLE 1Helical contents and PTHR-binding properties of PTH-(1–31)-NH2 analogsCD helical residuesIC50PTHR-WT (HKRK-B7 cells)PTHR-delNt (COS-7 cells)mmnmPTH-(1-31)-NH2868 ± 10 (23)3666 ± 431 (13)Ser17 → Ala1057 ± 12 (4)4678 ± 386 (4)Met18 → Ala1092 ± 14 (3)8799 ± 2334 (4)Glu19 → Ala949 ± 11 (4)2940 ± 833 (4)Arg20 → Ala10>10,000 (3)19,343 ± 8100 (4)Val21 → Ala10200 ± 18 (3)5686 ± 486 (4)Glu22 → Ala936 ± 2 (4)5857 ± 2237 (4)Trp23 → Ala71312 ± 139 (3)7357 ± 1832 (4)Leu24 → Ala7807 ± 165 (3)8764 ± 1167 (4)Arg25 → Ala7281 ± 67 (3)7177 ± 1083 (4)Lys26 → Ala7125 ± 8 (3)6210 ± 948 (4)Lys27 → Ala9217 ± 7 (3)7288 ± 615 (4)Leu28 → Ala8290 ± 63 (3)3459 ± 482 (4)Gln29 → Ala944 ± 11 (3)1931 ± 303 (4)Asp30 → Ala1063 ± 7 (4)1545 ± 215 (4)Val31 → Ala9176 ± 23 (4)4911 ± 1341 (4)Glu19 → Ala/Glu22 → Ala949 ± 9 (4)619 ± 75 (4)Glu19 → Arg/Glu22 → Arg854 ± 13 (4)360 ± 19 (4)Glu19 → Arg842 ± 6 (4)1226 ± 332 (4)Arg20 → Gln7>10,000 (3)10,284 ± 121 (3)Arg20 → Glu7>10,000 (3)14,747 ± 324 (3)Arg20 → Lys8>10,000 (3)7105 ± 1275 (3)Arg20 → Nle1215,404 ± 2334 (4)10,688 ± 1033 (3)Arg20 → Cit911,925 ± 2151 (4)10,624 ± 1612 (3)Arg20 → Orn6>10,000 (4)5530 ± 279 (3)Arg20 → ApaND>10,000 (3)5740 ± 775 (3)Arg20 → GphND>10,000 (3)2047 ± 442 (3)Arg20 → PipGly814135 ± 5345 (4)6605 ± 2407 (5)[Aib1,3,M]rPTH-(1-15)-NH2NDND2.2 ± 0.5 (8) Open table in a new tab We then assessed the effects of the alanine substitutions on binding to PTHR-delNt. For these experiments, we used membranes prepared from COS-7 cells transiently transfected with PTHR-delNt. As a tracer radioligand, we used 125I-[Aib1,3,M]rPTH-(1–15)-NH2, which we have shown binds exclusively to the PTHR J domain (30Shimizu N. Dean T. Tsang J.C. Khatri A. Potts Jr., J.T. Gardella T.J. J. Biol. Chem. 2005; 280: 1797-1807Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). To increase the total specific binding of 125I-[Aib1,3,M]rPTH-(1–15)-NH2 to these membranes, the cells were cotransfected with a negative-dominant mutant Gαs subunit (GαsND), which promotes ligand binding presumably by stabilizing high affinity receptor conformations (29Berlot C.H. J. Biol. Chem. 2002; 277: 21080-21085Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Unlabeled [Aib1,3,M]rPTH-(1–15)-NH2 (used as a control peptide) fully inhibited the binding of 125I-[Aib1,3,M]rPTH-(1–15)-NH2 to these membranes with high apparent affinity (IC50 = 2.2 ± 0.5 nm) (Fig. 1, C and D; and Table 1). As expected from the absence of ligand interactions with the PTHR N domain, unmodified PTH-(1–31)-NH2 bound to PTHR-delNt with relatively low affinity (IC50 = 3700 ± 400 nm) (Fig. 1, C and D; and Table 1). This binding was nevertheless sufficient to assess the effects of the alanine substitutions on the capacity of the ligand to interact with PTHR-delNt. None of the alanine substitutions altered binding to PTHR-delNt by >5-fold, including the Arg20 → Ala substitution, which had strongly diminished binding to the intact PTHR (Fig. 1, A versus C). Similarly, the Ala substitutions at Trp23 and Leu24, which reduced affinity for the PTHR by 19- and 12-fold, respectively, reduced affinity for PTHR-delNt by only ∼2-fold. These findings indicate that the mechanisms by which the Ala substitutions at Arg20, Trp23, and Leu24 impair binding to the intact PTHR are largely independent of interactions with the PTHR J domain. None of the alanine substitutions had a major impact on the secondary structure of the peptide as revealed by CD spectroscopy analysis. Thus, the CD spectrum of each analog exhibited clear negative deflections in the regions at 209 and 222 nm, which are indicative of α-helical structure (Fig. 2). The number of helical resides/peptide chain (calculated from the CD sign" @default.
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• W2009274925 title "Role of Amino Acid Side Chains in Region 17–31 of Parathyroid Hormone (PTH) in Binding to the PTH Receptor" @default.
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