Minimization of Parathyroid Hormone
2000; Elsevier BV; Volume: 275; Issue: 29 Linguagem: Inglês
10.1074/jbc.m909861199
ISSN1083-351X
AutoresMasaru Shimizu, John T. Potts, Thomas J. Gardella,
Tópico(s)Bone health and treatments
ResumoThe amino-terminal and carboxyl-terminal portions of the 1–34 fragment of parathyroid hormone (PTH) contain the major determinants of receptor activation and receptor binding, respectively. We investigated how the amino-terminal signaling portion of PTH interacts with the receptor by utilizing analogs of the weakly active fragment, rat (r) PTH(1–14)NH2, and cells transfected with the wild-type human PTH-1 receptor (hP1R-WT) or a truncated PTH-1 receptor which lacked most of the amino-terminal extracellular domain (hP1R-delNt). Of 132 mono-substituted PTH(1–14) analogs, most having substitutions in the (1.Kronenberg H. Abou-Samra A Bringhurst F. Gardella T. Jüppner H. Segre G. Thakker R. Genetics of Endocrine and Metabolic disorders. Chapman & Hall, London1997: 389-420Google Scholar, 2.Jüppner H. Abou-Samra A.-B. Freeman M. Kong X.-F. Schipani E. Richards J. Kolakowski Jr., L.F. Hock J. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Science. 1991; 254: 1024-1026Crossref PubMed Scopus (1144) Google Scholar, 3.Kolakowski L.F. Receptors Channels. 1994; 2: 1-7PubMed Google Scholar, 4.Iida-Klein A. Guo J. Xie L. Jüppner H. Potts Jr., J.T. Kronenberg H.M. Bringhurst F. Abou-Samra A.B. Segre G.V. J. Biol. Chem. 1995; 270: 8458-8465Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 5.Nussbaum S.R. Rosenblatt M. Potts Jr., J.T. J. Biol. Chem. 1980; 255: 10183-10187Abstract Full Text PDF PubMed Google Scholar, 6.Tregear 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, 7.Nutt R.F. Caulfield M.P. Levy J.J. Gibbons S.W. Rosenblatt M. McKee R.L. Endocrinology. 1990; 127: 491-493Crossref PubMed Scopus (62) Google Scholar, 8.Goltzman D. Peytremann A. Callahan E. Tregear G.W. Potts Jr., J.T. J. Biol. Chem. 1975; 250: 3199-3203Abstract Full Text PDF PubMed Google Scholar, 9.Carter P. Juppner H. Gardella T. Endocrinology. 1999; 140: 4972-4981Crossref PubMed Scopus (44) Google Scholar) region were inactive in assays of cAMP formation in LLC-PK1 cells stably expressing hP1R-WT, whereas most having substitutions in the (10.Dempster D.W. Cosman F. Parisien M. Shen V. Lindsay R. Endocr. Rev. 1993; 14 (Erratum (1994) Endocr. Rev. 15, 261): 690-709Crossref PubMed Scopus (666) Google Scholar, 11.Roe E. Sanchez S. del Puerto G. Pierini E. Bacchetti P. Cann C. Arnaud C. J. Bone Miner. Res. 1999; 14 Suppl. 1: S137Google Scholar, 12.Takasu H. Gardella T. Luck M. Potts Jr., J. Bringhurst F. Biochemistry. 1999; 38: 13453-13460Crossref PubMed Scopus (93) Google Scholar, 13.Neugebauer W. Barbier J.R. Sung W.L. Whitfield J.F. Willick G.E. Biochemistry. 1995; 34: 8835-8842Crossref PubMed Scopus (50) Google Scholar, 14.Luck M. Carter P. Gardella T. Mol. Endocrinol. 1999; 13: 670-680Crossref PubMed Google Scholar) region were active. Several substitutions (e.g.Ser3 → Ala, Asn10 → Ala or Gln, Leu11 → Arg, Gly12 → Ala, His14→ Trp) enhanced activity 2–10-fold. These effects were additive, as [Ala3,10,12,Arg11,Trp14] rPTH(1–14)NH2 was 220-fold more potent than rPTH(1–14)NH2 (EC50 = 0.6 ± 0.1 and 133 ± 16 μm, respectively). Native rPTH(1–11) was inactive, but [Ala3,10,Arg11]rPTH(1–11)NH2achieved maximal cAMP stimulation (EC50 = 17 μm). The modified PTH fragments induced cAMP formation with hP1R-delNt in COS-7 cells as potently as they did with hP1R-WT; PTH(1–34) was 6,000-fold weaker with hP1R-delNt than with hP1R-WT. The most potent analog, [Ala3,10,12,Arg11,Trp14]rPTH(1–14)NH2, stimulated inositol phosphate production with hP1R-WT. The results show that short NH2-terminal peptides of PTH can be optimized for considerable gains in signaling potency through modification of interactions involving the regions of the receptor containing the transmembrane domains and extracellular loops. The amino-terminal and carboxyl-terminal portions of the 1–34 fragment of parathyroid hormone (PTH) contain the major determinants of receptor activation and receptor binding, respectively. We investigated how the amino-terminal signaling portion of PTH interacts with the receptor by utilizing analogs of the weakly active fragment, rat (r) PTH(1–14)NH2, and cells transfected with the wild-type human PTH-1 receptor (hP1R-WT) or a truncated PTH-1 receptor which lacked most of the amino-terminal extracellular domain (hP1R-delNt). Of 132 mono-substituted PTH(1–14) analogs, most having substitutions in the (1.Kronenberg H. Abou-Samra A Bringhurst F. Gardella T. Jüppner H. Segre G. Thakker R. Genetics of Endocrine and Metabolic disorders. Chapman & Hall, London1997: 389-420Google Scholar, 2.Jüppner H. Abou-Samra A.-B. Freeman M. Kong X.-F. Schipani E. Richards J. Kolakowski Jr., L.F. Hock J. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Science. 1991; 254: 1024-1026Crossref PubMed Scopus (1144) Google Scholar, 3.Kolakowski L.F. Receptors Channels. 1994; 2: 1-7PubMed Google Scholar, 4.Iida-Klein A. Guo J. Xie L. Jüppner H. Potts Jr., J.T. Kronenberg H.M. Bringhurst F. Abou-Samra A.B. Segre G.V. J. Biol. Chem. 1995; 270: 8458-8465Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 5.Nussbaum S.R. Rosenblatt M. Potts Jr., J.T. J. Biol. Chem. 1980; 255: 10183-10187Abstract Full Text PDF PubMed Google Scholar, 6.Tregear 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, 7.Nutt R.F. Caulfield M.P. Levy J.J. Gibbons S.W. Rosenblatt M. McKee R.L. Endocrinology. 1990; 127: 491-493Crossref PubMed Scopus (62) Google Scholar, 8.Goltzman D. Peytremann A. Callahan E. Tregear G.W. Potts Jr., J.T. J. Biol. Chem. 1975; 250: 3199-3203Abstract Full Text PDF PubMed Google Scholar, 9.Carter P. Juppner H. Gardella T. Endocrinology. 1999; 140: 4972-4981Crossref PubMed Scopus (44) Google Scholar) region were inactive in assays of cAMP formation in LLC-PK1 cells stably expressing hP1R-WT, whereas most having substitutions in the (10.Dempster D.W. Cosman F. Parisien M. Shen V. Lindsay R. Endocr. Rev. 1993; 14 (Erratum (1994) Endocr. Rev. 15, 261): 690-709Crossref PubMed Scopus (666) Google Scholar, 11.Roe E. Sanchez S. del Puerto G. Pierini E. Bacchetti P. Cann C. Arnaud C. J. Bone Miner. Res. 1999; 14 Suppl. 1: S137Google Scholar, 12.Takasu H. Gardella T. Luck M. Potts Jr., J. Bringhurst F. Biochemistry. 1999; 38: 13453-13460Crossref PubMed Scopus (93) Google Scholar, 13.Neugebauer W. Barbier J.R. Sung W.L. Whitfield J.F. Willick G.E. Biochemistry. 1995; 34: 8835-8842Crossref PubMed Scopus (50) Google Scholar, 14.Luck M. Carter P. Gardella T. Mol. Endocrinol. 1999; 13: 670-680Crossref PubMed Google Scholar) region were active. Several substitutions (e.g.Ser3 → Ala, Asn10 → Ala or Gln, Leu11 → Arg, Gly12 → Ala, His14→ Trp) enhanced activity 2–10-fold. These effects were additive, as [Ala3,10,12,Arg11,Trp14] rPTH(1–14)NH2 was 220-fold more potent than rPTH(1–14)NH2 (EC50 = 0.6 ± 0.1 and 133 ± 16 μm, respectively). Native rPTH(1–11) was inactive, but [Ala3,10,Arg11]rPTH(1–11)NH2achieved maximal cAMP stimulation (EC50 = 17 μm). The modified PTH fragments induced cAMP formation with hP1R-delNt in COS-7 cells as potently as they did with hP1R-WT; PTH(1–34) was 6,000-fold weaker with hP1R-delNt than with hP1R-WT. The most potent analog, [Ala3,10,12,Arg11,Trp14]rPTH(1–14)NH2, stimulated inositol phosphate production with hP1R-WT. The results show that short NH2-terminal peptides of PTH can be optimized for considerable gains in signaling potency through modification of interactions involving the regions of the receptor containing the transmembrane domains and extracellular loops. parathyroid hormone rat human PTH-related peptide N-(9-fluorenyl)methoxycarbonyl high performance liquid chromatography In mammals, parathyroid hormone (PTH)1 plays a vital role in regulating blood calcium concentrations, and PTH-related peptide (PTHrP) plays a critical role in the development of the fetal skeleton (1.Kronenberg H. Abou-Samra A Bringhurst F. Gardella T. Jüppner H. Segre G. Thakker R. Genetics of Endocrine and Metabolic disorders. Chapman & Hall, London1997: 389-420Google Scholar). The biological actions of both of these peptides are mediated by the PTH/PTHrP receptor (or PTH-1 receptor) (2.Jüppner H. Abou-Samra A.-B. Freeman M. Kong X.-F. Schipani E. Richards J. Kolakowski Jr., L.F. Hock J. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Science. 1991; 254: 1024-1026Crossref PubMed Scopus (1144) Google Scholar), a family B G protein-coupled receptor (3.Kolakowski L.F. Receptors Channels. 1994; 2: 1-7PubMed Google Scholar) that strongly activates the adenylyl cyclase/protein kinase A-signaling cascade (2.Jüppner H. Abou-Samra A.-B. Freeman M. Kong X.-F. Schipani E. Richards J. Kolakowski Jr., L.F. Hock J. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Science. 1991; 254: 1024-1026Crossref PubMed Scopus (1144) Google Scholar), and more weakly the phospholipase C protein kinase C-signaling pathway (4.Iida-Klein A. Guo J. Xie L. Jüppner H. Potts Jr., J.T. Kronenberg H.M. Bringhurst F. Abou-Samra A.B. Segre G.V. J. Biol. Chem. 1995; 270: 8458-8465Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The mechanisms by which parathyroid hormone and PTHrP bind to the PTH-1 receptor and induce receptor activation are poorly understood but appear to involve multiple sites of intermolecular interaction. Early studies of PTH fragment analogs assigned the major determinants of receptor-binding affinity and cAMP-stimulating potency to the COOH-terminal and NH2-terminal portions of the fully active PTH(1–34) peptide, respectively (5.Nussbaum S.R. Rosenblatt M. Potts Jr., J.T. J. Biol. Chem. 1980; 255: 10183-10187Abstract Full Text PDF PubMed Google Scholar, 6.Tregear 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). PTH(1–34)-based analogs with NH2-terminal deletions, such as PTH(3–34) and PTH(7–34), bind efficiently to the receptor and are severely defective in stimulating a cAMP response; such peptides thus function as PTH-1 receptor antagonists (7.Nutt R.F. Caulfield M.P. Levy J.J. Gibbons S.W. Rosenblatt M. McKee R.L. Endocrinology. 1990; 127: 491-493Crossref PubMed Scopus (62) Google Scholar, 8.Goltzman D. Peytremann A. Callahan E. Tregear G.W. Potts Jr., J.T. J. Biol. Chem. 1975; 250: 3199-3203Abstract Full Text PDF PubMed Google Scholar, 9.Carter P. Juppner H. Gardella T. Endocrinology. 1999; 140: 4972-4981Crossref PubMed Scopus (44) Google Scholar). The dominant role that the NH2-terminal residues of PTH and PTHrP play in receptor activation is further reflected by their high level of evolutionary conservation. The anabolic effects of PTH on bone density (10.Dempster D.W. Cosman F. Parisien M. Shen V. Lindsay R. Endocr. Rev. 1993; 14 (Erratum (1994) Endocr. Rev. 15, 261): 690-709Crossref PubMed Scopus (666) Google Scholar, 11.Roe E. Sanchez S. del Puerto G. Pierini E. Bacchetti P. Cann C. Arnaud C. J. Bone Miner. Res. 1999; 14 Suppl. 1: S137Google Scholar) have prompted considerable interest in the development of new PTH-1 receptor agonist analogs. Recently PTH(1–28) was shown to be an effective agonist for cAMP production in cell-based assays, although potency was ∼10-fold reduced from that of PTH(1–34) (12.Takasu H. Gardella T. Luck M. Potts Jr., J. Bringhurst F. Biochemistry. 1999; 38: 13453-13460Crossref PubMed Scopus (93) Google Scholar, 13.Neugebauer W. Barbier J.R. Sung W.L. Whitfield J.F. Willick G.E. Biochemistry. 1995; 34: 8835-8842Crossref PubMed Scopus (50) Google Scholar). Recently we found that in COS-7 or LLC-PK1 cells transfected with high levels of rat or human PTH-1 receptors, a fragment as short as PTH(1–14) elicited ∼20-fold increases in cAMP formation levels (14.Luck M. Carter P. Gardella T. Mol. Endocrinol. 1999; 13: 670-680Crossref PubMed Google Scholar). Although the potency of PTH(1–14) in these transfected cells was weak compared with PTH(1–34) (EC50 = 1 nm and 100 μm, respectively), the response was sufficient for us to perform an initial structure-activity relationship analysis. In this previous study, we found that most alanine substitutions in the (1.Kronenberg H. Abou-Samra A Bringhurst F. Gardella T. Jüppner H. Segre G. Thakker R. Genetics of Endocrine and Metabolic disorders. Chapman & Hall, London1997: 389-420Google Scholar, 2.Jüppner H. Abou-Samra A.-B. Freeman M. Kong X.-F. Schipani E. Richards J. Kolakowski Jr., L.F. Hock J. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Science. 1991; 254: 1024-1026Crossref PubMed Scopus (1144) Google Scholar, 3.Kolakowski L.F. Receptors Channels. 1994; 2: 1-7PubMed Google Scholar, 4.Iida-Klein A. Guo J. Xie L. Jüppner H. Potts Jr., J.T. Kronenberg H.M. Bringhurst F. Abou-Samra A.B. Segre G.V. J. Biol. Chem. 1995; 270: 8458-8465Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 5.Nussbaum S.R. Rosenblatt M. Potts Jr., J.T. J. Biol. Chem. 1980; 255: 10183-10187Abstract Full Text PDF PubMed Google Scholar, 6.Tregear 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, 7.Nutt R.F. Caulfield M.P. Levy J.J. Gibbons S.W. Rosenblatt M. McKee R.L. Endocrinology. 1990; 127: 491-493Crossref PubMed Scopus (62) Google Scholar, 8.Goltzman D. Peytremann A. Callahan E. Tregear G.W. Potts Jr., J.T. J. Biol. Chem. 1975; 250: 3199-3203Abstract Full Text PDF PubMed Google Scholar, 9.Carter P. Juppner H. Gardella T. Endocrinology. 1999; 140: 4972-4981Crossref PubMed Scopus (44) Google Scholar) region severely diminished PTH(1–14)-signaling activity, whereas alanine substitutions in the (10.Dempster D.W. Cosman F. Parisien M. Shen V. Lindsay R. Endocr. Rev. 1993; 14 (Erratum (1994) Endocr. Rev. 15, 261): 690-709Crossref PubMed Scopus (666) Google Scholar, 11.Roe E. Sanchez S. del Puerto G. Pierini E. Bacchetti P. Cann C. Arnaud C. J. Bone Miner. Res. 1999; 14 Suppl. 1: S137Google Scholar, 12.Takasu H. Gardella T. Luck M. Potts Jr., J. Bringhurst F. Biochemistry. 1999; 38: 13453-13460Crossref PubMed Scopus (93) Google Scholar, 13.Neugebauer W. Barbier J.R. Sung W.L. Whitfield J.F. Willick G.E. Biochemistry. 1995; 34: 8835-8842Crossref PubMed Scopus (50) Google Scholar, 14.Luck M. Carter P. Gardella T. Mol. Endocrinol. 1999; 13: 670-680Crossref PubMed Google Scholar) region preserved activity (14.Luck M. Carter P. Gardella T. Mol. Endocrinol. 1999; 13: 670-680Crossref PubMed Google Scholar). We also showed that PTH(1–14) could activate a truncated PTH receptor that lacked most of the NH2-terminal domain (14.Luck M. Carter P. Gardella T. Mol. Endocrinol. 1999; 13: 670-680Crossref PubMed Google Scholar). These studies with PTH(1–14) and the truncated receptor were consistent with the hypothesis suggested by other mutational and cross-linking data (15.Bergwitz C. Jusseaume S. Luck M. Jüppner H. Gardella T. J. Biol. Chem. 1997; 272: 28861-28868Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 16.Lee C. Luck M. Jüppner H. Potts J. Kronenberg H. Gardella T. Mol. Endocrinol. 1995; 9: 1269-1278Crossref PubMed Google Scholar, 17.Bisello A. Adams A.E. Mierke D. Pellegrini M. Rosenblatt M. Suva L. Chorev M. J. Biol. Chem. 1998; 273: 22498-22505Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 18.Gardella T.J. Jüppner H. Wilson A.K. Keutmann H.T. Abou-Samra A.B. Segre G.V. Bringhurst F.R. Potts Jr., J.T. Nussbaum S.R. Kronenberg H.M. Endocrinology. 1994; 135: 1186-1194Crossref PubMed Scopus (70) Google Scholar, 19.Behar V. Bisello A. Bitan B. Rosenblatt M. Chorev M. J. Biol. Chem. 1999; 275: 9-17Abstract Full Text Full Text PDF Scopus (90) Google Scholar) that residues in the NH2-terminal portion of PTH(1–34) interact with the region of the receptor containing the seven transmembrane domains and extracellular loops. Other peptide hormones that bind family B receptors, such as calcitonin, secretin and glucagon, and are comparable in size to PTH(1–34) may utilize a similar topological arrangement in binding to their cognate receptors (20.Bergwitz C. Gardella T. Flannery M. Potts J.J. Kronenberg H. Goldring S. Jüppner H. J. Biol. Chem. 1996; 271: 26469-26472Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 21.Stroop S. Kuestner R. Serwold T. Chen L. Moore E. Biochemistry. 1995; 34: 1050-1057Crossref PubMed Scopus (93) Google Scholar); however, small NH2-terminal activating peptides for these other family B receptors have thus far not been reported. In the current study, we use PTH(1–14) as a starting scaffold for investigating whether amino acid modifications can be identified that both enhance the signaling potency of PTH(1–14) and enable further reductions in agonist peptide length. The results show that the NH2-terminal residues of PTH can be optimized, in that greater agonist potency can be achieved in peptides as short as 11 amino acids. Such minimized peptides serve as useful probes of the receptor-interaction mechanism, as we show that the activity-enhancing effects of the ligand modifications are mediated through the portion of the receptor containing the seven transmembrane domains and extracellular loops. The Massachusetts General Hospital Biopolymer Synthesis Facility (Boston, MA) prepared all peptides used in this study. Each peptide contained a carboxyl-terminal amide and a free amino group at the amino terminus, except for the PTH(1–14) analogs desN-G and desN-A, which contained the position 1 modifications of desamino-Ala and desamino-Gly, respectively (Fig. 1). All analogs of rPTH(1–14)NH2 (rPTH(1–14)) and shorter length rPTH peptides were synthesized on a multiple peptide synthesizer (Advanced Chemtech model 396 MBS) using Fmoc protecting group chemistry and trifluoroacetic acid-mediated cleavage/deprotection; and were desalted by adsorption on a C18-containing cartridge. [Nle8,21,Tyr34]rPTH(1–34)NH2(rPTH(1–34)), [Tyr34]hPTH(1–34)NH2((hPTH(1–34)), [Tyr34]hPTH(3–34)NH2((hPTH(3–34)), and [Ala1,3,10,12,Arg11,Tyr34]hPTH(1–34)NH2were prepared on an Applied Biosystems model 431A peptide synthesizer using the same Fmoc chemistry and trifluoroacetic acid-mediated cleavage/deprotection; after C18 desalting, these peptides were purified further by HPLC. All peptides were reconstituted in 10 mm acetic acid and stored at −80 °C. The purity, identity, and stock concentration of each compound were secured by analytical HPLC, matrix-assisted laser desorption/ionization mass spectrometry, and amino acid analysis. LLC-PK1-derived and COS-7 cells were cultured at 37 °C in T-75 flasks (75 mm2) in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum (10%), penicillin G (20 units/ml), streptomycin sulfate (20 μg/ml), and amphotericin B (0.05 μg/ml) in a humidified atmosphere containing 5% CO2. ROS 17/2.8 cells were cultured as above except that Ham's F-12 medium was used instead of Dulbecco's modified Eagle's medium and fetal bovine serum was at 5%. Stock solutions of EGTA/trypsin and antibiotics were from Life Technologies, Inc.; fetal bovine serum was from HyClone Laboratories (Logan, UT). Cells were subcultured in 24-well plates and, when confluent, were treated with fresh media and shifted to 33 °C for 12–24 h prior to assay. This shift to 33 °C was included as a means to potentially maximize cell surface expression of the PTH receptors and thus optimize signal sensitivity, since we found previously that the reduced temperature incubation resulted in small (10–50%) increases in surface expression of wild-type and mutant PTH receptors (15.Bergwitz C. Jusseaume S. Luck M. Jüppner H. Gardella T. J. Biol. Chem. 1997; 272: 28861-28868Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The ability of lower temperatures to improve the surface expression of the lutropin receptor and other membrane proteins has been discussed previously (22.Abell A. Liu X. Segaloff D. J. Biol. Chem. 1996; 271: 4518-4527Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The HKRK-B7 cell line (23.Takasu H. Guo J. Bringhurst F. J. Bone Miner. Res. 1999; 14: 11-20Crossref PubMed Scopus (84) Google Scholar) was derived by stable transfection of LLC-PK1 porcine kidney cells with the hPTH-1 receptor cDNA and express these receptors at a density of ∼950,000 receptors/cell. ROS 17/2.8 cells, a rat osteoblast-like cell line (24.Majeska R.J. Rodan S.B. Rodan G.A. Endocrinology. 1980; 107: 1494-1503Crossref PubMed Scopus (418) Google Scholar), express endogenous rat PTH-1 receptors at a density of ∼70,000 receptors/cell (25.Yamamoto I. Shigeno C. Potts Jr., J.T. Segre G.V. Endocrinology. 1988; 122: 1208-1217Crossref PubMed Scopus (100) Google Scholar). The pCDNA-1-based plasmid encoding the intact hPTH-1 receptor (HKrk in Ref. 26.Schipani E. Karga H. Karaplis A.C. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Abou-Samra A.B. Jüppner H. Endocrinology. 1993; 132: 2157-2165Crossref PubMed Scopus (153) Google Scholar and herein called hP1R-WT) was used for studies in COS-7 cells. The truncated human PTH-1 receptor (hP1R-delNt) was constructed from the hP1R-WT plasmid by oligonucleotide-directed mutagenesis (27.Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4900) Google Scholar). This mutant receptor is deleted for residues 24–181. The predicted signal peptidase cleavage of this receptor between Ala22 and Tyr23 (28.Nielsen H. Engelbrecht J. Brunak S. von Heijne G. Protein Eng. 1997; 10: 1-6Crossref PubMed Scopus (4934) Google Scholar) generates Tyr23 as the NH2-terminal residue, which is joined directly to Glu182 located at or near the boundary of the first transmembrane domain. A similarly truncated rat PTH receptor containing an NH2-terminal epitope tag (rP1R-delNt-HA) was described by us previously and shown by antibody binding experiments to be expressed at approximately 60% the level of the intact wild-type receptor (14.Luck M. Carter P. Gardella T. Mol. Endocrinol. 1999; 13: 670-680Crossref PubMed Google Scholar, 29.Carter P. Shimizu M. Luck M. Gardella T. J. Biol. Chem. 1999; 274: 31955-31960Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Transient transfections of COS-7 cells were performed using DEAE-dextran and 200 ng of cesium chloride-purified plasmid DNA per well of a 24-well plate, as described previously (15.Bergwitz C. Jusseaume S. Luck M. Jüppner H. Gardella T. J. Biol. Chem. 1997; 272: 28861-28868Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Stimulation of cells with peptide analogs was performed in 24-well plates. Cells were rinsed with 0.5 ml of 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) and treated with 200 μl of cAMP assay buffer (Dulbecco's modified Eagle's medium containing 2 mm3-isobutyl-1-methylxanthine, 1 mg/ml bovine serum albumin, 35 mm Hepes-NaOH, pH 7.4) and 100 μl of binding buffer containing varying amounts of peptide analog (final volume = 300 μl). The medium was removed after incubation for 1 h at room temperature, and the cells were frozen (−80 °C), lysed with 0.5 ml of 50 mm HCl, and refrozen (−80 °C). The cAMP content of the diluted lysate was determined by radioimmunoassay. Where possible, EC50 and corresponding maximum response values (E max) were calculated using nonlinear regression (see below). For inhibition studies, the hPTH(3–34) antagonist peptide was added to the rinsed cells in 100 μl of binding buffer immediately prior to the addition of 100 μl of cAMP assay buffer and 100 μl of cAMP assay buffer containing varying amounts of agonist peptide (final volume = 300 μl); the cells were then incubated for 60 min at room temperature and processed as described above. COS-7 cells transfected as above with hP1R-WT were treated with serum-free, inositol-free Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin and myo-[3H]inositol (NEN Life Science Products) (2 μCi/ml) for 16 h prior to assay. At the time of the assay, the cells were rinsed with binding buffer containing LiCl (30 mm) and treated with the same buffer with or without a PTH analog. The cells were then incubated at 37 °C for 40 min, after which the buffer was removed and replaced by 0.5 ml of ice-cold 5% trichloroacetic acid solution. After 3 h on ice, the lysate was collected and extracted twice with ethyl ether. The lysate was then applied to an ion exchange column (0.5-ml resin bed) and the total inositol phosphates were eluted as described previously (30.Berridge M. Dawson R. Downes C. Heslop J. Irvine R. Biochem. J. 1983; 212: 473-482Crossref PubMed Scopus (1541) Google Scholar), and counted in liquid scintillation mixture. Calculations were performed using Microsoft Excel. Nonlinear regression analysis of cAMP stimulation data was performed using four parameters, defined as the minimum (Min), maximum (Max, E max), midpoint (EC50), and slope of the response curve. The predicted response (y p) for a given dose (x) of peptide was calculated using the following equation:y p = Min + [(Max − Min)/(1 + (EC50/x)slope)]. The initial parameter values were estimated from the primary data, and the Excel "solver function" was then used to vary the four parameters in order to minimize the differences between the predicted and actual responses (least-squares method) (31.Bowen W. Jerman J. Trends Pharmacol. Sci. 1995; 16: 413-417Abstract Full Text PDF PubMed Scopus (152) Google Scholar). For each experiment, the maximum was constrained to within ±1 standard deviation of the maximum response observed in that experiment for rPTH(1–34) at a dose of 1 × 10−7m. The optimized equations were used to curve-fit the data shown in the graphs and to obtain the EC50 and corresponding maximum (E max(calc)) values reported in the tables. The observed maximum responses (E max(obs)) were those attained by each NH2-terminal fragment analog at a dose of 100 μm and by each PTH(1–34) analog at a dose of 100 nm, except for studies in cells expressing hP1R-delNT where the E max(obs) for rPTH(1–34) and hPTH(1–34) was determined at a dose of 10 μm and for [Ala1,3,10,12,Arg11,Tyr34]hPTH(1–34)NH2at dose of 20 μm. In some cases where the dose-response curves did not attain a true asymptotic maximum, as with native rPTH(1–14), the E max(calc) values are greater than the E max(obs) values. The statistical significance between two data sets was determined using a one-tailed Student's t test assuming unequal variances for the two sets. PTH(1–14) analogs having single substitutions (132 total) were tested for the ability to stimulate cAMP formation in HKRK-B7 cells. The substitutions were chosen such that at least one of each type of the 20 natural amino acids was introduced at each position, thus enabling a comparison of the effects of varied side chain chemistries (e.g. size, polarity, ionic charge, hydrophobicity, aromaticity, and proline) on receptor activation. The analogs and the control peptide (native rPTH(1–14)NH2) were tested at a single dose of 100 μm; rPTH(1–34) was tested at a maximum stimulatory dose (10−7m). In the assays shown in Fig. 1, native rPTH(1–14) and rPTH(1–34) stimulated 28- and 58-fold increases in cAMP formation, respectively, as compared with the cAMP level in unstimulated cells, which was less than 6 pmol/well. This response range ensured that both activity-enhancing and activity-impairing effects could be readily detected in the PTH(1–14) analogs. As shown in Fig. 1, most substitutions in the (1.Kronenberg H. Abou-Samra A Bringhurst F. Gardella T. Jüppner H. Segre G. Thakker R. 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