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• W2016123750 abstract "The parathyroid hormone (PTH)-2 receptor displays strong ligand selectivity in that it responds fully to PTH but not at all to PTH-related peptide (PTHrP). In contrast, the PTH-1 receptor (PTH/PTHrP receptor) responds fully to both ligands. Previously it was shown that two divergent residues in PTH and PTHrP account for PTH-2 receptor selectivity; position 23 (Trp in PTH and Phe in PTHrP) determines binding selectivity and position 5 (Ile in PTH and His in PTHrP) determines signaling selectivity. To identify sites in the PTH-2 receptor involved in discriminating between His5 and Ile5, we constructed PTH-2 receptor/PTH-1 receptor chimeras, expressed them in COS-7 cells, and tested for cAMP responsiveness to [Trp23] PTHrP-(1–36), and to the nondiscriminating peptide [Ile5,Trp23]PTHrP-(1–36) (the Phe23 → Trp modification enabled high affinity binding of each ligand to the PTH-2 receptor). The chimeras revealed that the membrane-spanning/loop region of the receptor determined His5/Ile5 signaling selectivity. Subsequent analysis of smaller cassette substitutions and then individual point mutations led to the identification of two single residues that function as major determinants of residue 5 signaling selectivity. These residues, Ile244 at the extracellular end of transmembrane helix 3, and Tyr318 at the COOH-terminal portion of extracellular loop 2, are replaced by Leu and Ile in the PTH-1 receptor, respectively. The results thus indicate a functional interaction between two residues in the core region of the PTH-2 receptor and residue 5 of the ligand. The parathyroid hormone (PTH)-2 receptor displays strong ligand selectivity in that it responds fully to PTH but not at all to PTH-related peptide (PTHrP). In contrast, the PTH-1 receptor (PTH/PTHrP receptor) responds fully to both ligands. Previously it was shown that two divergent residues in PTH and PTHrP account for PTH-2 receptor selectivity; position 23 (Trp in PTH and Phe in PTHrP) determines binding selectivity and position 5 (Ile in PTH and His in PTHrP) determines signaling selectivity. To identify sites in the PTH-2 receptor involved in discriminating between His5 and Ile5, we constructed PTH-2 receptor/PTH-1 receptor chimeras, expressed them in COS-7 cells, and tested for cAMP responsiveness to [Trp23] PTHrP-(1–36), and to the nondiscriminating peptide [Ile5,Trp23]PTHrP-(1–36) (the Phe23 → Trp modification enabled high affinity binding of each ligand to the PTH-2 receptor). The chimeras revealed that the membrane-spanning/loop region of the receptor determined His5/Ile5 signaling selectivity. Subsequent analysis of smaller cassette substitutions and then individual point mutations led to the identification of two single residues that function as major determinants of residue 5 signaling selectivity. These residues, Ile244 at the extracellular end of transmembrane helix 3, and Tyr318 at the COOH-terminal portion of extracellular loop 2, are replaced by Leu and Ile in the PTH-1 receptor, respectively. The results thus indicate a functional interaction between two residues in the core region of the PTH-2 receptor and residue 5 of the ligand. The parathyroid hormone (PTH 1The abbreviations used are: PTH, parathyroid hormone; rPTH, rat PTH; PTHrP, PTH-related peptide; P1R, PTH-1 receptor; P2R, PTH-2 receptor; WT, wild-type.)-2 receptor, a recently identified PTH receptor subtype, responds fully to PTH but not at all to PTHrP (1Usdin T. Gruber C. Bonner T. J. Biol Chem. 1995; 270: 15455-15458Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). This ligand selectivity profile of the PTH-2 receptor is dramatically different from that of the PTH-1 receptor (PTH/PTHrP receptor) which elicits a robust increase in cAMP formation in response to either ligand. The amino acid sequences of the two receptors are 51% identical, and each is a member of the subfamily of G protein-coupled receptors that bind peptide hormones of intermediate size including calcitonin, secretin, glucagon, vasoactive intestinal peptide, and several other peptides (2Segre G.V. Goldring S.R. Trends Endocrinol. Metab. 1993; 4: 309-314Abstract Full Text PDF PubMed Scopus (303) Google Scholar), in addition to PTH and PTHrP. These receptors are characterized by a relatively large amino-terminal extracellular domain of 100–200 amino acids, which contains six highly conserved cysteine residues, a “core” region with seven hydrophobic transmembrane helices and connecting loops, and a carboxyl-terminal tail of 150–200 amino acids. The molecular basis by which the peptide hormone receptors engage their respective receptors and trigger receptor activation is still largely unknown. This problem has been approached through strategies involving the construction of receptor chimeras and other types of receptor mutants (3Lee C. Gardella T. Abou-Samra A.-B. Nussbaum S. Segre G. Potts J. Kronenberg H. Jüppner H. Endocrinology. 1994; 135: 1488-1495Crossref PubMed Scopus (117) Google Scholar, 4Lee C. Luck M. Jüppner H. Potts J. Kronenberg H. Gardella T. Mol. Endocrinol. 1995; 9: 1269-1278Crossref PubMed Google Scholar, 5Jüppner H. Schipani E. Bringhurst F.R. McClure I. Keutmann H.T. Potts Jr., J.T. Kronenberg H.M. Abou-Samra A.B. Segre G.V. Gardella T. Endocrinology. 1994; 134: 879-884Crossref PubMed Scopus (118) Google Scholar, 6Gardella T. Luck M. Fan M. Lee C. J. Biol. Chem. 1996; 271: 12820-12825Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 7Gardella 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, 8Bergwitz C. Gardella T. Flannery M. Potts Jr., J.T. Kronenberg H. Goldring S. Jüppner H. J. Biol. Chem. 1996; 271: 26469-26472Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 9Holtmann M. Hadac E. Miller L. J. Biol. Chem. 1995; 270: 14394-14398Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 10Stroop S. Kuestner R. Serwold T. Chen L. Moore E. Biochemistry. 1994; 34: 1050-1057Crossref Scopus (93) Google Scholar, 11Turner P. Bambino T. Nissenson R. Mol. Endocrinol. 1996; 10: 132-139PubMed Google Scholar, 12Turner P.R. Bambino T. Nissenson R.A. J. Biol. Chem. 1996; 271: 9205-9208Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The data emerging from these studies suggest that multiple segments of the ligand and receptor contribute to the interaction. Recent studies with chimeric ligands acting on chimeric receptors suggest that the carboxyl-terminal portion of the ligand interacts with the amino-terminal extracellular domain, whereas the amino-terminal portion of the hormone interacts with the membrane-spanning/loop region of the receptor (8Bergwitz C. Gardella T. Flannery M. Potts Jr., J.T. Kronenberg H. Goldring S. Jüppner H. J. Biol. Chem. 1996; 271: 26469-26472Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). However, as yet, there are only limited data on the specific amino acids, either in the ligand or in the receptor, that contribute to the interaction. In other receptor systems, the availability of receptor subtypes that exhibit distinct pharmacological profiles for different ligands has facilitated the identification of receptor residues involved in ligand recognition (13Schwartz T.W. Gether U. Schambye H.T. Hjorth S.A. Current Pharmaceutical Design. 1995; 1: 325-342Google Scholar). The pronounced difference in the ligand selectivity profiles of the PTH-1 and PTH-2 receptors suggested that these two receptors could be used in such an analysis, since the difference in selectivity can most easily be explained by structural differences in the receptors at sites (or a site) that are involved in ligand recognition or ligand-induced receptor activation. Further, it suggests that the two ligands differ at residues that interact with these divergent receptor residues. Recently, two such residues in PTH and PTHrP that can account for their altered selectivity for the PTH-2 receptor were identified: residue 23, Phe in PTHrP and Trp in PTH, modulates ligand binding (14Gardella T. Luck M. Jensen G. Usdin T. Jüppner H. J. Biol. Chem. 1996; 271: 19888-19893Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar); and residue 5, His in PTHrP and Ile in PTH, modulates ligand-induced receptor activation (14Gardella T. Luck M. Jensen G. Usdin T. Jüppner H. J. Biol. Chem. 1996; 271: 19888-19893Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 15Behar V. Nakamoto C. Greenberg Z. Bisello A. Suva L.J. Rosenblatt M. Chorev M. Endocrinology. 1996; 137: 4217-4224Crossref PubMed Scopus (44) Google Scholar). Thus, the weak binding of PTHrP to the PTH-2 receptor can be explained by the presence of Phe23, and the weak signaling activity at this receptor can be explained by the histidine at position 5. In the present paper we use a receptor chimera and mutagenesis approach to search for sites in the PTH-2 receptor that are involved in His5/Ile5 signaling selectivity. The results reveal two divergent amino acids in the membrane-spanning and extracellular loop portion of the receptor that contribute strongly to this effect. The preparation and initial characterization of [Trp23,Tyr36]PTHrP-(1–36)amide and [Ile5,Trp23,Tyr36]PTHrP-(1–36)amide was described previously (14Gardella T. Luck M. Jensen G. Usdin T. Jüppner H. J. Biol. Chem. 1996; 271: 19888-19893Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Herein, these two peptides are referred to as [Trp23]PTHrP-(1–36) and [Ile5,Trp23]PTHrP-(1–36), respectively. These PTHrP analogs, and other peptides used in the study, were prepared by the biopolymer synthesis facility at Massachusetts General Hospital (Boston, MA), as were the DNA oligonucleotides used in receptor mutagenesis experiments. The PTH analog [Nle8,21,Tyr34]rPTH-(1–34)amide was radioiodinated by the chloramine-T procedure, and the product was purified by reverse phase high performance liquid chromatography (16Shigeno C. Hiraki Y. Westerberg D.P. Potts Jr., J.T. Segre G.V. J. Biol. Chem. 1988; 263: 3872-3878Abstract Full Text PDF PubMed Google Scholar).125I-Na (2,000 Ci/mmol) was purchased from NEN Life Science Products. Dulbecco's modified Eagle's medium, EGTA/trypsin, and concentrated antibiotic mixture (10,000 units/ml penicillin G and 10 mg/ml streptomycin) were from Life Technologies, Inc.; fetal bovine serum was from HyClone Laboratories (Logan, UT). DNA modifying reagents were from United States Biochemical Corp. (Cleveland, OH) or New England Biolabs, Inc. (Beverly, MA). The cDNAs encoding the human PTH-1 (PTH/PTHrP) receptor (17Schipani 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 the human PTH-2 receptor (1Usdin T. Gruber C. Bonner T. J. Biol Chem. 1995; 270: 15455-15458Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar) were carried on the expression vectors pcDNA-1 and pcDNAI/Amp (InVitrogen, San Diego, CA), respectively. The 1E2 and 2E1 receptor chimeras were constructed by utilizing the unique, naturally occurring EcoRI site in the PTH-2 receptor plasmid that overlaps codons 139/140. A matchingEcoRI site was introduced into the human PTH-1 receptor by oligonucleotide-directed mutagenesis at codons 182/183 to generate the receptor plasmid pHK-FE. The mutagenic oligonucleotide used to make pHK-FE also changed Val183 and Asp185 to Phe and Glu, respectively, which correspond to the amino acid sequence of the PTH-2 receptor. The HK-FE and WT PTH-2 receptor plasmids were cleaved with BamHI, which cuts in the 5′ polylinker region, and EcoRI, and then the appropriateEcoRI-BamHI DNA fragments were gel-purified and religated to yield the desired chimeras. The chimera 1E2 has residues 1–182 of the P1R joined to residues 140–550 of the P2R, and chimera 2E1 has residues 1–142 of the P2R joined to residues 186–593 of the P1R. All other cassette and point mutations were introduced by oligonucleotide-directed mutagenesis (18Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4900) Google Scholar). COS-7 cells were cultured at 37 °C in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum (10%), penicillin G (20 units/ml), and streptomycin (20 μg/ml) in a humidified atmosphere containing 5% CO2. Cells were transfected in 24-well plates using plasmid DNA (200 ng/well) that was purified by cesium chloride/ethidium bromide gradient centrifugation, except for the initial screening of the cassette mutants, in which phenol-extracted miniprep DNA was used. This miniprep DNA was quantified by ethidium bromide staining of agarose gels, and was transfected at a concentration of 100 ng/well. 48 h after transfection, the cell medium was replenished and the plates were shifted to a 33 °C humidified incubator for an additional 24–48 h, by which time the cell density reached 500,000 ± 100,000 cells/well. This shift to a lower temperature resulted in a general 10–50% increase in the number of receptors on the cell surface, as compared with cells maintained at 37 °C 2H. Jüppner and T. J. Gardella, unpublished observations. as has been found for other G protein-coupled receptors (19Abell A. Liu X. Segaloff D. J. Biol. Chem. 1996; 271: 4518-4527Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The cells were then used for binding and cAMP stimulation assays. Binding reactions were performed as described previously (18Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4900) Google Scholar). Each well (final volume = 300 μl) contained 26 fmol of125I-[Nle8,21,Tyr34]rPTH-(1–34)NH2(100,000 cpm) and various amounts (0.4–300 pmol) of unlabeled competitor ligand; peptides were diluted in binding buffer (50 mm Tris-HCl, pH 7.7, 100 mm NaCl, 5 mm KCl, 2 mm CaCl2, 5% heat-inactivated horse serum, 0.5% heat-inactivated fetal bovine serum). Incubations were at room temperature for 2 h, except for experiments performed for Scatchard analysis, which were performed at 4 °C for 6 h. At the end of the binding reactions the cells were rinsed 3 times with 0.5 ml of binding buffer, lysed with 0.5 ml of 5 m NaOH, and the entire lysate was counted. Nonspecific binding of tracer (NSB), determined in wells containing 1 μm[Nle8,21,Tyr34]rPTH-(1–34)NH2, was 1–1.5% of total counts added. Maximum specific binding (B0) was calculated as the total radioactivity bound to cells in the absence of unlabeled ligand minus NSB. IC50 values (dose of competing ligand that resulted in 50% inhibition of125I-[Nle8,21,Tyr34]rPTH-(1–34)NH2binding) were determined from plots of log(B/B0 − B) versuslog[competitor]. Cell surface receptor numbers were estimated from Scatchard analyses of homologous competition binding studies that were performed with125I-[Nle8,21,Tyr34]rPTH-(1–34)NH2(26 fmol/well) and varying amounts (1.2–300 pmol) of the same unlabeled ligand. Calculations of the number of receptors per cell assumed a single class of binding sites and a transfection efficiency of 20% (6Gardella T. Luck M. Fan M. Lee C. J. Biol. Chem. 1996; 271: 12820-12825Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 20Abou-Samra A.B. Jüppner H. Force T. Freeman M. Kong X.F. Schipani E. Urena P. Richards J. Bonventre J.V. Potts Jr, J.T. Kronenberg H.M. Segre G.V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2732-2736Crossref PubMed Scopus (1001) Google Scholar). Transfected COS-7 cells were rinsed with 500 μl of binding buffer, and 200 μl of IBMX 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 or binding buffer with various amounts of peptide added. The plates were incubated for 60 min at room temperature; the buffer was then withdrawn and the cells were lysed by adding 0.5 ml of 50 mm HCl and freezing. The diluted lysate (1:30 in distilled H20) was analyzed for cAMP content by radioimmunoassay. For the initial screening of the mutants, we compared the cAMP response of each receptor to a maximum dose (1 μm) of [Trp23]PTHrP-(1–36) to its response to the nonselective analog [Ile5,Trp23]PTHrP-(1–36) also at 1 μm. Dose-response analyses yielded EC50values (ligand dose resulting in 50% of maximum response (Emax) attained by that ligand), which were calculated from plots of log(E/E max − E)versus log[ligand], where E is the cAMP response measured at the corresponding dose of ligand (6Gardella T. Luck M. Fan M. Lee C. J. Biol. Chem. 1996; 271: 12820-12825Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The difference in the ligand selectivities of the PTH-1 receptor and the PTH-2 receptor can be seen in the cAMP response profiles shown in Fig. 1, panels A andB. The PTH-1 receptor responded fully and equally to both [Ile5, Trp23]PTHrP-(1–36) ([Ile5,Trp23,Tyr36]PTHrP-(1–36)amide) and [Trp23]PTHrP-(1–36) ([Trp23,Tyr36]PTHrP-(1–36)amide), which has histidine at position 5. In contrast, the PTH-2 receptor responded fully to [Ile5,Trp23]PTHrP-(1–36) but not at all to [Trp23]PTHrP-(1–36). The ligand selectivity of the PTH-2 receptor is not due to a difference in binding affinities because the two analogs exhibited comparable potencies in their ability to inhibit the binding125I-[Nle8,21,Tyr34]rPTH-(1–34)amide (Fig. 1 F). The high apparent binding affinity that the two PTHrP analogs displayed for the PTH-2 receptor in these experiments is due primarily to the Phe23 → Trp modification; this substitution of a PTHrP residue by the corresponding PTH residue markedly enhances binding potency at the PTH-2 receptor without affecting cAMP signaling (14Gardella T. Luck M. Jensen G. Usdin T. Jüppner H. J. Biol. Chem. 1996; 271: 19888-19893Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). A small improvement in binding potency was also seen for the His5 → Ile modification, however, this improvement was not receptor specific (Fig. 1, E andF) and was much smaller in magnitude than the effect of the substitution on PTH-2 receptor signaling. To localize the region of the PTH-2 receptor involved in His5/Ile5 signaling selectivity, we constructed a pair of chimeras in which the amino-terminal extracellular domains of the human PTH-1 and PTH-2 receptors were reciprocally interchanged and tested the chimeras in cAMP stimulation assays for responsiveness to [Trp23]PTHrP-(1–36) and to the nondiscriminating control peptide [Ile5,Trp23]PTHrP-(1–36). The 1E2 receptor chimera, which has the amino-terminal extracellular domain of the PTH-1 receptor connected (via an EcoRI site) to the mid- and carboxyl-terminal region of the PTH-2 receptor, discriminated between the two ligands (Fig. 1 C), whereas the reciprocal chimera 2E1 responded fully to each ligand (Fig. 1 D). These results indicated that the membrane-spanning and loop portion of the receptor determines His5/Ile5 signaling selectivity. To further localize the sites involved in residue 5 selectivity, we replaced most of the divergent residues in the membrane-spanning helices and extracellular connecting loops of the PTH-2 receptor with the corresponding residues of the PTH-1 receptor. As shown in Fig.2, these residues were replaced either by cassette substitution or, for two of the sites (Ala325 and Gly327), by single residue point mutation. All but 3 of the 21 mutant receptors were functional and adequately expressed on COS-7 cells, as judged by the cAMP response to [Ile5,Trp23]PTHrP-(1–36) and the binding of radioiodinated [Nle8,21,Tyr34]rPTH-(1–34)amide (TableI). The three cassette mutants showing poor cAMP responsiveness and little or no PTH binding, P2R-Csst#2, P2R-Csst#7 and P2R-Csst#18, were possibly not expressed on the cell surface and were considered uninformative. Of the functional mutant receptors, three displayed increased responsiveness to [Trp23]PTHrP-(1–36). These three cassettes are predicted to be located in the amino-terminal portion of extracellular loop 1 (P2R-Csst#5), the extracellular end of helix 3 (P2R-Csst#8), and at the COOH-terminal end of extracellular loop 2 (P2R-Csst#13) (Fig. 2). For each of these three receptor mutants the maximum binding of125I-[Nle8,21,Tyr34]rPTH-(1–34)amide was elevated by a factor of 1.8–3.2, in comparison to the binding observed for the WT PTH-2 receptor (Table I). This increase in maximum binding of radiolabeled PTH-(1–34) may indicate increased surface expression, enhanced PTH-(1–34) binding affinity, or both. It is unlikely, however, that such effects on surface expression or PTH-(1–34) binding affinity are the basis for the altered cAMP responsiveness to [Trp23]PTHrP-(1–36), because other mutants, such as P2R-Csst#16 and GV-327, caused comparable increases in radioligand-binding capacity without changing [Trp23]PTHrP-(1–36) signaling selectivity (TableI). We therefore focused on the receptor regions defined by cassette mutations 5, 8, and 13.Table ILocalization of PTHrP-signaling determinants in the PTH-2 receptorReceptorMaximum binding 125I-PTH-(1–34)cAMPBasal[Ile5]PTHrPPTHrPPTHrP/[Ile5]PTHrP% P2R-WTpmol/well%P2R-WT100 ± 16.1 ± 0.655.6 ± 1.86.9 ± 0.81.8 ± 1.0P1R-WT538 ± 574.4 ± 1.1148.5 ± 32.3142.6 ± 35.295.3 ± 8.6P2R cassette mutation 139 ± 43.9 ± 1.151.5 ± 2.53.1 ± 0.8< bsl 211 ± 31.9 ± 0.41.6 ± 0.42.1 ± 0.5ND 366 ± 23.8 ± 0.448.8 ± 5.64.8 ± 0.62.4 ± 1.0 4122 ± 44.8 ± 0.439.4 ± 4.65.9 ± 0.63.8 ± 1.8 5317 ± 254.5 ± 0.480.9 ± 8.336.0 ± 4.040.5 ± 1.5 621 ± 53.5 ± 0.432.5 ± 8.95.5 ± 0.39.2 ± 2.7 70.0 ± 0.72.2 ± 0.62.5 ± 0.82.1 ± 0.5ND 8181 ± 95.3 ± 0.654.9 ± 6.369.2 ± 8.7126.9 ± 4.5 996 ± 99.6 ± 1.190.8 ± 20.122.9 ± 4.116.3 ± 1.0 1092 ± 64.8 ± 0.681.2 ± 3.74.5 ± 1.0< bsl 1193 ± 104.0 ± 0.472.9 ± 8.911.9 ± 0.311.6 ± 0.5 12130 ± 93.3 ± 0.799.1 ± 9.916.3 ± 1.413.5 ± 0.4 13221 ± 141.6 ± 0.464.9 ± 23.537.9 ± 13.357.3 ± 0.9 1491 ± 44.3 ± 0.459.6 ± 9.43.9 ± 0.5< bsl 15157 ± 711.5 ± 0.890.3 ± 9.530.4 ± 2.424.3 ± 0.9 16203 ± 104.4 ± 0.364.2 ± 7.97.7 ± 0.46.1 ± 0.6 17129 ± 123.4 ± 0.775.2 ± 11.75.4 ± 0.23.1 ± 0.7 1827 ± 21.7 ± 0.22.2 ± 0.31.8 ± 0.2ND 1995 ± 24.1 ± 0.351.9 ± 5.94.4 ± 0.21.0 ± 0.6 AS-32589 ± 34.0 ± 0.739.3 ± 3.13.6 ± 0.5< bsl GV-327191 ± 117.6 ± 1.470.6 ± 15.111.0 ± 2.05.2 ± 1.2P2R point mutation parent cassette RA-1995104 ± 257.5 ± 0.9111.2 ± 22.39.7 ± 0.12.8 ± 1.1 VL-2015114 ± 53.4 ± 0.657.5 ± 21.16.3 ± 1.55.3 ± 0.9 HY-202553 ± 25.0 ± 1.449.8 ± 9.34.2 ± 0.2< bsl AS-2035105 ± 56.6 ± 1.062.3 ± 16.57.4 ± 0.72.5 ± 1.5 HG-2045128 ± 34.6 ± 0.946.1 ± 3.27.4 ± 0.76.7 ± 0.4 IA-205578 ± 45.9 ± 0.462.2 ± 12.86.4 ± 0.21.5 ± 1.0 GT-206584 ± 73.7 ± 0.337.7 ± 5.64.6 ± 0.83.3 ± 1.9 VL-20754.6 ± 2.81.5 ± 0.51.2 ± 0.41.5 ± 0.6ND KD-20858.6 ± 2.71.3 ± 0.41.9 ± 0.41.9 ± 0.5ND KR-237837 ± 45.3 ± 0.224.2 ± 4.44.5 ± 0.4< bsl IV-2388110 ± 44.3 ± 0.139.6 ± 6.55.0 ± 0.41.7 ± 0.9 VT-241875 ± 55.0 ± 0.882.4 ± 9.54.6 ± 0.2< bsl MF-242895 ± 75.4 ± 0.368.7 ± 10.96.0 ± 0.30.8 ± 0.4 IL-2448176 ± 36.0 ± 0.737.9 ± 9.040.3 ± 12.998.2 ± 22.2 IK-3141396 ± 64.8 ± 0.251.6 ± 8.54.3 ± 0.5< bsl YI-31813261 ± 176.8 ± 3.392.4 ± 6.466.3 ± 1.069.5 ± 3.9 AV-32113119 ± 65.2 ± 0.564.7 ± 6.79.8 ± 0.47.9 ± 0.8WT and mutant PTH receptors were expressed in COS-7 cells and evaluated for binding 125I-rPTH-(1–34) analog, and for intracellular cAMP levels, in the absence of added ligand (basal) or presence of [Trp23,Tyr36]hPTHrP-(1–36)NH2(PTHrP) or [Ile5,Trp23,Tyr36]hPTHrP(1–36)NH2([Ile5]PTHrP), each at a dose of 1 μm. The top portion of the table shows data from the 19 cassette mutations and 2 point mutations used in initial screening studies. The lower half shows the effect of point mutations made within regions defined by cassettes 5, 8, and 13. The far right column shows the ratio of the net responses to the two analogs. Data are the means ± S.E. of two experiments, each performed in duplicate. For some mutants, PTHrP responsiveness was not determined because of probable defects in expression (ND), or because the response to PTHrP was less than the basal cAMP level (< bsl). Open table in a new tab WT and mutant PTH receptors were expressed in COS-7 cells and evaluated for binding 125I-rPTH-(1–34) analog, and for intracellular cAMP levels, in the absence of added ligand (basal) or presence of [Trp23,Tyr36]hPTHrP-(1–36)NH2(PTHrP) or [Ile5,Trp23,Tyr36]hPTHrP(1–36)NH2([Ile5]PTHrP), each at a dose of 1 μm. The top portion of the table shows data from the 19 cassette mutations and 2 point mutations used in initial screening studies. The lower half shows the effect of point mutations made within regions defined by cassettes 5, 8, and 13. The far right column shows the ratio of the net responses to the two analogs. Data are the means ± S.E. of two experiments, each performed in duplicate. For some mutants, PTHrP responsiveness was not determined because of probable defects in expression (ND), or because the response to PTHrP was less than the basal cAMP level (< bsl). Replacement of each divergent residue in cassette region 5 by the corresponding residue of the PTH-1 receptor failed to identify a single residue affecting the cAMP response to [Trp23]PTHrP-(1–36) (Fig.3 B and Table I). It is possible that two or more sites in this region cooperatively contribute to His5/Ile5 signaling selectivity; however, two of the mutations in this set, Val207 → Leu (valine 207 changed to leucine, VL-207) and Lys208 → Asp (KD-208) were poorly expressed, as indicated by their very low responses to [Ile5,Trp23]PTHrP-(1–36) and minimal binding of125I-[Nle8,21,Tyr34]rPTH-(1–34)amide. Thus, the roles of these two residues could not be assessed. Within cassette region 8, one point mutation, Ile244 → Leu (IL-244), resulted in a strong increase in responsiveness to [Trp23]PTHrP-(1–36) (Fig. 3 B). Within cassette region 13, one other substitution Tyr318 → Ile (YI-318), also enhanced responsiveness to the PTHrP analog. Dose response analysis of these two mutant receptors demonstrated that each mutation by itself could account for a substantial component of the signaling selectivity inherent to the PTH-2 receptor (Fig.4). In fact, with the IL-244 receptor, the efficacy of [Trp23]PTHrP-(1–36) was equal to, if not slightly greater than, that of [Ile5,Trp23]PTHrP-(1–36) (Fig. 4 Band Table II). The effect of the YI-318 mutation was not as pronounced, and with this mutant [Trp23]PTHrP-(1–36) functioned as a partial agonist (Fig. 4 C). These two point mutations did not affect the ability of either PTHrP analog to inhibit the binding of125I-[Nle8,21,Tyr34]rPTH-(1–34)amide (Fig. 4 D-F). Scatchard analyses indicated that the number of PTH-(1–34) binding sites on the surface of COS-7 cells transfected with either mutant receptor did not differ significantly from the number of binding sites on cells expressing the WT PTH-2 receptor (Table III).Figure 4Ligand selectivity properties of wild-type and mutant PTH-2 receptors. The indicated wild-type and mutant PTH-2 receptors were expressed in COS-7 cells and examined for ligand-dependent stimulation of intracellular cAMP (A-C) and competition binding (D-F) using the analogs [Trp23,Tyr36]PTHrP(1–36)NH2(▵) and [Ile5,Trp23,Tyr36] PTHrP-(1–36)NH2(•). Intracellular cAMP and competition binding assays were performed as described under “Experimental Procedures.” Binding experiments used125I-[Nle8,21,Tyr34]rPTH-(1–34)NH2(100,000 cpm/well) as tracer. Each graph shows data combined from six to twelve independent experiments (mean ± S.E.) each performed in duplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIcAMP responses of WT and mutant PTH-1 and PTH-2 receptorsReceptorbasalcAMP[Trp23]PTHrP-(1–36)[Ile5,Trp23]PTHrP-(1–36)maximumEC50maximumEC50pmol/wellnmpmol/wellnmP2R-wt18 ± 219 ± 3134 ± 171.8 ± 0.4P2R-IL-24425 ± 4135 ± 241.9 ± 0.599 ± 242.5 ± 1.1P2R-YI-31810 ± 284 ± 242.9 ± 0.9122 ± 320.5 ± 0.1P1R-wt18 ± 4356 ± 550.6 ± 0.1287 ± 270.7 ± 0.2P1R-LI-28925 ± 8327 ± 690.5 ± 0.1288 ± 500.9 ± 0.3P1R-IY-36312 ± 3112 ± 260.9 ± 0.4103 ± 130.5 ± 0.1Values are means ± S.E. of six to twelve experiments performed in COS-7 cells. The peptides tested were [Trp23,Tyr36]PTHrP-(1–36)NH2 ([Trp23]PTHrP-(1–36)) and [Ile5,Trp23,Tyr36]PTHrP-(1–36)NH2 ([Ile5,Trp23]PTHrP-(1–36)). The maximum cAMP responses (basal not subtracted) were determined at a peptide dose of 1 μm. The low cAMP response of the WT PTH-2 receptor to [Trp23]PTHrP-(1–36) precluded the determination of an EC50 value. Open table in a new tab Table IIIScatchard analysis of WT and mutant PTH-1 and PTH-2 receptorsReceptorBinding affinity K d apparentSurface PTH-(1–34)binding sites/cellnm×10−6P2R-WT8.0 ± 1.70.5 ± 0.1P2R-IL-2443.5 ± 0.20.4 ± 0.1P2Rc-YI-3183.8 ± 0.81.5 ± 0.9P1R-WT10.1 ± 2.65.9 ± 2.0P1R-LI-2896.0 ± 0.73.0 ± 1.0P1R-IY-36311.3 ± 1.66.8 ± 1.5Homologous competition binding assays were performed in COS-7 cells for 6 h at 4 °C using125I-[Nle8,21,Tyr34]rPTH-(1–34)amide as a tracer radioligand and varying amounts of the same unlabeled peptide as a competitor ligand. Values are means ± S.E. of the mean of four separate experiments. Open table in a new tab Values are means ± S.E. of six to twelve experiments performed in COS-7 cells. The peptides tested were [Trp23,Tyr36]PTHrP-(1–36)NH2 ([Trp23]PTHrP-(1–36)) and [Ile5,Trp23,Tyr36]PTHrP-(1–36)NH2 ([Ile5,Trp23]PTHrP-(1–36)). The maximum cAMP responses (basal not subtracted) were determined at a peptide dose of 1 μm. The low cAMP response of the WT PTH-2 receptor to [Trp23]PTHrP-(1–36) precluded the de" @default.
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• W2016123750 title "Residues in the Membrane-spanning and Extracellular Loop Regions of the Parathyroid Hormone (PTH)-2 Receptor Determine Signaling Selectivity for PTH and PTH-related Peptide" @default.
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