Molecular Determinants of Tuberoinfundibular Peptide of 39 Residues (TIP39) Selectivity for the Parathyroid Hormone-2 (PTH2) Receptor

Abstract

Tuberoinfundibular peptide of 39 residues (TIP39) and the parathyroid hormone-2 (PTH2) receptor form part of an extended family of related signaling molecules that includes the PTH1 receptor, which responds to PTH and PTH-related protein. TIP39 does not appreciably activate the PTH1 receptor, but in this study it is shown to bind the receptor with moderate affinity (59 nm). In this study, we investigated the molecular determinants of both ligand and receptor for the PTH2 receptor selectivity of TIP39 and quantitatively evaluated the role of molecular elements in the binding of TIP39 to the PTH2 and PTH1 receptors. A chimeric receptor composed of the N-terminal extracellular domain of the PTH1 receptor and the remainder (juxtamembrane domain) of the PTH2 receptor (P2-NP1) was fully activated by TIP39 (E max = 98% of the rPTH-(1–34),E max, EC50 = 2.0 nm). This receptor chimera bound TIP39 with an equivalent affinity to the wild-type PTH2 receptor (2.3 and 2.0 nm, respectively). The reciprocal chimeric receptor (P1-NP2) was not activated by TIP39 and bound the ligand with an affinity equivalent to that of the PTH1 receptor. Thus, the juxtamembrane receptor domain specifies the signaling and binding selectivity of TIP39 for the PTH2 receptor over the PTH1 receptor. Removing six N-terminal residues of TIP39 eliminated activation of the PTH2 receptor and reduced binding affinity 70-fold. In contrast, this truncation increased affinity for the PTH1 receptor 10-fold, reversing the PTH2/PTH1 receptor binding selectivity and resulting in a high affinity interaction of TIP-(7–39) with the PTH1 receptor (6 nm). These findings can be explained by a strong interaction between the N-terminal region of TIP39 and the juxtamembrane domain of the PTH2 receptor, with the corresponding domain of the PTH1 receptor acting as a selectivity barrier against high affinity binding of TIP39. As a result, TIP-(7–39) is a highly potent, selective antagonist for the PTH1 receptor. Tuberoinfundibular peptide of 39 residues (TIP39) and the parathyroid hormone-2 (PTH2) receptor form part of an extended family of related signaling molecules that includes the PTH1 receptor, which responds to PTH and PTH-related protein. TIP39 does not appreciably activate the PTH1 receptor, but in this study it is shown to bind the receptor with moderate affinity (59 nm). In this study, we investigated the molecular determinants of both ligand and receptor for the PTH2 receptor selectivity of TIP39 and quantitatively evaluated the role of molecular elements in the binding of TIP39 to the PTH2 and PTH1 receptors. A chimeric receptor composed of the N-terminal extracellular domain of the PTH1 receptor and the remainder (juxtamembrane domain) of the PTH2 receptor (P2-NP1) was fully activated by TIP39 (E max = 98% of the rPTH-(1–34),E max, EC50 = 2.0 nm). This receptor chimera bound TIP39 with an equivalent affinity to the wild-type PTH2 receptor (2.3 and 2.0 nm, respectively). The reciprocal chimeric receptor (P1-NP2) was not activated by TIP39 and bound the ligand with an affinity equivalent to that of the PTH1 receptor. Thus, the juxtamembrane receptor domain specifies the signaling and binding selectivity of TIP39 for the PTH2 receptor over the PTH1 receptor. Removing six N-terminal residues of TIP39 eliminated activation of the PTH2 receptor and reduced binding affinity 70-fold. In contrast, this truncation increased affinity for the PTH1 receptor 10-fold, reversing the PTH2/PTH1 receptor binding selectivity and resulting in a high affinity interaction of TIP-(7–39) with the PTH1 receptor (6 nm). These findings can be explained by a strong interaction between the N-terminal region of TIP39 and the juxtamembrane domain of the PTH2 receptor, with the corresponding domain of the PTH1 receptor acting as a selectivity barrier against high affinity binding of TIP39. As a result, TIP-(7–39) is a highly potent, selective antagonist for the PTH1 receptor. tuberoinfundibular peptide of 39 residues bovine TIP39 parathyroid hormone rat PTH bovine PTH parathyroid hormone-related protein guanine nucleotide-binding regulatory protein receptor-G-protein guanosine 5′-3-O-(thio)triphosphate Tuberoinfundibular peptide of 39 residues (TIP39)1 is a recently discovered neuropeptide that was purified from bovine hypothalamus on the basis of its ability to activate the PTH2 receptor (1Usdin T.B. Hoare S.R.J. Wang T. Mezey É. Kowalak J.A. Nat. Neurosci. 1999; 2: 941-943Crossref PubMed Scopus (169) Google Scholar). TIP39 is a good candidate for the PTH2 receptor's endogenous ligand. It strongly activates the human, rat, and zebrafish 2Hoare, S. R. J., Rubin, D. A., Jüppner, H., and Usdin, T. B. (2000) Endocrinology, in press.2Hoare, S. R. J., Rubin, D. A., Jüppner, H., and Usdin, T. B. (2000) Endocrinology, in press. PTH2 receptors (1Usdin T.B. Hoare S.R.J. Wang T. Mezey É. Kowalak J.A. Nat. Neurosci. 1999; 2: 941-943Crossref PubMed Scopus (169) Google Scholar). PTH also strongly activates the human PTH2 receptor (2Usdin T.B. Gruber C. Bonner T.I. J. Biol. Chem. 1995; 270: 15455-15458Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar), but it is only a weak partial agonist for the rat (3Hoare S.R.J. Bonner T.I. Usdin T.B. Endocrinology. 1999; 140: 4419-4425Crossref PubMed Scopus (48) Google Scholar) and zebrafish2receptors. The physiological roles of TIP39 and the PTH2 receptor are currently being investigated. The PTH2 receptor is most abundant in the nervous system. Its expression is relatively high in the hypothalamus, where nerve terminals in the median eminence and cell bodies in the periventricular nucleus have particularly high receptor levels, suggesting a role in the modulation of pituitary function (1Usdin T.B. Hoare S.R.J. Wang T. Mezey É. Kowalak J.A. Nat. Neurosci. 1999; 2: 941-943Crossref PubMed Scopus (169) Google Scholar). PTH2 receptor concentration in the superficial lamina of the spinal cord dorsal horn suggests a role in the modulation of pain perception (1Usdin T.B. Hoare S.R.J. Wang T. Mezey É. Kowalak J.A. Nat. Neurosci. 1999; 2: 941-943Crossref PubMed Scopus (169) Google Scholar). In the periphery, the receptor is expressed by discrete cells in a number of tissues including pancreatic islet somatostatin cells, heart and vascular muscle cells, and cells within bronchioles and vasculature in the lung (4Usdin T.B. Hilton J. Vertesi T. Harta G. Segre S. Mezey É. Endocrinology. 1999; 140: 3363-3371Crossref PubMed Google Scholar). The PTH2 receptor and TIP39 form a part of an extended family of related receptors and ligands (1Usdin T.B. Hoare S.R.J. Wang T. Mezey É. Kowalak J.A. Nat. Neurosci. 1999; 2: 941-943Crossref PubMed Scopus (169) Google Scholar). The human PTH2 receptor shares 51% amino acid sequence identity with the human PTH1 receptor. The PTH1 receptor mediates the principal actions of PTH (elevation of blood calcium levels) and PTHrP (a locally acting autocrine/paracrine factor and developmental regulator) (5Martin T.J. Moseley J.M. Williams R.H. Wilson J.D. Foster D.W. Williams' Textbook of Endocrinology. Saunders, Philadelphia1995: 967-977Google Scholar, 6Potts J.T.J. Bringhurst F.R. Gardella T. Nussbaum S. Segre G. Kronenberg H. Williams R.H. Wilson J.D. Foster D.W. Williams' Textbook of Endocrinology. Saunders, Philadelphia1995: 920-966Google Scholar). Both PTH receptors belong to the type II family of G-protein-coupled receptors that respond to peptide modulators, including calcitonin, glucagon, secretin, and vasoactive intestinal polypeptide. The similarity identified for PTH receptors extends to their ligands (Fig. 1). Five residues are identical when the sequences of TIP39, PTH, and PTHrP are aligned. TIP39 is somewhat more similar to PTH. Seven of the 19 C-terminal amino acids are identical between bovine TIP39 and PTH from most species. The PTH2 and PTH1 receptors, together with their ligands, have presumably evolved to selectively mediate different physiological functions. In this regard, the PTH1 receptor mediates the responses to PTH and PTHrP (6Potts J.T.J. Bringhurst F.R. Gardella T. Nussbaum S. Segre G. Kronenberg H. Williams R.H. Wilson J.D. Foster D.W. Williams' Textbook of Endocrinology. Saunders, Philadelphia1995: 920-966Google Scholar) but does not respond to TIP39 (1Usdin T.B. Hoare S.R.J. Wang T. Mezey É. Kowalak J.A. Nat. Neurosci. 1999; 2: 941-943Crossref PubMed Scopus (169) Google Scholar), whereas the PTH2 receptor responds to TIP39 and perhaps PTH but not to PTHrP (1Usdin T.B. Hoare S.R.J. Wang T. Mezey É. Kowalak J.A. Nat. Neurosci. 1999; 2: 941-943Crossref PubMed Scopus (169) Google Scholar). The molecular basis of PTHrP selectivity for the PTH1 receptor over the PTH2 receptor has been studied extensively (7Behar V. Nakamoto C. Greenberg Z. Bisello A. Suva L.J. Rosenblatt M. Chorev M. Endocrinology. 1996; 137: 4217-4224Crossref PubMed Scopus (44) Google Scholar, 8Bergwitz C. Jusseaume S.A. Luck M.D. Jüppner H. Gardella T.J. J. Biol. Chem. 1997; 272: 28861-28868Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 9Clark J.A. Bonner T.I. Kim A.S. Usdin T.B. Mol. Endocrinol. 1998; 12: 193-206Crossref PubMed Google Scholar, 10Gardella T.J. Luck M.D. Jensen G.S. Usdin T.B. Jüppner H. J. Biol. Chem. 1996; 271: 19888-19893Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 11Turner P.R. Mefford S. Bambino T. Nissenson R.A. J. Biol. Chem. 1998; 273: 3830-3837Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). For the PTH1 receptor, these and other selectivity studies (12Bergwitz C. Gardella T.J. Flannery M.R. Potts Jr., J.T. 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Chem. 1996; 271: 12795-12800Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 21Stroop S.D. Kuestner R.E. Serwold T.F. Chen L. Moore E.E. Biochemistry. 1995; 34: 1050-1057Crossref PubMed Scopus (93) Google Scholar, 22Stroop S.D. Nakamuta H. Kuestner R.E. Moore E.E. Epand R.M. Endocrinology. 1996; 137: 4752-4756Crossref PubMed Scopus (62) Google Scholar). Receptor-ligand cross-linking studies have confirmed this binding orientation for ligand binding to the PTH1 receptor (17Mannstadt M. Jüppner H. Gardella T.J. Am. J. Physiol. 1999; 277: F665-F675Crossref PubMed Google Scholar, 23Bisello A. Adams A.E. Mierke D.F. Pellegrini M. Rosenblatt M. Suva L.J. Chorev M. J. Biol. Chem. 1998; 273: 22498-22505Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 24Mannstadt 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) and also for PTH binding to the human PTH2 receptor (25Behar V. Bisello A. Rosenblatt M. Chorev M. Endocrinology. 1999; 140: 4251-4261Crossref PubMed Google Scholar). The receptor interactions of TIP39 have not previously been examined, beyond the initial observation of selective activation of the PTH2 receptor. We have now begun investigating the molecular basis of TIP39 selectivity for the PTH2 receptor over the PTH1 receptor. Previous studies of PTH receptors and other type II G-protein-coupled receptors have demonstrated the involvement of particular domains or amino acid residues in binding or activation but have not taken into account the effects of receptor-G-protein coupling in evaluating their contribution to ligand binding affinity. In this study, we have considered the nature of receptor states in evaluating the contributions of binding determinants to receptor selectivity. This has enabled us to quantitatively evaluate the role of molecular elements in the binding of TIP39 to the PTH2 and PTH1 receptors. Coupled with the conformational characterization of TIP39 presented in the accompanying paper (41Piserchio A. Usdin T. Mierke D.F. J. Biol. Chem. 2000; 275: 27284-27290Abstract Full Text Full Text PDF PubMed Google Scholar), these data provide structural insight into the the molecular interactions of this new receptor-ligand system. The following peptides were purchased from Bachem (Torrance, CA) or Peninsula Laboratories (Belmont, CA): rPTH-(1–34), [Nle8,21,Tyr34]rPTH-(1–34) amide, [Nle8,18,Tyr34]bPTH-(3–34) amide, PTHrP-(1–34), and human glucagon-(1–29). bTIP39 was obtained from AnaSpec Inc. (San Jose, CA) or Biomolecules Midwest (Waterloo, IL). The letters “r” and “b” designate the peptide sequence as rat and bovine, respectively. The peptides were dissolved in 10 mmacetic acid, with the concentration calculated using the peptide content and weight provided by the supplier. Aliquots were stored at −80 °C and used once. N-terminally truncated TIP39 analogues were purchased from Biomolecules Midwest, purified by HPLC, and quantified using the copper bicinchoninic acid method (Pierce) with TIP39 as the standard. 125I-cAMP was obtained from NEN Life Science Products, and Na125I (2000 Ci/mmol) was from ICN Biomedicals (Costa Mesa, CA). [3-125I-iodotyrosyl10]glucagon (2000 Ci/mmol) was from Amersham Pharmacia Biotech. Lactose peroxidase was obtained from Sigma. Cell culture supplies were obtained from Life Technologies, Inc. except for Dulbecco's modified Eagle's medium, which was from Mediatech (Herndon, VA). The radioligands125I-[Nle8,21,Tyr34]rPTH-(1–34) and125I-[Nle8,18,Tyr34]bPTH-(3–34) were prepared using chloramine T as catalyst and the di-iodinated peptide (4000 Ci/mmol) purified by HPLC, as described previously (9Clark J.A. Bonner T.I. Kim A.S. Usdin T.B. Mol. Endocrinol. 1998; 12: 193-206Crossref PubMed Google Scholar).125I-TIP39 was prepared using the lactose-peroxidase method (27Marchalonis J.J. Biochem. J. 1969; 113: 299-305Crossref PubMed Scopus (1005) Google Scholar). TIP39 (5 μg in 5 μl of reaction buffer (0.1 msodium acetate buffer, pH 6.5)) was dispensed into a siliconized microcentrifuge tube, followed by the sequential addition of 0.5 mCi of Na125I, 5 μl of 20 μg/ml lactose peroxidase in reaction buffer, and 45 μl of reaction buffer. After mixing, 10 μl of 0.001% H2O2 was added. After 20 min at room temperature, the reaction was terminated by the addition of 0.5 ml of reaction buffer supplemented with 0.1% sodium azide. After a further 5 min, 0.5 ml of reaction buffer supplemented with 1 m NaCl, 0.1% bovine serum albumin, and 1% potassium iodide was added. The radioligand was then desalted using a C18 cartridge and purified by high pressure liquid chromatography. The radioactive peak fractions corresponded with a single peak of UV absorbance. The PTH2/PTH1 receptor chimeras have been described previously (9Clark J.A. Bonner T.I. Kim A.S. Usdin T.B. Mol. Endocrinol. 1998; 12: 193-206Crossref PubMed Google Scholar). Chimeric receptors and their parent wild-type receptors contain a sequence encoding a 12-residue hemagglutinin tag inserted at the 3′-end of the coding sequence. The chimeric receptors were constructed by exchanging residues 215–594 of the PTH1 receptor with residues 172–550 of the PTH2 receptor. Amino acids 62–106 (encoded by exon E2 of the PTH1 receptor gene) were removed from the PTH1 receptor used for construction of these chimeras to facilitate comparisons with the PTH2 receptor, which lacks a homologous sequence (9Clark J.A. Bonner T.I. Kim A.S. Usdin T.B. Mol. Endocrinol. 1998; 12: 193-206Crossref PubMed Google Scholar). TIP39 displayed an indistinguishable activation and ligand binding profile for the exon-deleted and full-length forms of the PTH1 receptor (data not shown). A slightly different chimeric receptor nomenclature was used in this study compared with the study of Clark et al. (9Clark J.A. Bonner T.I. Kim A.S. Usdin T.B. Mol. Endocrinol. 1998; 12: 193-206Crossref PubMed Google Scholar). P1-NP2 is the same construct as PrP-NP2, and P2-NP1 corresponds to P2-ΔNPrP. Chimeric PTH2/glucagon receptors were constructed by exchanging the N-terminal extracellular domain between the hemagglutinin-tagged PTH2 receptor in pcDNA1/Amp and the human glucagon receptor in pCI.neo (28MacNeil D.J. Occi J.L. Hey P.J. Strader C.D. Graziano M.P. Biochem. Biophys. Res. Commun. 1994; 198: 328-334Crossref PubMed Scopus (71) Google Scholar). A BstZ17I restriction site was engineered into the human glucagon receptor sequence by converting Cys435 to thymidine, using the GeneEditor Site-directed Mutagenesis System (Promega, Madison, WI) according to the manufacturer's protocol, allowing the first 443 base pairs of the coding sequence of the PTH2 receptor and the first 434 base pairs of the glucagon receptor to be exchanged as BstZ17I/XbaI fragments. COS-7 cells were grown as described previously (9Clark J.A. Bonner T.I. Kim A.S. Usdin T.B. Mol. Endocrinol. 1998; 12: 193-206Crossref PubMed Google Scholar). For cAMP accumulation assays, COS-7 cells were transfected as described previously (9Clark J.A. Bonner T.I. Kim A.S. Usdin T.B. Mol. Endocrinol. 1998; 12: 193-206Crossref PubMed Google Scholar) except that transfections were performed in 10-cm tissue culture dishes using 10 μg of plasmid DNA. The cells were transferred following trypsinization to 96-well plates at a density of 50,000 cells/well the following day. Cells were used for cAMP accumulation assays 3 days after transfection. For preparation of transfected COS-7 cell membranes, confluent 15-cm tissue culture plates were transfected with 30–100 μg of DNA, and cells were harvested 3 days after transfection. HEK293 cells stably expressing the human PTH2 or PTH1 receptors were grown as previously (29Usdin T.B. Endocrinology. 1997; 138: 831-838Crossref PubMed Scopus (54) Google Scholar) and transferred to polyornithine-coated 96-well tissue culture plates 1 day prior to assay. Ligand-stimulated accumulation of cAMP was measured as described previously (3Hoare S.R.J. Bonner T.I. Usdin T.B. Endocrinology. 1999; 140: 4419-4425Crossref PubMed Scopus (48) Google Scholar), using a radioimmunoassay to quantify cAMP (9Clark J.A. Bonner T.I. Kim A.S. Usdin T.B. Mol. Endocrinol. 1998; 12: 193-206Crossref PubMed Google Scholar). P2 membrane preparations from HEK293 cells expressing the human PTH2 and PTH1 receptors were isolated as described previously (30Hoare S.R.J. Usdin T.B. J. Pharmacol. Toxicol. Methods. 1999; 41: 83-90Crossref PubMed Scopus (15) Google Scholar). COS-7 cell membranes were prepared using a modified procedure. Cells were washed with 10 ml of PBS/plate and mechanically dislodged in 10 ml of 4 mm EDTA in phosphate-buffered saline. Cells were centrifuged at 1000 ×g for 10 min, and the cell pellet was suspended in lysis buffer (10 mm Tris, 2 mm EDTA, 6 mmMgCl2, and 100 μm(4-(2-aminoethyl))-benzenesulfonylfluoride, pH 7.5) using 32 ml of lysis buffer for five confluent 15-cm plates of cells. After 1 h at 4 °C, 8 ml of 1.25 m sucrose was added, and cells were immediately homogenized by 50 strokes with a Dounce homogenizer. The homogenate was then centrifuged at 1000 × g for 10 min to remove unbroken cells and larger debris. Cell membranes were collected by centrifugation, quantified, and stored as described previously (30Hoare S.R.J. Usdin T.B. J. Pharmacol. Toxicol. Methods. 1999; 41: 83-90Crossref PubMed Scopus (15) Google Scholar). In these assays, the binding of a range of concentrations of an unlabeled ligand was measured by displacement of radioligand binding. Three methods were employed. An assay employing centrifugation to separate bound and free radioligand was used to accurately measure ligand binding parameters (30Hoare S.R.J. Usdin T.B. J. Pharmacol. Toxicol. Methods. 1999; 41: 83-90Crossref PubMed Scopus (15) Google Scholar). A higher through-put method employing rapid filtration was used to generate comparative ligand binding data (30Hoare S.R.J. Usdin T.B. J. Pharmacol. Toxicol. Methods. 1999; 41: 83-90Crossref PubMed Scopus (15) Google Scholar). Whole-cell binding assays (9Clark J.A. Bonner T.I. Kim A.S. Usdin T.B. Mol. Endocrinol. 1998; 12: 193-206Crossref PubMed Google Scholar) were used to measure radioligand binding to chimeric PTH2/glucagon receptors, since this assay provides the highest total binding/nonspecific binding ratio, important for detecting lower affinity binding of radioligands. In all these assays, a very low concentration of radioligand was used so that the IC50closely approximates the ligand affinity. In the centrifugation assay, cell membranes (45–50 μg), radioligand (100,000–300,000 cpm), and unlabeled ligand were incubated in a final volume of 1 ml of assay buffer (20 mm HEPES, 100 mm NaCl, 1 mm EDTA, 3 mmMgSO4, pH 7.5, supplemented with 0.3% nonfat dried milk powder, 100 μm(4-(2-aminoethyl))-benzenesulfonylfluoride, and 1 μg/ml bacitracin) for 2 h at 21 °C. Membranes were collected at 18,000 × g, the surface of the pellet was gently washed, and the radioactivity was counted as described previously (30Hoare S.R.J. Usdin T.B. J. Pharmacol. Toxicol. Methods. 1999; 41: 83-90Crossref PubMed Scopus (15) Google Scholar). For the PTH1 receptor,125I-[Nle8,18,Tyr34]bPTH-(3–34) was used as radioligand at a final concentration of approximately 20–32 pm. The PTH2 receptor was labeled of TIP39 using the lactose peroxidase of the total radioligand was bound within the membrane For binding to the PTH2 receptor in HEK293 membranes, this the of μg of membrane protein from transfected cells, to 45 μg with membranes from HEK293 cells. of the total radioligand was bound all of the membrane in the was from transfected In the filtration assay μg of membrane of radioligand for and and unlabeled ligand were incubated for 2 h at 21 °C. Membranes were harvested as described (30Hoare S.R.J. Usdin T.B. J. Pharmacol. Toxicol. Methods. 1999; 41: 83-90Crossref PubMed Scopus (15) Google Scholar). binding was of the total of radioactivity added. The binding assay was performed as described previously (9Clark J.A. Bonner T.I. Kim A.S. Usdin T.B. Mol. Endocrinol. 1998; 12: 193-206Crossref PubMed Google Scholar). data for cAMP accumulation and displacement of radioligand binding were using the following using 1 where the of the ligand concentration and the For cAMP the of cAMP at a peptide min is the cAMP in the of and max is the For of radioligand is the bound at a unlabeled ligand min is binding in the of a high concentration of the unlabeled of the and max is total binding in the of unlabeled of was performed by single factor of followed by with the of was performed using a In HEK293 cells, the stably expressed human PTH2 receptor is activated by TIP39 = and by = max = of the to whereas is (Fig. 2 The human PTH1 receptor stably expressed in HEK293 cells is activated by and of and nm, but is not appreciably activated by TIP39 TIP39 selectively activates the PTH2 receptor in HEK293 cells. This activation profile that of the receptors expressed in COS-7 cells (1Usdin T.B. Hoare S.R.J. Wang T. Mezey É. Kowalak J.A. Nat. Neurosci. 1999; 2: 941-943Crossref PubMed Scopus (169) Google Scholar). It is that TIP39 to the PTH1 receptor but to activate It is also not related are the concentration of TIP39 activation and binding. We measured the binding of TIP39 to PTH1 and PTH2 receptors. The binding assays were performed in the and of 10 to ligand binding was to receptor-G-protein of PTH1 receptor activation and binding by TIP39 and The receptor was used for these of cAMP in cells by TIP39 and binding to isolated cell membranes by TIP39 in the and of 10 using the centrifugation binding assay described [Nle8,18,Tyr34]bPTH-(3–34) binding was as the between total binding unlabeled ligand and binding in the of unlabeled has been used previously as a radioligand for the PTH2 receptor (9Clark J.A. Bonner T.I. Kim A.S. Usdin T.B. Mol. Endocrinol. 1998; 12: 193-206Crossref PubMed Google Scholar, 10Gardella T.J. Luck M.D. Jensen G.S. Usdin T.B. Jüppner H. J. Biol. Chem. 1996; 271: 19888-19893Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), but we that the was low in membrane binding assays and in the and of respectively). TIP39 is a agonist for the PTH2 receptor, we evaluated it as a TIP39 a at that can be as as a at that can be TIP39 labeled in a reaction not bind to the PTH2 receptor. prepared in a lactose reaction bound to the PTH2 receptor in HEK293 membranes with a higher and in the and of and binding was in membranes prepared from HEK293 cells. TIP39 binding to the PTH2 receptor with high = nm, 2 The of 10 a of the binding suggesting that TIP39 with higher affinity to the to the receptor (Fig. 2 The for TIP39 was of the was by or the (data not shown). the PTH1 receptor, TIP39 binding with a moderate affinity of nm, the binding described by a of was to that the ligand with indistinguishable affinity to the and states of the receptor (Fig. 3 TIP39 selectively to the PTH2 receptor over the PTH1 receptor. The peptide is a high affinity agonist of the PTH2 receptor and a moderate affinity antagonist of the PTH1 receptor. selectivity was in the of that this selectivity from a interaction with the PTH2 receptor and is not a of receptor-G-protein coupling ligand affinity for this of TIP39 and to PTH2 and PTH1 receptors in HEK293