CDB15:0001270 PTH2 — PTH2R

ABSTRACT: The tuberoinfundibular peptide TIP39 [TIP-(1-39)], which exhibits only limited amino acid sequence homology with PTH and PTH-related peptide (PTHrP), stimulates cAMP accumulation in cells expressing the PTH2 receptor (PTH2R), but it is inactive at the PTH/PTHrP receptor (PTH1R). However, when using either (125)I-labeled rat [Nle(8,21),Tyr(34)]PTH-(1-34)amide (rPTH) or (125)I-labeled human [Tyr(36)]PTHrP-(1-36)amide [PTHrP-(1-36)] for radioreceptor studies, TIP-(1-39) bound to LLCPK(1) cells stably expressing the PTH1R (HKrk-B7 cells), albeit with weak apparent affinity (243 +/- 52 and 210 +/- 64 nM, respectively). In comparison to the parent peptide, the apparent binding affinity of TIP-(3-39) was about 3-fold higher, and that of TIP-(9-39) was about 5.5-fold higher. However, despite their improved IC(50) values at the PTH1R, both truncated peptides failed to stimulate cAMP accumulation in HKrk-B7 cells. In contrast, the chimeric peptide PTHrP-(1-20)/TIP-(23-39) bound to HKrk-B7 cells with affinities of 31 +/- 8.2 and 11 +/- 4.0 nM when using radiolabeled rPTH and PTHrP-(1-36), respectively, and it stimulated cAMP accumulation in HKrk-B7 and SaOS-2 cells with potencies (EC(50), 1.40 +/- 0.3 and 0.38 +/- 0.12 nM, respectively) and efficacies (maximum levels, 39 +/- 8 and 31 +/- 3 pmol/well, respectively) similar to those of PTH-(1-34) and PTHrP-(1-36). In both cell lines, TIP(9-39) and, to a lesser extent, TIP-(1-39) inhibited the actions of the three agonists with efficiencies similar to those of [Leu(11),D-Trp(12),Trp(23),Tyr(36)]PTHrP-(7-36)amide, an established PTH1R antagonist. Taken together, the currently available data suggest that the carboxyl-terminal portion of TIP-(1-39) interacts efficiently with the PTH1R, at sites identical to or closely overlapping those used by PTH-(1-34) and PTHrP-(1-36). The amino-terminal residues of TIP-(1-39), however, are unable to interact productively with the PTH1R, thus enabling TIP-(1-39) and some of its truncated analogs to function as an antagonist at this receptor.

ABSTRACT: The parathyroid hormone (PTH) 2 receptor is potently activated by tuberoinfundibular peptide (TIP39). Rat and human PTH2 receptors differ considerably in their PTH responsiveness. PTH weakly stimulates cAMP accumulation via the rat receptor, and here we show it did not detectably increase intracellular calcium ([Ca2+]i) and bound with low affinity (450 nM). For the human PTH2 receptor PTH was a full agonist for increasing cAMP, a partial agonist for increasing [Ca2+]i, and bound with high affinity (18 nM). In addition, the antagonists PTH(7-34) and TIP(7-39) bound with 10- to 49-fold lower affinity to the rat receptor. We investigated the molecular basis of differential PTH and antagonist interaction with human and rat PTH2 receptors by using chimeric human/rat PTH2 receptors. PTH cAMP-signaling efficacy (Emax) was determined by extracellular loop (EL) 1 and a region including EL2 and EL3. The N-terminal domain determined PTH binding selectivity at the inactive receptor state. Multiple regions throughout the receptor are required for the PTH-PTH2 receptor complex to adopt a high-affinity active state: inserting the rat receptor's N-terminal domain, EL1 or EL2/3, into the human receptor increased PTH's EC50 and reciprocal exchanges did not reduce EC50. This suggests the global receptor conformation prevents the rat receptor from adopting a high-affinity state when in complex with PTH. N-terminal ligand truncation, producing the antagonists PTH(7-34) and TIP(7-39), altered ligand interaction with the membrane-embedded domain of the receptor, eliminating EL2/3 as a specificity determinant and lowering binding affinity. These insights should contribute to the development of a high-affinity PTH2 receptor antagonist, for investigating the receptor's physiological role.