The human PTH2 receptor: Binding and signal transduction properties of the stably expressed recombinant receptor

We have generated a series of stably transferred HEK-293 cell lines expressing the newly identified alternate human PTH receptor (hPTH2 receptor). This receptor subtype is selectively activated by N-terminal PTH- (1-34) and not the corresponding N-terminal (1-34) region of the functionally and structurally related hormone, PTH-related protein (PTHrP). A total of 20 distinct clones displaying different levels of PTH-responsive cAMP production were analyzed. None responded to PTHrP-(1-34). One of these clones (BP-16), displaying maximal PTH responsiveness, was chosen for more detailed evaluation. The BP-16 clone (and the parental HEK-293 cell line lacking both the hPTH/PTHrP receptor and the hPTH2 receptor) were examined for PTH binding, PTH-stimulated cAMP accumulation, PTH-stimulated changes in intracellular calcium ([Ca²⁺](i)) levels, and hPTH2 receptor messenger RNA expression. In addition, we studied the photomediated cross-linking of a potent PTH agonist, namely [Nle^8,18,Lys¹³(ε-pBz₂),2-L- Na]²³,Tyr³⁴]bPTH(1-34)NH₂ (K13), to the hPTH2 receptor on BP-16 cells. Photoaffinity cross-linking identified an ~90-kDa cell membrane component that was specifically competed by PTH-(1-34) and other receptor-interacting ligands, PTH-(1-34) and K13 are potent stimulators of both cAMP accumulation and increases in [Ca²⁺](i) levels, and both bind to the hPTH2 receptor with high affinity (apparent K(d), 2.8 ± 0.9 x 10^-8 and 8.5 ± 1.7 x 10^-8 M, respectively). There was no apparent binding, cAMP-stimulating activity, or [Ca²⁺](i) signaling observed, nor was specific competition vs. binding of a PTH-(1-34) radioligand ([¹²⁵I]PTH) with PTHrP-(134)NH₂ found. PTHrP-(1- 34) failed to inhibit cross-linking of the hPTH2 receptor by radiolabeled K13 ([¹²⁵I]K13). However, effective competition vs. [[¹²⁵I]PTH and [[¹²⁵I]K13 binding and [[¹²⁵I]K13 cross-linking were observed with the potent PTH/PTHrP receptor antagonists, PTHrP-(7-34)NH₂ and PTH-(7-34)NH₂. PTHrP-(7-34)NH₂ was shown to be a partial agonist that weakly stimulates both cAMP accumulation and increases in [Ca²⁺](i) levels in BP-16 cells. These data suggest that the hPTH2 receptor is distinct from the hPTH/PTHrP receptor in the structural features it requires for ligand binding in the family of PTH and PTHrP peptides.