We have generated a series of stably transfected 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 [Nle8,18,Lys13 (epsilon-pBz2), 2-L-Nal23,Tyr34]bPTH(1-34)NH2 (K13), to the hPTH2 receptor on BP-16 cells. Photoaffinity cross-linking identified an approximately 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 Kd, 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 [Ca 2+]i signaling observed, nor was specific competition vs. binding of a PTH-(1-34) radioligand ([125I]PTH) with PTHrP-(1-34)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 [Ca 2+]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.