Behar, V., Div. of Bone and Mineral Metabolism, Harvard-Thorndike Charles A. Dana L., Beth Israel Hospital, Boston, MA 02215, United States
Pines, M., Div. of Bone and Mineral Metabolism, Harvard-Thorndike Charles A. Dana L., Beth Israel Hospital, Boston, MA 02215, United States
Nakamoto, C., Div. of Bone and Mineral Metabolism, Harvard-Thorndike Charles A. Dana L., Beth Israel Hospital, Boston, MA 02215, United States
Greenberg, Z., Div. of Bone and Mineral Metabolism, Harvard-Thorndike Charles A. Dana L., Beth Israel Hospital, Boston, MA 02215, United States
Bisello, A., Div. of Bone and Mineral Metabolism, Harvard-Thorndike Charles A. Dana L., Beth Israel Hospital, Boston, MA 02215, United States
Stueckle, S.M., Div. of Bone and Mineral Metabolism, Harvard-Thorndike Charles A. Dana L., Beth Israel Hospital, Boston, MA 02215, United States
Bessalle, R., Div. of Bone and Mineral Metabolism, Harvard-Thorndike Charles A. Dana L., Beth Israel Hospital, Boston, MA 02215, United States
Usdin, T.B., Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, MD 20892-4090, United States
Chorev, M., Div. of Bone and Mineral Metabolism, Harvard-Thorndike Charles A. Dana L., Beth Israel Hospital, Boston, MA 02215, United States
Rosenblatt, M., Div. of Bone and Mineral Metabolism, Harvard-Thorndike Charles A. Dana L., Beth Israel Hospital, Boston, MA 02215, United States
Suva, L.J., Div. of Bone and Mineral Metabolism, Harvard-Thorndike Charles A. Dana L., Beth Israel Hospital, Boston, MA 02215, United States, Div. Bone and Mineral Metab. HIM946, Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215, United States

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 [Nle8,18,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.