THE HUMAN PTH2 RECEPTOR - BINDING AND SIGNAL-TRANSDUCTION PROPERTIES OF THE STABLY EXPRESSED RECOMBINANT RECEPTOR

We have generated a series of stably transfected HEK-293 cell lines ex pressing the newly identified alternate human PTH receptor (hPTH2 rece ptor). This receptor subtype is selectively activated by N-terminal PT H-(1-34) and not the corresponding N-terminal (1-34) region of the fun ctionally and structurally related hormone, PTH-related protein (PTHrP ). A total of 20 distinct clones displaying different levels of PTH-re sponsive 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 pare ntal HEK-293 cell line lacking both the hPTH/PTHrP receptor and the hP TH2 receptor) were examined for PTH binding, PTH-stimulated cAMP accum ulation, PTH-stimulated changes in intracellular calcium ([Ca2+](i)) l evels, and hPTH2 receptor messenger RNA expression. In addition, we st udied the photomediated cross-linking of a potent PTH agonist, namely (epsilon-pBz(2)),2-L-Nal(23),Tyr(34)]bPTH(1-34)NH2 (K13), to the hPTH2 receptor on BP-16 cells. Photoaffinity cross-linking identified an si milar to 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 affini ty (apparent K-d, 2.8 +/- 0.9 x 10(-8) and 8.5 +/- 1.7 x 10(-8) M, res pectively). There was no apparent binding, cAMP-stimulating activity, or [Ca2+](i) signaling observed, nor was specific competition vs. bind ing of a PTH-(1-34) radioligand ([I-125]PTH) with PTHrP-(1-34)-NH2 fou nd. PTHrP-(1-34) failed to inhibit cross-linking of the hPTH2 receptor by radiolabeled K13 ([I-125]K13). However, effective competition vs. [I-125]PTH and [I-125]K13 binding and [125I]K13 cross-linking were obs erved 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 t hat 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 r equires for ligand binding in the family of PTH and PTHrP peptides.