The mechanisms of ligand binding and receptor activation for G-protein-coupled receptors in the secretin/ parathyroid hormone (PTH) receptor subfamily are not understood. The PTH1 receptor (PTH1R) signals in response to both PTH and parathyroid hormone-related peptide (PTHrP), whereas the PTH2 receptor (PTH2R) responds only to PTH, not to PTHrP. To locate PTHrP discriminatory domains in the PTH2R, we generated PTH1R/PTH2R chimeras in which the extracellular amino-terminal domains were exchanged. Production of cAMP in response to 1 M PTHrP or PTH was identical in cells expressing the PTH1R with the PTH2R amino terminus and in cells expressing the PTH2R with the PTH1R amino terminus. The ability of the chimeric receptor with the PTH2R amino terminus to respond fully to PTHrP showed that the body of the PTH2R must contain sites that limit the response to PTHrP. Mutations to PTH1R sequence were therefore made in each of the seven transmembrane domains of the PTH2R. Mutations in transmembrane domains 3 and 7 resulted in receptors able to respond to PTHrP. Thus, residues in more than one domain form a barrier or filter, allowing the receptor to discriminate between different ligands.The mechanisms that allow G-protein-coupled receptors (GPCRs) 1 to distinguish between different ligands are not known. The GPCR for parathyroid hormone (PTH) and for parathyroid hormone-related peptide (PTHrP) binds and signals via adenylate cyclase and phospholipase C in response to both PTH and the structurally very different ligand PTHrP (1). Both ligands activate this receptor (PTH1R) with equal potency. A recently identified member of this secretin/PTH/calcitonin subfamily (2) of GPCRs, the PTH2 receptor (PTH2R), signals only in response to PTH, not to PTHrP (3, 4). What are the structural features of the PTH2R required for this effective discrimination?Features of the ligand required for this selective response have been identified. A single modification of Phe 23 in hPTHrP-(1-34) to Trp, the residue in PTH (see Fig. 1), allows the ligand to bind to, but not activate, the PTH2R (5). A modification of a residue in the N-terminal region of the ligand (His 5 ) to the residue found at this position in PTH (Ile) allows activation as measured by cAMP production (5, 6). These data are consistent with the idea that the C-terminal region of the peptide hormone is important for binding to the receptor and that the N terminus of the ligand is important for receptor activation (7). Unanswered by these studies are the structural features of the PTH2R that limit the response to PTHrP. The PTH1R and PTH2R share a high percent sequence homology in their seven transmembrane regions (ϳ70%), with little homology present in the amino-terminal extracellular domain (3), making them well suited for studies to determine the location and nature of selectivity and activation mechanisms.Previous work with chimeric receptors of between species homologues of the PTH1R (7) or with secretin and VIP receptors (8) has shown that the amino-terminal domain is ...

The present studies were carried out to evaluate the mechanisms by which PTH/PTHrP receptor (PTHR) activation influences cell viability. In 293 cells expressing recombinant PTHRs, PTH treatment markedly reduced the number of viable cells. This effect was associated with a marked apoptotic response including DNA fragmentation and the appearance of apoptotic nuclei. Similar effects were evidenced in response to serum withdrawal or to the addition of tumor necrosis factor (TNFalpha). Addition of caspase inhibitors or overexpression of bcl-2 partially abrogated apoptosis induced by serum withdrawal. Caspase inhibitors also protected cells from PTH-induced apoptosis, but overexpression of bcl-2 did not. The effects of PTH on cell number and apoptosis were neither mimicked by activators of the cAMP pathway (forskolin, isoproterenol) nor blocked by an inhibitor (H-89). However, elevation of Ca(i)2+ by addition of thapsigargin induced rapid apoptosis, and suppression of Ca(i)2+ by overexpression of the calcium- binding protein, calbindin D28k, inhibited PTH-induced apoptosis. The protein kinase C inhibitor GF 109203X partially inhibited PTH-induced apoptosis. Regulator of G protein signaling 4 (RGS4) (an inhibitor of the activity of the alpha-subunit of Gq) suppressed apoptotic signaling by the PTHR, whereas the C-terminal fragment of GRK2 (an inhibitor of the activity of the beta(gamma)-subunits of G proteins) was without effect. Chemical mutagenesis allowed selection of a series of 293 cell lines resistant to the apoptotic actions of PTH; a subset of these were also resistant to TNFalpha. These results suggest that 1) apoptosis produced by PTHR and TNF receptor signaling involve converging pathways; and 2) Gq-mediated phospholipase C/Ca2+ signaling, rather than Gs-mediated cAMP signaling, is required for the apoptotic effects of PTHR activation.