AGONIST-STIMULATED PHOSPHORYLATION OF THE G-PROTEIN COUPLED RECEPTOR FOR PARATHYROID-HORMONE (PTH) AND PTH-RELATED PROTEIN

The objectives of the present study were to determine whether the G protein-coupled receptor for PTH and PTH-related protein (PTHrP) is subject to agonist-specific phosphorylation and to characterize the relevant kinase(s). The opossum kidney PTH/PTHrP receptor stably expressed in human embryonic kidney 293 cells was coupled to adenylyl cyclase, with half-maximal activation occurring in the presence of 0.1 nM bovine (b) PTH-(1-34). Immunoprecipitation of extracts of P-32-labeled cells using a monoclonal antibody to the PTH/PTHrP receptor revealed the presence of a major P-32-labeled protein of approximately 85 kilodaltons that was not evident in untransfected 293 cells. bPTH-(1-34) treatment produced a rapid dose-dependent increase in phosphorylation of the 85-kilodalton receptor, with a maximal effect that was 3.5 +/- 0.7-fold (n = 4) over basal. Half-maximal phosphorylation occurred with 10 nM bPTH-(1-34), similar to the hormone concentration required for 50% receptor occupancy. Activation of protein kinase A or protein kinase C with forskolin or phorbol 12-myristate 13-acetate also increased PTH/PTHrP receptor phosphorylation, but to a lesser degree than PTH. Neither of these kinases mediated the effect of PTH, as blockade of the protein kinase A pathway (with H-89) or the protein kinase C pathway (with the bisindolylmaleimide GF 109203X) did not inhibit bPTH-(1-34)-induced PTH/PTHrP receptor phosphorylation. These results suggest that agonist-stimulated PTH/PTHrP receptor phosphorylation may involve a nonsecond messenger-activated kinase, such as a member of the G protein-coupled receptor kinase family.