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 32P-labeled cells using a monoclonal antibody to the PTH/PTHrP receptor revealed the presence of a major 32P-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.
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Endocrine Unit, Veterans Administration Medical Center, San Francisco, California 94121, USA.
Bambino
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Nissenson
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D000818:Animals; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D019204:GTP-Binding Proteins / Q000032:analysis; D009893:Opossums; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000378:metabolism*
D010281:Parathyroid Hormone; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019204:GTP-Binding Proteins