title: Agents that elevate cyclic AMP induce receptor phosphorylation primarily at serine 331 in HEK 293 cells overexpressing human thromboxane receptor alpha.
abstract: Human embryonic kidney (HEK) 293 cells stably transfected with the His-tagged thromboxane receptor alpha (TPalpha) were used to study the phosphorylation and desensitization of the receptor induced by prostaglandin E1 (PGE1) or forskolin. These agents are known to increase the intracellular level of cyclic AMP (cAMP) and activate cAMP-dependent protein kinase (PKA). Pretreatment of cells with either agent significantly attenuated Ca2+ release induced by the agonist [1S-[1alpha,2alpha(Z),3beta(1E,3S),4alpha]]-7-[3-[3-hydroxy-4-(4-indophenoxy)-1-butenyl]-7-oxabicyclo[2,2,1]hept-2-yl]-5-heptenoic acid (I-BOP). These agents also induced concentration-dependent phosphorylation of TPalpha as demonstrated by increased 32P-labeling of the receptor from cells prelabeled with 32P(i). To facilitate the identification of the intracellular domains involved in phosphorylation, glutathione S-transferase (GST)-intracellular domain fusion proteins were used as substrates for purified PKA. It was found that only the C-terminal tail fusion protein could serve as a substrate for PKA. To identify the specific serine/threonine residues in the C-terminal tail that are involved in phosphorylation, various alanine mutants of these residues were checked for their ability to serve as substrates. Ser-331 was found to be involved in PKA-mediated phosphorylation. The S331A mutant receptor overexpressed in HEK 293 cells was not phosphorylated significantly following stimulation by PGE1 or forskolin, indicating that Ser-331 was the major site of phosphorylation. Furthermore, cells overexpressing the mutant receptor became responsive to I-BOP-induced Ca2+ mobilization even after pretreatment with PGE1 or forskolin. These results indicate that Ser-331 is the primary site responsible for the phosphorylation and desensitization of the human TPalpha induced by agents that activate PKA.
authors.affiliation: Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Yamamoto
authors.forename: Fengxiang
authors.initials: F
authors.lastname: Yan
authors.forename: Huiping
authors.initials: H
authors.lastname: Zhou
authors.forename: Hsin-Hsiung
authors.initials: HH
authors.lastname: Tai
mesh_terms: D000527:Alprostadil / Q000494:pharmacology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D011464:Epoprostenol / Q000494:pharmacology; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D015230:Prostaglandin D2 / Q000494:pharmacology; D017482:Receptors, Thromboxane / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*
chemical_list: D010975:Platelet Aggregation Inhibitors; D017482:Receptors, Thromboxane; D012694:Serine; D011464:Epoprostenol; D000242:Cyclic AMP; D000527:Alprostadil; D015230:Prostaglandin D2