Serine 331 is major site of phosphorylation and desensitization induced by protein kinase C in thromboxane receptor...

To identify the specific serine/threonine residues in the C-terminal tail of thromboxane receptor alpha (TPalpha) being phosphorylated and desensitized, and various alanine mutants of these serine/threonine residues were checked for their ability to serve as substrates.

To facilitate the identification of the intracellular domains involved in phosphorylation, glutathione S-transferase (GST)-intracellular domain fusion proteins were used as substrates for the purified PKC, and then the cDNA of phosphorylated protein was mutagenized to localize the major site of receptor phosphorylation induced by protein kinase C. Human embryonic kidney (HEK) 293 cells stably transfected with the His-tagged wild type or mutant TPalpha were used to study the phosphorylation and desensitization.

Only the C-terminal tail can be used as a substrate for the purified PKC. Ser-331 (mP4) was demonstrated to be heavily phosphorylated, Ser-324 (mP1) was shown to be slightly phosphorylated, Ser-329 was illustrated to be faintly phosphorylated, and other Ser/Thr residues were not found to be phosphorylated. Phorbol-12-myristate-13-acetate (PMA) induced receptor phosphorylation in HEK 293 cells expressing the wild type TPalpha. However, PMA did not significantly trigger receptor phosphorylation in HEK 293 cells expressing the S331A mutant receptor. Pretreatment of the cells expressing the wild type with PMA inhibited I-BOP induced Ca2+ release, however, pretreatment of the cells expressing the S331A mutant receptor with PMA did not abolish I-BOP induced Ca2+ release.

Ser-331 is the major and crucial site of receptor phosphorylation and desensitization.