Original Article

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

Authors: Feng-Xiang YAN, Shuichi YAMAMOTO, Hui-Ping ZHOU, Hsin-Hsiung TAI, Duan-Fang LIAO

Abstract

AIM: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSION: Ser-331 is the major and crucial site of receptor phosphorylation and
desensitization.
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