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Serine 331 Is the Major Site of Receptor Phosphorylation Induced by Agents That Activate Protein Kinase G in HEK 293 Cells Overexpressing Thromboxane Receptor Alpha

Serine 331 Is the Major Site of Receptor Phosphorylation Induced by Agents That Activate Protein Kinase G in HEK 293 Cells Overexpressing Thromboxane Receptor Alpha

S Yamamoto, F Yan, H Zhou, H H Tai

Arch Biochem Biophys. 2001 Sep 1;393(1):97-105.

PMID: 11516166

Abstract:

Human embryonic kidney (HEK)293 cells stably transfected with the His-tagged thromboxane receptor alpha (TPalpha) was used to study the phosphorylation and desensitization of the receptor induced by 8-bromo-cyclic GMP (8-Br-cGMP), sodium nitroprusside (SNP), or S-nitroso-glutathione (SNG). These agents are known to activate cGMP-dependent protein kinase (PKG). Pretreatment of cells with these agents attenuated significantly agonist I-BOP induced Ca(2+) release. These agents also induced dose-dependent phosphorylation of the TPalpha as demonstrated by increased (32)P-labeling of the receptor from cells prelabeled with (32)Pi. 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 PKG. It was found that only the GST-C-terminal tail fusion protein could serve as a substrate for the PKG. To identify the specific serine/threonine residues in the C-terminal tail being phosphorylated, various alanine mutants of these serine/threonine residues were checked for their ability to serve as substrates. It was found that the Ser-331 of the C-terminal tail was primarily involved in the PKG-mediated phosphorylation. That Ser-331 is a predominant site of phosphorylation was supported by in vivo studies in which HEK293 cells expressing the S331A mutant receptor showed little phosphorylation induced by any of the above three agents. Furthermore, HEK293 cells expressing the S331A mutant receptor pretreated with any of the above three agents became responsive to the agonist I-BOP-induced Ca(2+) release. These results indicate that Ser-331 of the TPalpha is the primary site responsible for the phosphorylation and the desensitization of the receptor induced by agents that activate the PKG.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42412867	CyclinC, GST tagged human			Price

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