PHOSPHORYLATION OF THE INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR BY CYCLIC GMP-DEPENDENT PROTEIN-KINASE

Cyclic GMP (cGMP) inhibits intracellular calcium ([Ca2+](i)) mobilizat ion in vascular smooth muscle cells by a mechanism that is not well un derstood. Because several studies suggest that cGMP inhibits inositol 1,4,5-trisphosphate (IP3) action, we examined the effects of cGMP-depe ndent protein kinase on IP3 receptor phosphorylation. The purified IP3 receptor was phosphorylated using either the cGMP- or cAMP-dependent protein kinase in vitro. Phosphorylation was time-dependent and stoich iometric using both kinases. Two-dimensional phosphopeptide mapping hi gh performance liquid chromatography analysis, and amino acid analysis showed that identical sites were phosphorylated using either kinase, and identified serine 1755 as the site of phosphorylation. The synthet ic peptide corresponding to serine 1755 (GRRESLTSFG) was phosphorylate d with a K-m in the range of 30-40 mu m by both kinases. The kinetic a nalysis revealed that this peptide substrate is the best substrate des cribed for cGMP kinase to date. Vascular smooth muscle cells prelabele d with [P-32]orthophosphate and treated with atrial natriuretic peptid e or sodium nitroprusside to elevate cGMP also resulted in increased l abeling of the IP3 receptor. Phosphorylation of IP3 receptor by cGMP k inase may regulate the function of IP3 receptor in vascular smooth mus cle cells and contribute to the effect of cGMP to regulate intracellul ar calcium levels.