Regulation of intracellular calcium by a signalling complex of IRAG, IP3 receptor and cGMP kinase I beta

Calcium release from the endoplasmic reticulum controls a number of cellula r processes, including proliferation and contraction of smooth muscle and o ther cells(1,2). Calcium release from inositol 1,4,5-trisphosphate (IP3)-se nsitive stores is negatively regulated by binding of calmodulin to the IP3 receptor (IP3R)(3,4) and the NO/cGMP/cGMP kinase I (cGKI) signalling pathwa ys(5,6). Activation of cGKI decreases IP3-stimulated elevations in intracel lular calcium(7), induces smooth muscle relaxation(8) and contributes to th e antiproliferative(9) and pro-apoptotic effects of NO/cGMP(10). Here we sh ow that, in microsomal smooth muscle membranes, cGKI beta phosphorylated th e IP3R and cGKI beta, and a protein of relative molecular mass 125,000 whic h we now identify as the IP3R-associated cGMP kinase substrate (IRAG). Thes e proteins were co-immunoprecipitated by antibodies directed against cGKI, IP3R or IRAG. IRAG was found in many tissues including aorta, trachea and u terus, and was localized perinuclearly after heterologous expression in COS -7 cells. Bradykinin-stimulated calcium release was not affected by the exp ression of either IRAG or cGKI beta, which we tested in the absence and pre sence of cGMP. However, calcium release was inhibited after co-expression o f IRAG and cGKI beta in the presence of cGMP. These results identify IRAG a s an essential NO/cGKI-dependent regulator of IP3-induced calcium release.