Function of IRAG for cGMP kinase signalling in smooth muscle and platelets

Intracellular signalling by NO/cGMP/cGMP-dependent protein kinase type I (cGKI) regulates various physiological processes including smooth muscle contractility and platelet aggregation. An important mediator of this signalling cascade is the inositol 1,4,5-trisphosphate receptor I (IP₃RI) associated protein cGMP kinase substrate (IRAG). This protein forms a trimeric complex together with the cGMP kinase Iβ (cGKIβ) and the IP₃RI. Targeted deletion of exon 12 of IRAG coding for the N-terminal part of the coiled-coil domain disrupted in vivo the IRAG-IP₃RI interaction. The resulting IRAG^Δ12/Δ12 mice showed an increased mortality and a severely reduced gastrointestinal motility. The relaxation of hormone-contracted aortic and longitudinal colonic smooth muscle by cGMP was abolished in IRAG^Δ12/Δ12 mice, whereas cAMP-mediated relaxation was not altered. In contrast to WT mice, norepinephrine-induced increases in [Ca²⁺]_i were not reduced by cGMP in aortic smooth muscle cells from IRAG^Δ12/Δ12 mice. These data suggest, that IRAG is involved in the cGMP-dependent decrease of [Ca²⁺]_i in vivo and is essential for cGMP-dependent relaxation of hormone-induced vascular and colonic muscle contraction. However, cGMP-mediated relaxation of small intestinal smooth muscles was only partially affected in IRAG^Δ12/Δ12 mice suggesting tissue specific selectivity of cGKI mechanisms.

In addition, IRAG is highly expressed in platelets. To study the effect of IRAG signalling in platelets, we analysed the aggregation of IRAG^Δ12/Δ12 platelets. Nitric oxide and the cGMP analogue 8-pCPT-cGMP did not inhibit the aggregation of IRAG^Δ12/Δ12 platelets in contrast to wild type platelets, whereas the shape change of platelets was not affected in both mutant and wild type platelets. Furthermore, tail bleeding was abbreviated in IRAG^Δ12/Δ12 mice suggesting a defect in the regulation of coagulation in vivo. Therefore, cGKI/IRAG/IP₃RI signalling might be crucial for the NO/cGMP-dependent inhibition of platelet aggregation.