Signal transduction via NO/cGMP/cGKI 1 is involved in a variety of cellular mechanisms including smooth muscle contractility and platelet aggregation (1-4). cGKI affects smooth muscle tone by either decreasing the release of calcium from InsP 3 -sensitive stores (5-9) or by reducing calcium sensitivity of the contractile elements (10, 11). During the last years, several mechanisms were proposed for the action of cGKI mediating these effects. A decrease of the cytosolic calcium concentration by cGKI might involve reduced InsP 3 synthesis (12-15), enhanced calcium re-uptake by intracellular stores via CaATPase (16), or inhibition of calcium release via the InsP 3 R (17). The molecular mechanisms for these different possible intracellular calcium regulation pathways were only partly resolved up to now.Recently, we identified a 125-kDa cGKI substrate protein which was designated as inositol 1,4,5-trisphophate receptorassociated cGMP kinase substrate (IRAG). IRAG which is phosphorylated by cGKI is associated in a macromolecular complex with cGKI␤ and InsP 3 RI in smooth muscle (9). The observed perinuclear localization of heterologously expressed IRAG suggested the potential role of IRAG as a modulator of calcium release from intracellular stores. Indeed, functional studies revealed that IRAG inhibits InsP 3 -induced calcium release after activation of cGKI␤ with 8-pCPT-cGMP in COS-7 cells (9). However, the precise mechanism by which IRAG influences calcium release is still unknown.In the present study we investigated the molecular determinants for the interaction of IRAG and cGKI. It is shown that IRAG interacts specifically with the amino-terminal region containing the leucine zipper of cGKI␤. Phosphorylation of Ser 696 of IRAG is essential for the inhibition of InsP 3 -induced calcium release. EXPERIMENTAL PROCEDURESMaterials-The yeast strain EGY48 (MAT␣, his3, trp1, ura3, lex-A opx6 Leu2) and the yeast expression plasmids pEG202, pJG4-5, and pSH18-34 were used for the two-hybrid screen. Yeast media and dropout media lacking the appropriate amino acids were obtained from CLONTECH (Heidelberg, Germany) and Difco (Hamburg, Germany), respectively. The full-length rat cDNAs of the neuronal InsP 3 RI (S1Ϫ/ S2ϩ) (18) and the peripheral InsP 3 RI (S1Ϫ/S2Ϫ) (19) were a gift from Dr. Ilya Bezprozvanny (Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX).Antibodies-A polyclonal antibody specific for IRAG, raised in rabbits against recombinant IRAG 53-499 expressed in bacteria, was used for Western blot analysis at a dilution of 1:1000. Further antibodies were directed against cGKI (8) and InsP 3 RI (ABR Biochemicals).Plasmid Construction of Baits and Preys-The bovine IRAG cDNA was the template of all IRAG constructs (baits) which were synthesized by PCR. The generated IRAG amplicons were purified and inserted into the BamHI/EcoRI-digested expression plasmid pEG202 (20) in-frame with the DNA-binding domain of LexA, yielding pEG202-LexA/IRAG. The full-length and truncated PCR amplicons of bo...

Signalling by cGMP-dependent protein kinase type I (cGKI) relaxes various smooth muscles modulating thereby vascular tone and gastrointestinal motility. cGKIdependent relaxation is possibly mediated by phosphorylation of the inositol 1,4,5-trisphosphate receptor I (IP 3 RI)-associated protein (IRAG), which decreases hormone-induced IP 3 -dependent Ca 2 þ release. We show now that the targeted deletion of exon 12 of IRAG coding for the N-terminus of the coiled-coil domain disrupted in vivo the IRAG-IP 3 RI interaction and resulted in hypomorphic IRAG D12/D12 mice. These mice had a dilated gastrointestinal tract and a disturbed gastrointestinal motility. Carbachol-and phenylephrine-contracted smooth muscle strips from colon and aorta, respectively, of IRAG D12/D12 mice were not relaxed by cGMP, while cAMP-mediated relaxation was unperturbed. Norepinephrine-induced increases in [Ca 2 þ ] i were not decreased by cGMP in aortic smooth muscle cells from IRAG D12/D12 mice. In contrast, cGMP-induced relaxation of potassium-induced smooth muscle contraction was not abolished in IRAG D12/D12 mice. We conclude that cGMP-dependent relaxation of hormone receptor-triggered smooth muscle contraction essentially depends on the interaction of cGKI-IRAG with IP 3 RI.

cGMP kinase I (cGKI) signaling modulates multiple physiological processes including smooth muscle relaxation. The expression of cGKI and its substrate IRAG (Inositol 1,4,5-trisphosphate receptor associated cGMP kinase substrate) was studied. IRAG and cGKI were colocalized in the smooth muscle of aorta and colon. IRAG was present in the thalamus and in most of the myenteric plexus in the absence of cGKI. Coexpression of IRAG and cGKIb or cGKIa in COS-7 cells revealed that IRAG recruits cGKIb but not cGKIa to the endoplasmic reticulum. These results suggest that IRAG may be involved in cGKI-dependent and -independent pathways.

Smooth muscle tone is predominately regulated by the rise and fall of free intracellular cytosolic calcium concentration (1). Thus, modulation of intracellular calcium handling is one important mechanism by which inhibitory neurotransmitters induce smooth muscle relaxation (2). In the human colon, nitric oxide has been shown to be the principal inhibitory transmitter in nerve-muscle interaction (3). NO 1 -dependent relaxation of gastrointestinal smooth muscle has been demonstrated throughout the gut of various species (4 -9) and has been established as a crucial event contributing to the maintenance of normal gut function (10, 11).The molecular target of NO in smooth muscle is the soluble guanylate cyclase, which is strongly activated by NO (12, 13). Increased levels of cGMP lead to smooth muscle relaxation predominately via activation of cGMP-dependent protein kinase (cGK) (14, 15). The crucial role of cGK for gastrointestinal smooth muscle function became apparent in cGKI-deficient mice showing a selective lack of NO-dependent smooth muscle relaxation associated with severe gastrointestinal dysfunction and marked hypertrophy of gastrointestinal smooth muscle (16). The mechanisms of cGKI-dependent smooth muscle cell relaxation, however, have not been fully understood (for a review, see Ref. 14).In a recent study (see Ref. 26), a new protein has been identified as molecular target of cGKI in smooth muscle microsomal membranes and has been termed IRAG (InsP 3 R-associated cGMP kinase substrate). IRAG precipitated together with cGKI␤ and InsP 3 receptor type I in a complex which was found to be localized at the endoplasmic reticulum membrane. In transfected COS cells, the coexpression of IRAG with cGKI was essential for the cGMP-dependent inhibition of InsP 3 -dependent calcium release. This inhibition has further been demonstrated to depend on the cGKI-mediated phosphorylation of IRAG at Ser 696 . No interaction between cGKI and the InsP 3 receptor was observed when IRAG was absent (17).To date, only little is known about IRAG expression and its function in gastrointestinal tissues. Furthermore, evidence for a functional role of IRAG in smooth muscle tissue is lacking. We show here that IRAG is expressed in human colon and that the suppression of IRAG protein expression in human colonic smooth muscle cells is sufficient to abolish the inhibitory effect of sodium nitroprusside and 8-pCPT-cGMP on bradykinin-induced calcium release in these cells. EXPERIMENTAL PROCEDURESTissue Preparation-Tissues from human colon and rectum were obtained from surgical resections for colorectal malignant disease. The tissues were macroscopically and microscopically free of tumor.

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