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The inositol triphosphate-associated proteins IRAG1 and IRAG2 are cGMP kinase substrate proteins that regulate intracellular Ca 2+ . Previously, IRAG1 was discovered as a 125 kDa membrane protein at the endoplasmic reticulum, which is associated with the intracellular Ca 2+ channel IP 3 R-I and the PKGIβ and inhibits IP 3 R-I upon PKGIβ-mediated phosphorylation. IRAG2 is a 75 kDa membrane protein homolog of IRAG1 and was recently also determined as a PKGI substrate. Several (patho-)physiological functions of IRAG1 and IRAG2 were meanwhile elucidated in a variety of human and murine tissues, e.g., of IRAG1 in various smooth muscles, heart, platelets, and other blood cells, of IRAG2 in the pancreas, heart, platelets, and taste cells. Hence, lack of IRAG1 or IRAG2 leads to diverse phenotypes in these organs, e.g., smooth muscle and platelet disorders or secretory deficiency, respectively. This review aims to highlight the recent research regarding these two regulatory proteins to envision their molecular and (patho-)physiological tasks and to unravel their functional interplay as possible (patho-)physiological counterparts.

Variants in genes encoding the soluble guanylyl cyclase (sGC) in platelets are associated with coronary artery disease (CAD) risk. Here, by using histology, flow cytometry and intravital microscopy, we show that functional loss of sGC in platelets of atherosclerosis-prone Ldlr -/- mice contributes to atherosclerotic plaque formation, particularly via increasing in vivo leukocyte adhesion to atherosclerotic lesions. In vitro experiments revealed that supernatant from activated platelets lacking sGC promotes leukocyte adhesion to endothelial cells (ECs) by activating ECs. Profiling of platelet-released cytokines indicated that reduced platelet angiopoietin-1 release by sGC-depleted platelets, which was validated in isolated human platelets from carriers of GUCY1A1 risk alleles, enhances leukocyte adhesion to ECs. I mp or ta ntly, p ha rm ac ol ogical sGC stimulation increased platelet angiopoietin-1 release in vitro and reduced leukocyte recruitment and atherosclerotic plaque formation in atherosclerosis-prone Ldlr -/- mice. Therefore, pharmacological sGC stimulation might represent a potential therapeutic strategy to prevent and treat CAD., Competing Interests: Competing interests H.S. has received personal fees from MSD Sharp & Dohme, Amgen, Bayer Vital GmbH, Boehringer Ingelheim, Daiichi Sankyo, Novartis, Servier, Brahms, Bristol Myers Squibb, Medtronic, Sanofi Aventis, Synlab, Pfizer and Vifor T as well as grants and personal fees from AstraZeneca that are unrelated to the submitted work. H.S. and T.K. are named inventors on a patent application for the prevention of restenosis after angioplasty and stent implantation (patent applicants: Klinikum rechts der Isar, German Heart Centre Munich; inventors: T. Kessler, A. Kastrati, H. Schunkert; application no. PCT/EP2021/053116; status: pending), which is unrelated to the submitted work. T.K. received lecture fees from Bayer HealthCare Pharmaceuticals. L.D., F.W. and P.S. are full-time employees of Bayer HealthCare Pharmaceuticals. The other authors declare no competing interests.

Inositol 1,4,5-triphosphate receptor-associated 2 (IRAG2) is a type II membrane protein located at the endoplasmic reticulum. It is a homologue of inositol 1,4,5-triphosphate receptor-associated cGMP kinase substrate 1 (IRAG1), a substrate protein of cGMP-dependent protein kinase I (PKGI), and is among others expressed in platelets. Here, we studied if IRAG2 is also located in platelets and might be a substrate protein of PKGI. IRAG2 was detected in platelets of IRAG2-WT animals but not in those of IRAG2-KO animals. Next, we validated by co-immunoprecipitation studies that IRAG2 is associated with IP 3 R1-3. No direct stable interaction with PKGIβ or with IRAG1 was observed. Phosphorylation of IRAG2 in murine platelets using a Ser/Thr-specific phospho-antibody was found in vitro and ex vivo upon cGMP stimulation. To gain insight into the function of IRAG2, platelet aggregation studies were performed using thrombin and collagen as agonists for treatment of isolated IRAG2-WT or IRAG2-KO platelets. Interestingly, platelet aggregation was reduced in the absence of IRAG2. Pretreatment of wild type or IRAG2-KO platelets with sodium nitroprusside (SNP) or 8-pCPT-cGMP revealed a further reduction in platelet aggregation in the absence of IRAG2. These results show that IRAG2 is a substrate of PKGI in murine platelets. Furthermore, our results indicate that IRAG2 is involved in the induction of thrombin- or collagen-induced platelet aggregation and that this effect is enhanced by cGMP-dependent phosphorylation of IRAG2. As IRAG1 was previously shown to inhibit platelet aggregation in a cGMP-dependent manner, it can be speculated that IRAG2 exerts an opposing function and might be an IRAG1 counterpart in murine platelets.

Background and Purpose: Diabetic nephropathy is the leading cause for end-stage renal disease worldwide. Until now, there is no specific therapy available. Standard treatment with inhibitors of the renin-angiotensin system just slows down progression. However, targeting the NO/sGC/cGMP pathway using sGC activators does prevent kidney damage. Thus, we investigated if the sGC activator cinaciguat was beneficial in a mouse model of diabetic nephropathy, and we analysed how mesangial cells (MCs) were affected by related conditions in cell culture., Experimental Approach: Type 1 diabetes was induced with streptozotocin in wild-type and endothelial NOS knockout (eNOS KO) mice for 8 or 12 weeks.. Half of these mice received cinaciguat in their chow for the last 4 weeks. Kidneys from the diabetic mice were analysed with histochemical assays and by RT-PCR and western blotting. . Additionally, primary murine MCs under diabetic conditions were stimulated with 8-Br-cGMP or cinaciguat to activate the sGC/cGMP pathway., Key Results: The diabetic eNOS KO mice developed most characteristics of diabetic nephropathy, most marked at 12 weeks. Treatment with cinaciguat markedly improved GFR, serum creatinine, mesangial expansion and kidney fibrosis in these animals. We determined expression levels of related signalling proteins. Thrombospondin 1, a key mediator in kidney diseases, was strongly up-regulated under diabetic conditions and this increase was suppressed by activation of sGC/cGMP signalling., Conclusion and Implications: Activation of the NO/sGC/PKG pathway with cinaciguat was beneficial in a model of diabetic nephropathy. Activators of sGC might be an appropriate therapy option in patients with Type 1 diabetes., Linked Articles: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

The inositol 1,4,5-triphosphate receptor-associated 2 (IRAG2) is also known as Jaw1 or lymphoid-restricted membrane protein (LRMP) and shares homology with the inositol 1,4,5-triphosphate receptor-associated cGMP kinase substrate 1 (IRAG1). IRAG1 interacts with inositol trisphosphate receptors (IP 3 receptors /IP 3 R) via its coiled-coil domain and modulates Ca 2+ release from intracellular stores. Due to the homology of IRAG1 and IRAG2, especially in its coiled-coil domain, it is possible that IRAG2 has similar interaction partners like IRAG1 and that IRAG2 also modulates intracellular Ca 2+ signaling. In our study, we localized IRAG2 in pancreatic acinar cells of the exocrine pancreas, and we investigated the interaction of IRAG2 with IP 3 receptors and its impact on intracellular Ca 2+ signaling and exocrine pancreatic function, like amylase secretion. We detected the interaction of IRAG2 with different subtypes of IP 3 R and altered Ca 2+ release in pancreatic acinar cells from mice lacking IRAG2. IRAG2 deficiency decreased basal levels of intracellular Ca 2+ , suggesting that IRAG2 leads to activation of IP 3 R under unstimulated basal conditions. Moreover, we observed that loss of IRAG2 impacts the secretion of amylase. Our data, therefore, suggest that IRAG2 modulates intracellular Ca 2+ signaling, which regulates exocrine pancreatic function.