Function of IRAG2 Is Modulated by NO/cGMP in Murine Platelets

Abstract

Inositol 1,4,5-triphosphate receptor-associated 2 (IRAG2) is a type ІІ 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 IP3R1-3. 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.

Novel Functional Features of cGMP Substrate Proteins IRAG1 and IRAG2

“…Furthermore, expression of IRAG2 is seen in sinoatrial nodes [ 18 ] and in intestinal tuft cells [ 63 ], where it might exert important physiological functions due to its interaction with different ion channels or receptors. We recently detected the expression of IRAG2 in platelets and pancreatic acinar cells [ 9 , 64 ]. In these cells, IRAG2 engages in Ca 2+ signaling, platelet aggregation, and enzymatic secretion ( Section 3.4 , Section 3.6.4 and Section 3.6.5 ).…”

“…Later, an interaction of IRAG2 was also detected with the IP 3 R-II in mouse embryo fibroblast (MEF) cells, and with IP 3 R-I, IP 3 R-II, and IP 3 R-III in HEK cells that were transfected with IRAG2 and one IP 3 receptor subtype each [ 63 , 83 ]. Furthermore, we recently reported the interaction of IRAG2 with all IP 3 receptor subtypes in the murine pancreas as well as in murine platelets [ 9 , 64 ]. The interaction with these receptors occurs through the coiled-coil domain of IRAG2 and impacts the release of Ca 2+ from intracellular stores [ 8 , 83 ].…”

“…The interaction site of IRAG1 with PKGIβ is located between amino acids 152 and 184; however, this site is missing in IRAG2 [ 1 , 14 ]. Furthermore, we could not detect a stable interaction between IRAG2 and PKGIβ in murine platelets [ 64 ]. Despite the absence of a stable interaction site with PKGI, a quantitative phosphoproteomics study revealed phosphorylation of human LRMP at amino acids Ser363, threonine (Thr) 375, and Ser418 upon stimulation with NO donors or riociguat in platelets [ 85 ].…”

“…Remarkably, the phosphorylation sites Ser363 and Thr375 in IRAG2 (consensus sequences: RSAS363 and RRVT375) ( Figure 2 ) and the identified phosphorylation sites Ser664 and Ser677 in human IRAG1 (consensus sequences: RSMS664 and RRVS677) are very homologous. Moreover, we detected cGMP-dependent phosphorylation of IRAG2 in murine platelets [ 64 ]. As cGMP predominantly activates PKGI [ 35 , 86 ], we assume that phosphorylation of IRAG2 after cGMP-stimulation is achieved through PKGI ( Figure 3 ).…”

“…In the circumvallate papillae, coexpression with the IP 3 R-III was detected [ 8 ]. Moreover, the direct interaction of IRAG2 and IP 3 R-III was shown in several cell lines and tissues [ 8 , 9 , 64 , 83 ]. The IP 3 /Ca 2+ signal cascade plays a significant role in sweet, bitter, and umami taste signal transduction.…”