Inositol 1,4,5-Trisphosphate-Associated cGMP Kinase Substrate
Inositol 1,4,5-trisphosphate (IP3) receptor-associated cyclic GMP (cGMP) kinase substrate (IRAG) is a 125-kD type 2 integral endoplasmic reticulum (ER) membrane protein. IRAG is 900 amino acids long and encoded by the Mrvi1 gene. Two splice variants exist in both mouse and humans with the IRAGa isoform containing an extra 84 amino acids in the N-terminus. Both variants contain a C-terminal hydrophobic domain, α-helix coiled-coil domain, basic N-terminal region, and multiple glycosylation and phosphorylation sites (Ammendola et al. 2001; Schlossmann et al. 2000; Shaughnessy et al. 1999). IRAG forms a complex with the inositol 1,4,5-trisphosphate receptor (IP3R) and cGMP kinase I-β (cGKIβ) and is involved in nitric oxide (NO), cGMP, and cGKIβ signaling pathways. IRAG plays a role in transcription modulation, smooth muscle contraction, platelet aggregation, cell growth, differentiation, and possibly tumorigenesis.
Expression of IRAG is found in the heart, lungs, liver, pancreas, smooth muscle, colon, small intestines, pancreas, trachea, hippocampus, nucleus motorius, and nucleus trigemini (Graham et al. 2008). Perinuclear immunostaining and immunoprecipitation with resident ER proteins suggests IRAG localization to the ER membrane (Ammendola et al. 2001; Fritsch et al. 2004; Geiselhöringer et al. 2004a; Schlossmann et al. 2000; Shaughnessy et al. 1999). Downregulation of IRAG coincides with siRNA knockdown of the transcription factor BTF3 in pancreatic cancer cell lines (Kusumawidjaja et al. 2007), and knockout of Ca2+-dependent K+(BK) channel in trachea causes an upregulation in IRAG protein concentration (Sausbier et al. 2007). Deletion of exon 12 coding for the N-terminus coiled-coil domain of IRAG resulted in a 25% decrease in protein concentration in mouse intestine (Geiselhöringer et al. 2004a). Alternative splicing of the human IRAG/MRVI1 gene resulted in distinct protein variants with and without the sites for cGKI phosphorylation and/or IP3R binding, which results in the expression of IRAG variants that function as negative modulators of IRAG-mediated NO, cGMP, and cGKIβ signaling pathways (Werder et al. 2011).
Interactions with Ligands and Other Proteins
IRAG co-immunoprecipitates with both IP3R1 and cGKIβ/cGKI in smooth muscle from the bovine trachea (Schlossmann et al. 2000). Interaction with IP3R1 possibly occurs at the IRAG N-terminus coiled-coil domain. Deletion of this region of the protein in mice shows disruption in IRAG/IP3R coupling in aortic and intestinal smooth muscle (Geiselhöringer et al. 2004). Interactions between IRAG and cGKIβ occur between amino acids 152 and 184 of IRAG, through possible electrostatic interactions, and the N-terminal leucine zipper motif, amino acids 1–53, of cGKIβ but not cGKIα or cGKII (Ammendola et al. 2001; Casteel et al. 2005). Upon cGMP stimulation, cGKIβ phosphorylates IRAG at ser 644 and ser 677 in human platelets and ser 696 in bovine trachea cells (Schlossmann et al. 2000; Antl et al. 2007). Phosphorylation of IRAG causes a decrease in IP3R1 function (Schlossmann et al. 2000), with ser 696 phosphorylation being essential for this response (Schlossmann et al. 2000). Phosphorylation of IRAG by cGMP-dependent kinase attenuated IP3 receptor activity, and this reduction in activity could not be counteracted by simultaneously occurring activation of IP3 receptors through PKA-mediated phosphorylation (Masuda et al. 2010). In COS-7 cells, expression of phospholamban, IP3R1, cGKIβ, and IRAG resulted in co-immunoprecipitation of these proteins in a complex (Koller et al. 2003).
As previously stated, upon phosphorylation, IRAG decreases calcium release from IP3R-mediated stores (Schlossmann et al. 2000). The interaction of IRAG and cGKIβ has been shown to mediate translocation of cGKIβ to the nucleus, thus attenuating cAMP response element-dependent genes in hamster kidney cells (Casteel et al. 2008). Deletion of the IRAG/IP3R association or IRAG deficiency in platelets caused NO/cGMP inhibition of platelet aggregation and control of thrombosis to be lost (Antl et al. 2007; Schinner et al. 2011), and this loss of association in smooth muscle caused a loss in cGMP-dependent relaxation of hormone receptor mediated smooth muscle contraction (Geiselhöringer et al. 2004a). In colonic smooth muscle, siRNA interference of IRAG caused a loss of NO-induced relaxation (Fritsch et al. 2004), and deletion ablated cGMP relaxation (Frei et al. 2009). Deletion of IRAG causes a loss in atrial natriuretic peptide (ANP) and NO relaxation of smooth muscle tone (Desch et al. 2010). Osteoclast attachment is impaired in siRNA knockouts of the IRAG protein (Yaroslavskiy et al. 2010).
IRAG is involved directly or indirectly in many signaling pathways. IRAG regulates the intracellular calcium concentration through an interaction with IP3R (Schlossmann et al. 2000), affects transcription through its anchoring of cGKIβ (Casteel et al. 2008), and controls NO, ANP, and cGMP relaxation of smooth muscle in multiple tissues throughout the body (Hoffman et al. 2004; Fritsch et al. 2004; Desch et al. 2010). Due to its essential role in NO/cGMP signaling, IRAG provides an interesting target for diseases involving cardiovascular dysfunction.
This chapter is part of the Encyclopedia of Signaling Molecules, 2nd Edition, and is based on a chapter of the same name previously published in the first edition of Encyclopedia of Signaling Molecules. This study was supported in part by grants EY014227 and EY022774 from NIH/NEI, RR022570 and RR027093 from NIH/NCRR and NIH/NIGMS, and AG010485, AG022550, and AG027956 from NIH/NIA, by the Felix and Carmen Sabates Missouri Endowed Chair in Vision Research (PK). We thank Margaret, Richard, and Sara Koulen for their generous support and encouragement.
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