IRAG is essential for relaxation of receptor-triggered smooth muscle contraction by cGMP kinase

Geiselhöringer, Angela; Werner, Matthias; Sigl, Katja; Smital, Petra; Wörner, René; Acheo, Linda; Stieber, Juliane; Weinmeister, Pascal; Feil, Robert; Feil, Susanne; Wegener, Jörg W.; Hofmann, Franz; Schlossmann, Jens

doi:10.1038/sj.emboj.7600440

Cited by 130 publications

(160 citation statements)

References 43 publications

(61 reference statements)

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“…the inositol-1,3,4-phosphat receptor (IP 3 R), the cGMP-dependent protein kinase (cGK), and, as a linker protein between both, the inositol-1,3,4-phosphat receptor associated G-kinase substrate (IRAG [1]). Recently, mutant mice were created which lack the part of the IRAG protein that is supposed to mediate the association between IRAG and the IP 3 R. In these mice (IRAG ∆12 mice), cGMP failed to attenuate both, hormone-induced Ca2+ signals and hormone-induced contractions in vascular preparations [2]. These result confirm the concept that IRAG is an essential mediator of relaxation mediated by cGMP/cGK signalling in vascular smooth muscle.…”

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confidence: 70%

IRAG is involved in cGMP/cGK-mediated relaxation of contractions induced by calcium entry

et al. 2005

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Inhibition of hormone-induced Ca 2+ release from intracellular stores by cGMP involves a protein complex which consists of at least 3 proteins, i.e. the inositol-1,3,4-phosphat receptor (IP 3 R), the cGMP-dependent protein kinase (cGK), and, as a linker protein between both, the inositol-1,3,4-phosphat receptor associated G-kinase substrate (IRAG [1]). Recently, mutant mice were created which lack the part of the IRAG protein that is supposed to mediate the association between IRAG and the IP 3 R. In these mice (IRAG ∆12 mice), cGMP failed to attenuate both, hormone-induced Ca2+ signals and hormone-induced contractions in vascular preparations [2]. These result confirm the concept that IRAG is an essential mediator of relaxation mediated by cGMP/cGK signalling in vascular smooth muscle.In the present study, we tested this hypothesis in two other smooth muscle types, i.e. from jejunum and colon. In line with the concept above, cGMP failed to relax hormone-induced contractions in colon muscle from IRAG ∆12 mice. In contrast, cGMP clearly relaxed hormoneinduced contractions in jejunum muscle from these mutant mice. Thus, Rho/Rho kinase rather than IRAG seems to be the key player in mediating cGMP/cGK signalling in this particular type of smooth muscle.Next, we questioned whether the mutation of IRAG affects the relaxant effects of cGMP on hormone-independent contractions. For this purpose, muscles were contracted by activation of Ca 2+ influx, either by depolarisation with high K + or by initiating capacitative Ca 2+ entry with thapsigargin. Since IRAG is mainly associated with sarcoplasmatic membrane proteins, we expected that the IRAG mutation would not affect cell membrane Ca 2+ signalling. Surprisingly, the relaxant effects of cGMP on these types of contraction were attenuated in intestinal muscles from IRAG ∆12 mice.The results suggests that (1) the association of IRAG/IP 3 R determines cGMP/cGK-mediated relaxations only in distinct types of smooth muscle, and (2) IRAG participates in cGMP/cGK-mediated relaxation not only during hormone-induced Ca 2+ release but also during hormoneindependent Ca 2+ entry. Thus, cGMP/cGK may target via IRAG, either alone or with the IP 3 R, to, at present, unidentified elements of the Ca 2+ entry machinery in certain types of smooth muscle. Geiselhoringer A, Werner M, Sigl K, Smital P, Worner R, Acheo L, Stieber J, Weinmeister P, Feil R, Feil S, Wegener J, Hofmann F, Schlossmann J: IRAG is essential for relaxation of receptortriggered smooth muscle contraction by cGMP kinase. Embo

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IRAG is involved in cGMP/cGK-mediated relaxation of contractions induced by calcium entry

et al. 2005

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“…These mice are terminally ill when born (the majority die before 6 weeks of age) and have multiple cardiovascular, gastrointestinal, hematopoietic, and neurological defects that preclude complete investigation of most cardiovascular functions, including blood pressure (13,23). PKGI␣ and PKGI␤ both regulate VSMC tone through interactions with key VSMC contractile proteins mediated by their respective LZ domains (10,16,17,24). PKGI␣ promotes vascular relaxation by regulation of the two major VSMC pathways regulating contractile state.…”

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High blood pressure arising from a defect in vascular function

Michael

Surks

Wang

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Hypertension, a major cardiovascular risk factor and cause of mortality worldwide, is thought to arise from primary renal abnormalities. However, the etiology of most cases of hypertension remains unexplained. Vascular tone, an important determinant of blood pressure, is regulated by nitric oxide, which causes vascular relaxation by increasing intracellular cGMP and activating cGMPdependent protein kinase I (PKGI). Here we show that mice with a selective mutation in the N-terminal protein interaction domain of PKGI␣ display inherited vascular smooth muscle cell abnormalities of contraction, abnormal relaxation of large and resistance blood vessels, and increased systemic blood pressure. Renal function studies and responses to changes in dietary sodium in the PKGI␣ mutant mice are normal. These data reveal that PKGI␣ is required for normal VSMC physiology and support the idea that high blood pressure can arise from a primary abnormality of vascular smooth muscle cell contractile regulation, suggesting a new approach to the diagnosis and therapy of hypertension and cardiovascular diseases.cyclic nucleotides ͉ hypertension ͉ nitric oxide ͉ vascular biology ͉ vascular smooth muscle E levated blood pressure is a major risk factor for cardiovascular diseases and is responsible for widespread morbidity and mortality (1). Blood pressure is regulated by a variety of complex neurohumoral and mechanical signals that together determine systemic vascular tone and resistance (2, 3). The prevailing model for elevated blood pressure states that renal abnormalities of sodium handling cause volume expansion, increased systemic vascular resistance, and hypertension, and a large number of physiologic and genetic studies support this model and the central role of the renal renin-angiotensinaldosterone system in blood pressure regulation (4-8). Changes in vascular morphology and tone can increase vascular resistance and blood pressure (5), but the hypothesis that primary abnormalities of vascular smooth muscle tone can cause hypertension has not been sufficiently tested (6).Vascular smooth muscle contraction is initiated by both calcium-dependent and -independent mechanisms. Increases in intracellular calcium from receptor-or ion channel-activated pathways (2) lead to activation of myosin light chain kinase, which phosphorylates myosin light chains, activating myosin ATPase and increasing vascular smooth muscle cell (VSMC) contraction and vascular tone. The central calcium-independent pathway regulating VSMC tension is mediated by the GTPase RhoA and Rho kinase, which promote VSMC differentiation, stress fiber formation, and contraction, also increasing vascular tone (2, 7). Conversely, VSMC relaxation is mediated by activation of myosin light chain phosphatase (MLCP), which dephosphorylates myosin light chains to cause relaxation. The relative proportion of phosphorylated and dephosphorylated myosin light chains thus determines the state of VSMC tone (reviewed in ref.2). Nitric oxide, the most important endogenous vasodilator, cause...

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“…Specific binding partners for PKG I␤ include the inositol triphosphate receptor-associated PKG substrate (IRAG) and the transcriptional regulator TFII-I (5, 6). Phosphorylation of IRAG by PKG I␤ inhibits 1,4,5-inositol triphosphate receptordependent calcium release from the endoplasmic reticulum, contributing to smooth muscle relaxation (7). Phosphorylation of TFII-I by PKG I␤ modulates the co-activator functions of TFII-I for serum response factor and Smad transcription factors (6,8).…”

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A Crystal Structure of the Cyclic GMP-dependent Protein Kinase Iβ Dimerization/Docking Domain Reveals Molecular Details of Isoform-specific Anchoring*

Casteel

Smith-Nguyen

Sankaran

et al. 2010

Journal of Biological Chemistry

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Cyclic GMP-dependent protein kinase (PKG) is a key mediator of the nitric oxide/cGMP signaling pathway and plays a central role in regulating cardiovascular and neuronal functions. The N-terminal ϳ50 amino acids of the kinase are required for homodimerization and association with isoform-specific PKGanchoring proteins (GKAPs), which target the kinase to specific substrates. To understand the molecular details of PKG dimerization and gain insight into its association with GKAPs, we solved a crystal structure of the PKG I␤ dimerization/docking domain. Our structure provides molecular details of this unique leucine/isoleucine zipper, revealing specific hydrophobic and ionic interactions that mediate dimerization and demonstrating the topology of the GKAP interaction surface.As the main effector of the nitric oxide/cGMP signaling cascade, cGMP-dependent protein kinase (PKG) regulates smooth muscle tone, inhibits platelet activation, and modulates neuronal functions (1). In mammalian cells, two different genes encode a soluble type I PKG and a membrane-anchored type II PKG (1). Both enzymes form homodimers through an N-terminal leucine/isoleucine zipper domain. PKG I has two splice variants (␣ and ␤) that differ in the first ϳ100 amino acids, resulting in unique dimerization and autoinhibitory domains. The leucine/isoleucine zipper domain mediates interaction with isotype-specific G-kinase-anchoring proteins (GKAPs), 2 targeting PKG I␣ and I␤ to different subcellular compartments and intracellular substrates (2); therefore, we refer to this region as the dimerization and docking (D/D) domain. The domain organization of PKG is shown in Fig. 1. The N-terminal D/D domain is followed by an inhibitory sequence (IS), tandem cyclic nucleotide binding pockets, and the catalytic domain. The D/D domain contains a distinct primary sequence, with a repeating pattern of leucines and isoleucines every seven residues (Fig. 1). This pattern is referred to as a heptad repeat, and the positions of residues are labeled a-g.Specific binding partners for PKG I␣ include the myosin phosphatase targeting subunit (MYPT1) of myosin light chain phosphatase and the regulator of G-protein signaling-2 (RGS-2) (3, 4). Phosphorylation of MYPT1 by PKG I␣ activates its phosphatase activity, leading to dephosphorylation of myosin light chain, which desensitizes the contractile apparatus response to calcium, resulting in vasorelaxation (3). RGS-2 functions as a GTPase-activating protein for G␣ q subunits of heterotrimeric G-protein complexes, and phosphorylation of RGS-2 by PKG I␣ increases its activity toward G␣ q , uncoupling downstream signaling from G␣ qlinked receptors for vasoconstrictive agents (4). Specific binding partners for PKG I␤ include the inositol triphosphate receptor-associated PKG substrate (IRAG) and the transcriptional regulator TFII-I (5, 6). Phosphorylation of IRAG by PKG I␤ inhibits 1,4,5-inositol triphosphate receptordependent calcium release from the endoplasmic reticulum, contributing to smooth muscle relaxation (7). Phosphor...

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IRAG is essential for relaxation of receptor-triggered smooth muscle contraction by cGMP kinase

Cited by 130 publications

References 43 publications

IRAG is involved in cGMP/cGK-mediated relaxation of contractions induced by calcium entry

IRAG is involved in cGMP/cGK-mediated relaxation of contractions induced by calcium entry

High blood pressure arising from a defect in vascular function

A Crystal Structure of the Cyclic GMP-dependent Protein Kinase Iβ Dimerization/Docking Domain Reveals Molecular Details of Isoform-specific Anchoring*

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