Inhibitory Phosphorylation of Soluble Guanylyl Cyclase by Muscarinic m2 Receptors via Gβγ-Dependent Activation of c-Src Kinase

Abstract

In gastrointestinal smooth muscle, cGMP levels in response to relaxant agonists are regulated by activation of phosphodiesterase 5 and inhibition of soluble guanylyl cyclase (sGC) in a feedback mechanism via cGMP-dependent protein kinase. The aim of the present study was to determine whether contractile agonists modulate cGMP levels by cross-regulating sGC activity. In gastric muscle cells, acetylcholine (ACh) stimulated Src activity and induced sGC phosphorylation. Concurrent stimulation of cells with ACh attenuated sGC activity and cGMP formation in response to the nitric oxide (NO) donor, S-nitrosoglutathione (GSNO). The effect of ACh on Src activity, sGC phosphorylation, and on GSNO-stimulated sGC activity and cGMP formation were blocked by the m2 receptor antagonist (methoctramine), pertussis toxin, and by inhibitors of phosphatidylinositol 3 kinase, LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride], or Src kinase, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine, in dispersed muscle cells and in cells expressing G␣ i minigene or G␤␥-scavenging peptide, whereas the m3 receptor antagonist, N-(2-chloroethyl)-4-piperidinyl diphenylacetate, or expression of the G␣ q minigene had no effect. ACh also attenuated sGC activity and cGMP formation in response to the NOindependent activator, YC-1 [3-(5Ј-hydroxymethyl-2Јfuryl)-1-benzylindazole]. The pattern implied that phosphorylation of sGC by c-Src kinase inhibits NO-sensitive sGC activity, and the inhibition was not due to a decrease in the binding of NO but probably due to decrease in catalytic activity. We conclude that cGMP levels are cross-regulated by contractile agonists via a mechanism that involves c-Src-dependent phosphorylation of sGC, leading to inhibition of sGC activity and cGMP formation. The finding highlights a novel mechanism for attenuation of the NO/sGC/cGMP signal by G i -coupled contractile agonists, in addition to their inhibitory effect on adenylyl cyclase and cAMP formation.In the gastrointestinal smooth muscle, the relaxant neurotransmitters vasoactive intestinal peptide (VIP) and pituitary adenylyl cycle-activating peptide, which are colocalized with nitric oxide (NO) synthase, are coreleased with NO in response to neural activation resulting in the generation of cAMP and cGMP and activation of both cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) (Kunze and Furness, 1999;Bornstein et al., 2004;Murthy, 2006). The levels of cAMP and cGMP are determined by the activities of cyclases and phosphodiesterases.

Insulin-like growth factor-binding protein-5 stimulates growth of human intestinal muscle cells by activation of Gαi3

“…The participation of G␣i-and G␤␥-subunits was investigated by transient transfection expression of either a cDNA minigene encoding the last COOH-terminal 11 amino acids, which block the effects mediated via G␣i or expression of cDNA encoding a G␤␥-scavenging peptide, which blocks the effects mediated via G␤␥ as described previously (21,28). The oligonucleotide sequence corresponding to the COOH-terminal 11-amino-acid residues of G␣i in random order was synthesized and ligated into pcDNA 3.1(ϩ) as a control scrambled sense minigene.…”

“…Activation of specific G proteins was determined from IGFBP-5-induced increase in G␣ binding to guanosine 5Ј-O-(3-thiotriphosphate) (GTP␥S) as described previously (16,21). Cells were homogenized in 20 mM HEPES (pH 7.4); the homogenates were centrifuged at 4°C for 30 min at 30,000 g; and the membranes were solubilized in 20 mM HEPES buffer containing 0.…”

Regulation of PP2AC Carboxylmethylation and Cellular Localisation by Inhibitory Class G-Protein Coupled Receptors in Cardiomyocytes

“…Evidence [41], [50] suggests that Gβγ subunits initiate intracellular signalling cascades via the activation of PI3K [38]. It has been considered for some time that class IB PI3Ks can be activated by Gβγ dimers [51], [52].…”

Inhibition of Gαiactivity by Gβγ is mediated by PI 3-kinase-γ- and cSrc-dependent tyrosine phosphorylation of Gαiand recruitment of RGS12

“…1A). The COOH-terminal sequence of GRK2 acts as a specific G␤␥ (but not G␣) antagonist by binding to G␤␥ and blocking its interaction with effectors such as GRK2 (14,16,18,38). Treatment of freshly dispersed smooth muscle cells with DPDPE also stimulated cSrc phosphorylation, which was abolished in smooth muscle cells pretreated with the cSrc inhibitor PP2 or the PI 3-kinase inhibitor LY294002 (Fig.…”

“…Previous studies using freshly dispersed or cultured gastric smooth muscle cells, a model system used extensively to explore G protein-coupled receptor signaling in visceral smooth muscle, have shown that receptors coupled to G i1 [e.g., somatostatin (sstr 3 )] (21), G i2 (e.g., -, ␦-, and -opioid) (23), or G i3 (e.g., adenosine A 1 ) (22) induce contraction by activating dual signaling pathways initiated by G␤␥ (8): the first pathway involves activation of PLC␤ 3 , stimulation of inositol 1,4,5-trisphosphate-dependent Ca 2ϩ release, and phosphorylation of myosin light chain 20 (MLC 20 ), resulting in a transient smooth muscle contraction. The second pathway involves sequential activation of PI 3-kinase and ILK: the latter acts as a Ca 2ϩ -independent MLC kinase and inhibits MLC phosphatase (PP1c␦), resulting in sustained MLC 20 phosphorylation and smooth muscle contraction (5,8). In the present study we examined the significance of a parallel pathway initiated by these receptors and by G i3 -coupled muscarinic m 2 receptors involving sequential activation of G␤␥, PI 3-kinase-␥, and cSrc.…”