Inhibition of Gαq-dependent PLC-β1 activity by PKG and PKA is mediated by phosphorylation of RGS4 and GRK2

2007 ◽  
Vol 292 (1) ◽  
pp. C200-C208 ◽  
Author(s):  
Jiean Huang ◽  
Huiping Zhou ◽  
Sunila Mahavadi ◽  
Wimolpak Sriwai ◽  
Karnam S. Murthy
Keyword(s):  
Smooth Muscle ◽  
G Protein ◽  
Phosphorylation Site ◽  
Type I ◽  
Gtpase Activity ◽  
Protein Signaling ◽  
Mechanism Of Inhibition ◽  
Rgs Domain ◽  
Pi Hydrolysis ◽  
In Cells

In smooth muscle of the gut, Gq-coupled receptor agonists activate preferentially PLC-β1 to stimulate phosphoinositide (PI) hydrolysis and inositol 1,4,5-trisphosphate (IP3) generation and induce IP3-dependent Ca2+ release. Inhibition of Ca2+ mobilization by cAMP- (PKA) and cGMP-dependent (PKG) protein kinases reflects inhibition of PI hydrolysis by both kinases and PKG-specific inhibitory phosphorylation of IP3 receptor type I. The mechanism of inhibition of PLC-β1-dependent PI hydrolysis has not been established. Neither Gq nor PLC-β1 was directly phosphorylated by PKA or PKG in gastric smooth muscle cells. However, both kinases 1) phosphorylated regulator of G protein signaling 4 (RGS4) and induced its translocation from cytosol to plasma membrane, 2) enhanced ACh-stimulated association of RGS4 and Gαq·GTP and intrinsic Gαq·GTPase activity, and 3) inhibited ACh-stimulated PI hydrolysis. RGS4 phosphorylation and inhibition of PI hydrolysis were blocked by selective PKA and PKG inhibitors. Expression of RGS4(S52A), which lacks a PKA/PKG phosphorylation site, blocked the increase in GTPase activity and the decrease in PI hydrolysis induced by PKA and PKG. Blockade of PKA-dependent effects was only partial. Selective phosphorylation of G protein-coupled receptor kinase 2 (GRK2), which contains a RGS domain, by PKA augmented ACh-stimulated GRK2:Gαq·GTP association; both effects were blocked in cells expressing GRK2(S685A), which lacks a PKA phosphorylation site. Inhibition of PI hydrolysis induced by PKA was partly blocked in cells expressing GRK2(S685A) and completely blocked in cells coexpressing GRK2(S685A) and RGS4(S52A) or Gαq(G188S), a Gαq mutant that binds GRK2 but not RGS4. The results demonstrate that inhibition of PLC-β1-dependent PI hydrolysis by PKA is mediated via stimulatory phosphorylation of RGS4 and GRK2, leading to rapid inactivation of Gαq·GTP. PKG acts only via phosphorylation of RGS4.

Phosphorylation of GRK2 by PKA augments GRK2-mediated phosphorylation, internalization, and desensitization of VPAC2 receptors in smooth muscle

2008 ◽  
Vol 294 (2) ◽  
pp. C477-C487 ◽  
Author(s):  
Karnam S. Murthy ◽  
Sunila Mahavadi ◽  
Jiean Huang ◽  
Huiping Zhou ◽  
Wimolpak Sriwai
Keyword(s):  
Smooth Muscle ◽  
G Protein ◽  
Prolonged Exposure ◽  
Phosphorylation Site ◽  
Receptor Expression ◽  
Receptor Internalization ◽  
Receptor Phosphorylation ◽  
G Protein Coupled ◽  
Homologous Desensitization ◽  
In Cells

The smooth muscle of the gut expresses mainly Gs protein-coupled vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide receptors (VPAC2 receptors), which belong to the secretin family of G protein-coupled receptors. The extent to which PKA and G protein-coupled receptor kinases (GRKs) participate in homologous desensitization varies greatly among the secretin family of receptors. The present study identified the novel role of PKA in homologous desensitization of VPAC2 receptors via the phosphorylation of GRK2 at Ser685. VIP induced phosphorylation of GRK2 in a concentration-dependent fashion, and the phosphorylation was abolished by blockade of PKA with cell-permeable myristoylated protein kinase inhibitor (PKI) or in cells expressing PKA phosphorylation-site deficient GRK2(S685A). Phosphorylation of GRK2 increased its activity and binding to Gβγ. VIP-induced phosphorylation of VPAC2 receptors was abolished in muscle cells expressing kinase-deficient GRK2(K220R) and attenuated in cells expressing GRK2(S685A) or by PKI. VPAC2 receptor internalization (determined from residual 125I-labeled VIP binding and receptor biotinylation after a 30-min exposure to VIP) was blocked in cells expressing GRK2(K220R) and attenuated in cells expressing GRK2(S685A) or by PKI. Finally, VPAC2 receptor degradation (determined from residual 125I-labeled VIP binding and receptor expression after a prolonged exposure to VIP) and functional VPAC2 receptor desensitization (determined from the decrease in adenylyl cyclase activity and cAMP formation after a 30-min exposure to VIP) were abolished in cells expressing GRK2(K220R) and attenuated in cells expressing GRK2(S685A). These results demonstrate that in gastric smooth muscle VPAC2 receptor phosphorylation is mediated by GRK2. Phosphorylation of GRK2 by PKA enhances GRK2 activity and its ability to induce VPAC2 receptor phosphorylation, internalization, desensitization, and degradation.

scholarly journals Regulators of G protein Signaling (RGS) proteins (version 2020.4) in the IUPHAR/BPS Guide to Pharmacology Database

2020 ◽  
Vol 2020 (4) ◽  
Author(s):  
Katelin E. Ahlers-Dannen ◽  
Mohammed Alqinyah ◽  
Christopher Bodle ◽  
Josephine Bou Dagher ◽  
Bandana Chakravarti ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Signaling Cascades ◽  
G Protein Signaling ◽  
Rgs Proteins ◽  
Protein Signaling ◽  
Heterotrimeric G Proteins ◽  
Gtp Hydrolysis ◽  
Rgs Domain ◽  
G Protein Coupled

Regulator of G protein Signaling, or RGS, proteins serve an important regulatory role in signaling mediated by G protein-coupled receptors (GPCRs). They all share a common RGS domain that directly interacts with active, GTP-bound Gα subunits of heterotrimeric G proteins. RGS proteins stabilize the transition state for GTP hydrolysis on Gα and thus induce a conformational change in the Gα subunit that accelerates GTP hydrolysis, thereby effectively turning off signaling cascades mediated by GPCRs. This GTPase accelerating protein (GAP) activity is the canonical mechanism of action for RGS proteins, although many also possess additional functions and domains. RGS proteins are divided into four families, R4, R7, R12 and RZ based on sequence homology, domain structure as well as specificity towards Gα subunits. For reviews on RGS proteins and their potential as therapeutic targets, see e.g. [160, 377, 411, 415, 416, 512, 519, 312, 6].

scholarly journals Rgs5 Targeting Leads to Chronic Low Blood Pressure and a Lean Body Habitus

2008 ◽  
Vol 28 (8) ◽  
pp. 2590-2597 ◽  
Author(s):  
Hyeseon Cho ◽  
Chung Park ◽  
Il-Young Hwang ◽  
Sang-Bae Han ◽  
Dan Schimel ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
G Protein ◽  
Muscle Cells ◽  
Cardiovascular Development ◽  
Protein Signaling ◽  
Body Habitus ◽  
Low Blood Pressure

ABSTRACT RGS5 is a potent GTPase-activating protein for Giα and Gqα that is expressed strongly in pericytes and is present in vascular smooth muscle cells. To study the role of RGS5 in blood vessel physiology, we generated Rgs5-deficient mice. The Rgs5 −/− mice developed normally, without obvious defects in cardiovascular development or function. Surprisingly, Rgs5 −/− mice had persistently low blood pressure, lower in female mice than in male mice, without concomitant cardiac dysfunction, and a lean body habitus. The examination of the major blood vessels revealed that the aortas of Rgs5 −/− mice were dilated compared to those of control mice, without altered wall thickness. Isolated aortic smooth muscle cells from the Rgs5 −/− mice exhibited exaggerated levels of phosphorylation of vasodilator-stimulated phosphoprotein and extracellular signal-regulated kinase in response to stimulation with either sodium nitroprusside or sphingosine 1-phosphate. The results of this study, along with those of previous studies demonstrating that RGS5 stability is under the control of nitric oxide via the N-end rule pathway, suggest that RGS5 may balance vascular tone by attenuating vasodilatory signaling in vivo in opposition to RGS2, another RGS (regulator of G protein signaling) family member known to inhibit G protein-coupled receptor-mediated vasoconstrictor signaling. Blocking the function or the expression of RGS5 may provide an alternative approach to treat hypertension.

Modulation of Transducin GTPase Activity by Chimeric RGS16 and RGS9 Regulators of G Protein Signaling and the Effector Molecule†

Biochemistry ◽  
1999 ◽  
Vol 38 (16) ◽  
pp. 4931-4937 ◽  
Author(s):  
Randall L. McEntaffer ◽  
Michael Natochin ◽  
Nikolai O. Artemyev
Keyword(s):  
G Protein ◽  
G Protein Signaling ◽  
Gtpase Activity ◽  
Protein Signaling ◽  
Effector Molecule

scholarly journals Inhibition of Gαi activity by Gβγ is mediated by PI 3-kinase-γ- and cSrc-dependent tyrosine phosphorylation of Gαi and recruitment of RGS12

2014 ◽  
Vol 306 (9) ◽  
pp. G802-G810 ◽  
Author(s):  
Jiean Huang ◽  
Ancy D. Nalli ◽  
Sunila Mahavadi ◽  
Divya P. Kumar ◽  
Karnam S. Murthy
Keyword(s):  
Smooth Muscle ◽  
Tyrosine Phosphorylation ◽  
G Protein ◽  
Muscle Relaxation ◽  
P38 Map Kinase ◽  
Smooth Muscle Relaxation ◽  
Inhibitory Influence ◽  
Protein Signaling ◽  
P21 Activated Kinase ◽  
Δ Opioid Receptor

Others and we have characterized several Gβγ-dependent effectors in smooth muscle, including G protein-coupled receptor kinase 2 (GRK2), PLCβ3, and phosphatidylinositol (PI) 3-kinase-γ, and have identified various signaling targets downstream of PI 3-kinase-γ, including cSrc, integrin-linked kinase, and Rac1-Cdc42/p21-activated kinase/p38 MAP kinase. This study identified a novel mechanism whereby Gβγ acting via PI 3-kinase-γ and cSrc exerts an inhibitory influence on Gαi activity. The Gi2-coupled δ-opioid receptor agonist d-penicillamine ( 2 , 5 )-enkephalin (DPDPE) activated cSrc, stimulated tyrosine phosphorylation of Gαi2, and induced regulator of G protein signaling 12 (RGS12) association; all three events were blocked by PI 3-kinase (LY294002) and cSrc (PP2) inhibitors and by expression of the COOH-terminal sequence of GRK2-(495–689), a Gβγ-scavenging peptide. Inhibition of forskolin-stimulated cAMP and muscle relaxation by DPDPE was augmented by PP2, LY294002 , and a selective PI 3-kinase-γ inhibitor, AS-605420. Expression of tyrosine-deficient (Y69F, Y231F, or Y321F) Gαi2 mutant or knockdown of RGS12 blocked Gαi2 phosphorylation and Gαi2-RGS12 association and caused greater inhibition of cAMP. Parallel studies using somatostatin, cyclopentyl adenosine, or ACh to activate, respectively, Gi1-coupled somatostatin (sstr3) receptors, and Gi3-coupled adenosine A1 or muscarinic m2 receptors elicited cSrc activation, Gαi1 or Gαi3 phosphorylation, Gαi1-RGS12 or Gαi3-RGS12 association, and inhibition of cAMP. Inhibition of cAMP and muscle relaxation was greatly increased by AS-605240 and PP2. The results demonstrate that Gβγ-dependent tyrosine phosphorylation of Gαi1/2/3 by cSrc facilitated recruitment of RGS12, a Gαi-specific RGS protein with a unique phosphotyrosine-binding domain, resulting in rapid deactivation of Gαi and facilitation of smooth muscle relaxation.

Sign in / Sign up

Export Citation Format

Share Document