p63RhoGEF Couples G _αq/11 -Mediated Signaling to Ca ²⁺ Sensitization of Vascular Smooth Muscle Contractility

Abstract

Rationale In normal and diseased vascular smooth muscle (SM), the RhoA pathway, which is activated by multiple agonists through G protein-coupled receptors (GPCRs), plays a central role in regulating basal tone and peripheral resistance. This occurs through inhibition of myosin light chain phosphatase, leading to increased phosphorylation of the myosin regulatory light chain. While it is thought that specific agonists and GPCRs may couple to distinct RhoA guanine nucleotide exchange factors (GEFs), thus raising the possibility of selective targeting of specific GEFs for therapeutic use, this notion is largely unexplored for SM contraction. Objective We examine whether p63RhoGEF, known to couple specifically to Gαq/11 in vitro, is functional in blood vessels as a mediator of RhoA activation, and if it is selectively activated by Gαq/11 coupled agonists. Methods and Results We find that p63RhoGEF is present across SM tissues and demonstrate that silencing of the endogenous p63RhoGEF in mouse portal vein inhibits contractile force induced by endothelin-1 to a greater extent than the predominantly Gα12/13 mediated thromboxane analogue, U46619. This is because endothelin-1 acts on Gαq/11 as well as Gα12/13. Introduction of the exogenous isolated pleckstrin-homology (PH) domain of p63RhoGEF (residues 331–580) into permeabilized rabbit portal vein inhibited Ca2+ sensitized force and activation of RhoA, when phenylephrine was used as an agonist. This reinforces the results based on endothelin-1, because phenylephrine is thought to act exclusively through Gαq/11. Conclusion We demonstrate that p63RhoGEF selectively couples Gαq/11, but not Gα12/13, to RhoA activation in blood vessels and cultured cells, and thus mediates the physiologically important Ca2+ sensitization of force induced with Gαq/11 coupled agonists. Our results suggest that signaling through p63RhoGEF provides a novel mechanism for selective regulation of blood pressure.

GPR116, an Adhesion G-Protein–Coupled Receptor, Promotes Breast Cancer Metastasis via the Gαq-p63RhoGEF-Rho GTPase Pathway

“…One such Rho GEF is p63RhoGEF (also called GEFT), which is known as an effector of the heterotrimeric guanine nucleotide-binding protein Gaq and thereby links Gaq-coupled receptors (GPCR) to the activation of the Rho GTPases (6). Gaqp63RhoGEF-Rho GTPase has been reported as a protein complex (6), which is involved in multiple physiologic functions such as vascular smooth muscle contractility (7) and cell movement (8).…”

The blocking of angiotensin II type 1 receptor and RhoA/Rho kinase activity in hypertensive patients: Effect of olmesartan medoxomil and implication with cardiovascular-renal remodeling

“…1,9 A critical role for the transduction of Ang II signaling to directly activate the RhoA/ Rho kinase pathway has been provided for p63RhoGEF as demonstrated in vitro by studies of loss of function and functional responses 5,7 and supported by reports of its increased level in animal models of hypertension 20 and in hypertensive patients. 8,21 One of the processes triggered by RhoA/Rho kinase activation is the Ang II-mediated increase of oxidative stress via increased expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 10 with subsequent endothelial dysfunction, production of proinflammatory cytokines and growth factors ultimately leading to cardiovascular and renal remodeling, target organ damages of hypertension.…”

“…p63RhoGEF has been reported as a specific mediator transducing the Ang II message from activated AT1R via Gq protein to RhoA/ Rho kinase activation 5 via binding of the α subunit of Gq protein. 6,7 This leads to effects of Ang II-mediated activation of RhoA/Rho kinase on the cardiovascular system, such as vascular contraction, proliferation and cardiovascular remodeling. 1,2 The involvement of RhoA/Rho kinase in these processes is based on the following evidence: (i) the increased level of p63RhoGEF and increased MYPT-1 phosphorylation in hypertensive patients; 8,9 (ii) the RhoA/Rho kinase activation of NAD(P)H oxidase, 10 essential for the induction of oxidative stress; (iii) the demonstration of RhoA/ Rho kinase signaling involvement in the induction of atherothrombogenesis 1,10 and (iv) the Rho kinase inhibition, which leads to cardiovascular protection and the prevention of atherogenesis.…”

“…19 This study aims to evaluate in mononuclear cells from essential hypertensive patients the effect of a relatively short-term treatment with olmesartan on the RhoA/Rho kinase pathway. To this end we have evaluated the protein expression of p63RhoGEF, a guanine nucleotide exchange factor, key mediator of the RhoA/Rho kinase activation by Ang II, [5][6][7] and the phosphorylation state of MYPT-1, a marker of Rho kinase. 1,2 …”

Agonist-induced Ca2+ Sensitization in Smooth Muscle

“…It is widely believed that the Ca 2ϩ -sensitizing function of TXA2 is mediated by G␣ 12/13 , but it is also known that G q/11 may be activated by the TXA2 receptor in which case the p63RhoGEF could be activated. However, this is unlikely as we have previously shown that the silencing of p63RhoGEF in mouse portal vein had no effect of U46619-induced Ca 2ϩ -sensitized force but did suppress the ET-1 response (11). Similarly, ET-1 has been shown to cause transient Ca 2ϩ -induced contraction through G q -coupled receptors, whereas the Ca 2ϩ -sensitized force is mediated by G␣ 13 coupling (39).…”