p63RhoGEF: a new switch for G(q)-mediated activation of smooth muscle

Abstract

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. Multiple RhoA GTP exchange factors (GEFs) are expressed in SM, raising the possibility that specific agonists coupled to specific GPCRs may couple to distinct RhoGEFs and provide novel therapeutic targets. This review focuses on the function and mechanisms of activation of p63RhoGEF (Arhgef 25; GEFT) recently identified in SM and its possible role in selective targeting of RhoA-mediated regulation of basal blood pressure through agonists that couple through G(αq/11).

Fig. 1

Crystal structure of G_αq-p63RhoGEF-RhoA complex (PDB ID:2RGN) showing how G_αq allosterically activates and relieves autoinhibition of p63RhoGEF. In this structure G_αq •GDP•AlF^-₄ allosterically relieves the autoinhibition of the catalytic DH domain by the PH domain. The DH and PH domains are colored purple and pink respectively. The α6-αN linker (shown in green) between the DH and PH domain acts as a conformational switch with different residues controlling the autoinhibitory mechanisms and the activation by G_αq. RhoA is shown in blue with gold switch 1 and switch 2 regions. G_αq is colored grey with Mg²⁺•GDP•AlF^-₄ in the active site shown as a ball and stick model. Residues Ala253 and Trp263 in G_αq at an interface that contacts the PH domain are critical for the ability of G_αq to activate p63RhoGEF. These residues contribute to the selectivity of p63RhoGEF. Tyr356 is another critical residue in G_αq that interfaces with the DH domain (Lutz et al. 2007; Rojas et al. 2007; Shankaranarayanan et al. 2010)) and when mutated results in loss of activation of p63RhoGEF. The relative positions of Arg341 and Gly340, in the α6-αN linker region are necessary for the basal inhibited state and interact with Arg68 in switch 2 of RhoA. Modified from (Shankaranarayanan et al. 2010).

Fig. 2

Scheme illustrating signaling pathways for p63RhoGEF, p115RhoGEF, LARG and PDZRhoGEF leading to RhoA mediated vasoconstriction. GPCRs for phenylephrine (PE), angiotensin (AngII), endothelin (ET-1) and thromboxane (TXA2) couple through G_αq/11 and or G_α12/13 to activate varieties RhoGEFs. Activated RhoGEFs differently or synergistically catalyze GTP loading and activation of RhoA. RhoA activates Rho kinase (ROCK) to phosphorylate and inhibit myosin light chain phosphatase (MLCP) activity resulting in an increase in myosin regulatory light chain phosphorylation (MLC20) and vasoconstriction. G_αq/11 selectively activates p63RhoGEF and phospholipase C β (PLCβ) that may compete for G_αq/11. PLCβ increases cytosolic Ca²⁺ to activate myosin light chain kinase (MLCK) and to activate Jak2 that in turn activates p115RhoGEF to activate RhoA.

Fig. 3

Inhibition of ET1-induced Ca²⁺-sensitized force and phosphorylation of MCLP (RhoA/ROCK substrate) when p63RhoGEF is partially silenced in mouse portal vein. A. ET1-, GTPγS- (i.e. maximal activation of RhoA/Ca²⁺-sensitized force) and Ca²⁺-induced force in permeabilized mouse portal veins following treatment with shRNA targeting p63RhoGEF or a non-targeting control. Note that ET1-induced Ca²⁺-sensitized force (shaded component) is inhibited by partial silencing of p63RhoGEF at constant Ca²⁺ concentration (pCa 6.3). GTPγS-induced force is a measure of the residual component of Ca²⁺-sensitization. pCa 4.5: maximal force. B. Summary of changes in ET1- (50 nM and subsequent addition of 100 nM; i.e. 150 nM final concentration) induced Ca²⁺-sensitized force as shown in panels A, in thromboxane analogue U46619- (150 nM) induced Ca²⁺-sensitized force, and in phosphorylation of the RhoA/ROCK substrate MLCP (i.e. phosphorylation of the MLCP subunit MYPT1 at Thr853) using the protocol shown in panel A. The reduction in force, normalized to maximal force induced by pCa 4.5, for 50 nM ET-1 (IC₅₀ ) was 29.0±3.4 vs 19.7±2.6 respectively and at maximal ET-1 (150 nM) was 38.1±4.0% vs 23.0±2.5% respectively; no significant change in U46619-induced force between control and treated samples (mean values ± S.E. n = 12–14, * P < 0.01). Relative phosphorylations of MLCP: mean values ± S.E. n = 4, *** P < 0.02. From (Momotani et al. 2011).