Regulation of Rho-activating proteins by heterotrimeric G proteins: Sensitivity of Gα RhoGEF interaction is determined by dissociation kinetics

Activation of RhoGTPases downstream of G protein coupled receptors is important for many physiological functions, such as blood pressure regulation. The subfamilies Rho, Rac and Cdc42 are the best understood RhoGTPases and the present study focused on signaling towards the Rho subfamily member RhoA. In its active state RhoA regulates the cytoskeleton by its influence on actin dynamics, activates important signal transducers such as Rho-associated coiled-coil kinase, which phosphorylates and thereby inactivates myosin light chain phosphatase and induces gene transcription via serum response factor. Most RhoGTPases cycle between a GDP-bound inactive and a GTP-bound active state. The exchange of GTP for GDP and therefore activation is mediated through Rho guanine nucleotide exchange factors (RhoGEFs). In the case of RhoA the largest family of RhoGEFs is functionally and structurally characterized by a DH domain adjunct to a PH domain. The DH domain holds GEF activity and the PH domain has mainly regulatory functions. Some of these RhoGEFs can be activated by Gαq/11 and/or Gα12/13 and the present work focused on their regulation: Downstream of Gα13 RH-RhoGEFs are activated. This group of RhoGEFs shares a regulator of G protein signaling homology domain (RH) in addition to the DH-PH domain, which is also present in the Gαq-activated p63RhoGEF. Knock-out of the RH-RhoGEF leukemia-associated RhoGEF (LARG) protects against salt-induced hypertension in mice and the acute response of vascular smooth muscle cells to angiotensin II treatment is mediated mainly by p63RhoGEF. For both proteins several other physiological functions have been described. Nevertheless, little had been known about why RhoGEFs are activated downstream of two Gα subfamilies and the temporal as well as spatial dynamics of their receptor-mediated activation. Therefore we developed FRET-based assays monitoring RhoGEF activation in living cells for the first time. The Förster resonance energy transfer (FRET) occurs between two fluorophores - in the present study fused to the proteins of interest - with a distance of less than 10nm. Thus an increase in FRET upon stimulation with the agonist reflects convergence of the proteins of interest. Changes in FRET were recorded in single, living cells with a high-speed CCD-camera. The interaction between LARG and Gα13 was monitored in cells transfected with Gα13-mTur2 and YFP-LARG. The stimulation of thromboxane A2 receptor induced a robust increase in FRET. Surprisingly, as shown by the slow decrease in FRET between LARG and Gα13, the interaction of LARG and Gα13 dissociated very slowly (estimated t1/2>5min) compared to the Gα13 inactivation (t1/2=17.50s). This observation was also reflected in the kinetics of LARG translocation to the plasma membrane. Thus LARG and Gα13 interact rapidly upon activation of Gα13, but either LARG inhibits Gα13 inactivation or stays in a complex with Gα13 after inactivation of the same. In our opinion the prolonged interaction is most likely the reason for the almost 100-fold higher sensitivity towards stimulation with a thromboxane agonist of the Gα13 LARG interaction compared with the Gα13 activation. The p63RhoGEF activation was studied by monitoring the interaction of Gαq-CFP and Venus-p63RhoGEF. A robust increase in FRET was observed upon stimulation of Gαq coupled receptors. In contrast to the LARG Gα13 interaction, the p63RhoGEF Gαq interaction mirrored closely the Gαq activation as well as inactivation. In addition also the sensitivity of p63RhoGEF Gαq interaction and Gαq activation was in the same range (EC50 of 500nM histamine). Both observations were also true in a trimeric complex of p63RhoGEF and Gαq with the regulator of G protein signaling RGS2. RGS2 was previously shown to accelerate Gαq inactivation in vitro and consequently we observed an accelerated dissociation of p63RhoGEF and Gαq in the presence of RGS2. Additionally, we could monitor an increase in FRET between p63RhoGEF and RGS2, which is the first evidence for such a trimeric complex in living cells. Thus our data strongly support the concept of a functional activation-dependent p63RhoGEF Gαq RGS2 complex. In this complex RGS2 inhibits downstream signaling. This could be an explanation for severe hypertension, which has been observed in RGS2 knock-out mice (Tang et al., 2003). In summary, LARG as well as p63RhoGEF are both activated upon stimulation of G protein coupled receptors. Nevertheless LARG´s sensitivity towards receptor activation and duration of signaling seems to be remarkably higher and longer than p63RhoGEF´s. The inactivation of p63RhoGEF is further accelerated by RGS2, which also decreases downstream signaling