Diverse cellular functions are controlled by RhoA-GTPases, which are activated by trimeric G proteins RhoGEFs, among others. In this study, we focused on the signaling from GPCRs to RhoA Gα and leukemia-associated RhoGEF (LARG). The activation of Gα was elucidated in living cells with high temporal and spatial resolution by means of FRET. The inactivation after agonist withdrawal occurred in the same range ( = 25.3 ± 2.2 s; mean ± sem; 22) as described for other Gα proteins. The interaction of Gα and LARG and the thereby-induced LARG translocation to the plasma membrane were at least 1 order of magnitude more stable after agonist withdrawal, exceeding Gα deactivation in the absence of LARG several fold. Consequently, we observed an almost 100-fold higher agonist sensitivity of the Gα LARG interaction compared to the Gα activation in the absence of LARG.-Bodmann, E.-L., Krett, A.-L., Bünemann, M. Potentiation of receptor responses induced by prolonged binding of Gα and leukemia-associated RhoGEF.

G protein-coupled receptors (GPCRs) are the largest class of transmembrane receptors and serve as signal mediators to transduce information from extracellular signals such as neurotransmitters, hormones, or drugs to cellular responses. They are exposed to the strong electrical field of the plasma membrane. In the last decade voltage modulation of ligand-induced GPCR activity has been reported for several GPCRs. Using Foerster resonance energy transfer-based biosensors in patch clamp experiments, we discovered a robust voltage dependence of the thromboxane receptor (TP receptor) on the receptor level as well as on downstream signaling. TP receptor activity doubled upon depolarization from 290 to 160 mV in the presence of U46619, a stable analog of prostaglandin H 2. Half-maximal effective potential (V 0.5) determined for TP receptor was 246 mV, which is within the physiologic range. We identified that depolarization affected the agonist affinity for the TP receptor. Depolarization enhanced responses of several structural analogs of U46619 with modifications to a similar extent all around the molecule, indicating that voltage modulates the general conformation of TP receptor. By means of site direct mutagenesis, we identified TP receptor R295 7.40 , which showed alteration of voltage sensitivity of TP receptor upon mutation. Voltage sensitivity was not limited to TP receptor because prostaglandin F receptor activated with U46619 and prostaglandin E 2 receptor subtype 3 activated with iloprost showed a similar reaction to depolarization as TP receptor. However, prostacyclin receptor activated with iloprost showed no detectable voltage dependence. SIGNIFICANCE STATEMENT Prostanoids mediate many of their physiological effects via transmembrane receptors expressed in the plasma membrane of excitable cells. We found that agonist-mediated activation of prostaglandin F receptors and prostaglandin E 2 receptors as well as thromboxane receptors are activated upon depolarization, whereas prostacyclin receptors are not. The voltage-induced modulation of thromboxane receptor activity was observed on the level of receptor conformation and downstream signaling. The range of voltage dependence was restricted by R295 7.40 in the agonist-binding pocket.

The three RH-RhoGEFs (Guanine nucleotide exchange factors) p115-RhoGEF, LARG (leukemia-associated RhoGEF) and PDZ-RhoGEF link G-protein coupled receptors (GPCRs) with RhoA signaling through activation of Gα12/13. In order to find functional differences in signaling between the different RH-RhoGEFs we examined their interaction with Gα13 in high spatial and temporal resolution, utilizing a FRET-based single cell assay. We found that p115-RhoGEF interacts significantly shorter with Gα13 than LARG and PDZ-RhoGEF, while narrowing the structural basis for these differences down to a single amino acid in the rgRGS domain of p115-RhoGEF. The mutation of this amino acid led to an increased interaction time with Gα13 and an enhanced agonist sensitivity, comparable to LARG, while mutating the corresponding amino acid in Gα13 the same effect could be achieved. While the rgRGS domains of RH-RhoGEFs showed GAP (GTPase-activating protein) activity towards Gα13 in vitro, our approach suggests higher GAP activity of p115-RhoGEF in intact cells.