Determinierung funktioneller Unterschiede der Interaktion von G-Proteinen mit den Mitgliedern der RH-RhoGEF-Familie

GPCRs represent the largest group of transmembrane receptors and fulfill countless physiological tasks. The three RH-RhoGEFs (Guanine nucleotide exchange factors) p115-RhoGEF, LARG (leukemia-associated RhoGEF) and PDZ (postsynaptic density 95, disc large, zona occludens-1)-RhoGEF link G-protein coupled receptors (GPCRs) with RhoA signalling through being activated by the Gα12/13 subfamily of G proteins. While the RhoA signaling cascade regulates cell shape, cell differentiation and cell growth, RH-RhoGEFs provide a way for GPCRs to regulate these physiological processes. While the three RH-RhoGEFs are expressed in different organs and tissues, it is not known whether they also exhibit functional differences with respect to their activation via GPCRs. Therefore, the interaction of the various RH-RhoGEFs with Gα13 regarding their interaction kinetics and agonist sensitivity has been examined. In a second project of this work the three RH-RhoGEFs were also examined with respect to their potential interaction with Gβγ subunits. A FRET-based single cell approach has been used to address these scientific questions that allowed the observation of the G protein-RH-RhoGEF interaction, upon the stimulation of the thromboxane receptor, in a high spatial and temporal resolution. In addition different cloning strategies have been used to create numerous mutants and chimeras to address the problems at hand. Confocal imaging, BRET and the Dual-Luciferase Reporter Assay-System have been used as supporting methods. I was able to show the specific interactions of the RH-RhoGEFs with Gα13 subunits. Here p115-RhoGEF displayed a significantly shorter interaction with Gα13 than LARG and PDZ-RhoGEF. The interactions also showed a causative connection between interaction kinetics and agonist sensitivity. Through the use of mutants and chimeras it was possible to narrow the structural basis for these differences down to a single amino acid in the rgRGS domain of p115-RhoGEF. The mutation of this single amino acid led to an increased interaction time with Gα13 comparable to LARG and PDZ-RhoGEF, while also enhancing the agonist sensitivity. This amino acid in the rgRGS domain of p115-RhoGEF forms a polar interaction with an amino acid in the alpha helical domain of Gα13. Mutation of this amino acid in Gα13 showed the same results in altering the interaction dynamics of the Gα13-RH-RhoGEF interaction. The measurements of the Gβγ-RH-RhoGEF interactions by means of FRET displayed robust signals with kinetics that resembled the Gα13-RH-RhoGEF interaction, while no signals could be observed when Gα subunits other than Gα13 were co-transfected. While the rgRGS domains of p115-RhoGEF, LARG (Suzuki et al., 2009) and a PDZ-RhoGEF chimera (Chen et al., 2008) showed in vitro GAP (GTPase-activating protein) activity towards Gα13, the results of the intact single cell approach of this work suggests that only p115-RhoGEF displays in vivo GAP activity towards Gα13, as the GAP activity determines the length of the interaction between the two proteins. In contrast, LARG slowed down Gα13 reassembly (Bodmann et al., 2017), suggesting that an important functional difference between the PDZ domain containing RH-RhoGEFs and p115-RhoGEF is based on a functional GAP activity in living cells only detectable in p115-RhoGEF. The results of the Gβγ-RH-RhoGEF interaction study indicate that the Gα13 and Gβγ subunits do not fully dissociate upon activation and bind the Gα13 effectors together. Giving the plethora of diseases the Gα13-RhoGEF-RhoA signaling axis is involved in, it would be important to understand the functional role of the different RH-RhoGEFs in vivo. The p115-RhoGEF and Gα13 point mutations, as well as the numerous chimeras created in this work provide useful tools to study the physiological consequences of the functional differences in the RH-RhoGEF interactions with Gα13 that were uncovered in this work.