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Abstract
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 Gα
12/13
. In order to find functional differences in signaling between the different RH-RhoGEFs we examined their interaction with Gα
13
in high spatial and temporal resolution, utilizing a FRET-based single cell assay. We found that p115-RhoGEF interacts significantly shorter with Gα
13
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 Gα
13
and an enhanced agonist sensitivity, comparable to LARG, while mutating the corresponding amino acid in Gα
13
the same effect could be achieved. While the rgRGS domains of RH-RhoGEFs showed GAP (GTPase-activating protein) activity towards Gα
13
in vitro, our approach suggests higher GAP activity of p115-RhoGEF in intact cells.