MECHANISM OF GALPHA-13 REGULATED RHOGEF ACTIVITY OF P115RHOGEF

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. p115RhoGEF (p115) represents a direct link between the G12 class heterotrimeric G proteins and the small GTPase Rho. p115 is both a negative regulator and a downstream effector for Galpha-13 (Ga13). At its N-terminus, p115 possesses a RhoGEF-RGS (rgRGS) domain that functions as a GTPase activating protein (GAP) towards Ga13. Binding to Ga13 also stimulates its Guanine Nucleotide Exchange (GEF) activity towards RhoA. The GEF activity of p115 is carried out by the tandem DH and PH domains, which are separated from the N-terminal rgRGS domain by a 150 residue linker region. Crystal structures of the complex of the rgRGS domain with Ga13, and the DH-PH domains in complex with RhoA have been determined. There is evidence for an inhibitory mechanism within p115 that is released upon binding to Ga13. There is also evidence that Ga13 interacts with a second binding site in p115 that resides outside of the N-terminal rgRGS domain, possibly within the DH and PH domains. We propose that the binding of Ga13 to p115 causes substantial conformational changes in the latter, possibly domain rotation at one or more hinge points. Such conformational changes are required for stimulation of the GEF activity of p115 towards RhoA. We have recently conducted small angle X-ray scattering experiments at BioCAT to determine the molecular envelope of p115, and that of its complex with Ga13. Initial data analysis indicates that p115 or its complex with Ga13 remains a dimer in solution. Detailed modeling is currently underway. 4