Specificity and functional modulation of RHOGDIs: An in vitro reconstitution approach

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Signal transduction pathways mediated by RHO family proteins require tight temporal and spatial control. Guanine nucleotide dissociation inhibitors (GDIs; three members known) are evolutionarily conserved key regulators, which bind selectively to prenylated RHO proteins and control their cycle between cytosol and membrane. Cell membrane association is a prerequisite for the function of RHO proteins. Despite considerable efforts over the last decades, the control mechanisms underlying spatiotemporal regulation of RHO protein activity by GDIs have not been completely explored. The DFG research grant allowed us to investigate the function and specificity of GDIs. Our published results have provided first unprecedented mechanistic insights into GDI interactions with purified RHO proteins and their variants using the advantages of both liposome-based in-vitro reconstitution systems and fluorescence-based approaches that are established in our lab. Three very recently published studies investigated different aspects of RHO-specific GDIs, describing electrostatic forces mediate the specificity of RHO GTPase-GDI, a novel disorder involving dyshematopoiesis, inflammation, and hemophagocytic lymphohistiocytosis due to aberrant CDC42-GDI1 interaction, and the identification of the pseudo-natural product Rhonin as a first small-molecule inhibitor that binds to GDI1 and interferes with its function. Moreover, we have a substantial set of unpublished preliminary data enabling us to elucidate a distinct and specific mode of GDI functions that holds true for only a subset of RHO GTPases. Our in vitro liposome reconstitution experiments as a `bottom-up´ approach have provided additional mechanistic insights into interaction of the three GDI paralogs with different RHO GTPases. Here, we took advantage of the in vitro liposome sedimentation/flotation and liposome-immobilized SPR sensor chips, established in our lab. Key issues to be investigated extensively in the next future are the regulatory elements of specificity and functional modulation of the GDI paralogs as proposed in the following two work packages, (i) in-depth analysis of the interaction selectivity of the GDI paralogs, and (ii) elucidation of modulatory mechanisms controlling the GDI function. Addressing these goals is of fundamental importance in understanding how GDIs are integrated in the signal transduction and control membrane localization of RHO proteins.