Kontrollmechanismen von GTPasen der Rho-Familie

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Small guanosine triphosphatases (or GTPases) of the Rho family are key mediators of signal transduction in a multitude of diverse (patho)biochemical processes. Loss or gain of function of regulators of Rho GTPases is generally accepted as potential cause for an altered signaling strength and highly likely the reason for their dysfunctions. Much progress has been made towards understanding the structure-function relationship in respect to the regulation of Rho GTPases, however, it is still unclear how these regulators are controlled themselves. What emerged out of our analysis of DLC1 and OPHN1 proteins within the first funding period of this project is very engaging and encouraging. We described the BAR domain of the X-linked mental retardation protein OPHNl as a module with dual functions, a membrane binding and bending function, and a cis-acting RhoGAP domain binding and autoinhibiting function. Contrary to OPHNl, we found that the RhoGAP function of the tumor suppressor protein DLC1 is efficiently shut down not via a cis-inhibitory (by SAM or START domains) but via a trans-inhibitory mechanism by the SH3 domain of the Ras-specific p120RasGAP. Further analysis of these data will provide several important insights in deciphering structure function relationships of these critical Rho GTPase regulators and will expand our understanding of (patho)biochemical mechanisms. Thus, we aim at investigating the following points within the second (year 4 to 6) funding period: (i) finalizing our investigations of the molecular basis of the BAR-mediated autoinhibition of OPNH1 by structural and mutational analysis, (ii) functional monitoring and imaging of OPHN1 wild-type and constitutive active mutants in cells and on liposomes, (iii) identification and characterization of an 'OPHN1 activating factor' (OAF) using purified synaptosomal fractions, (iv) analysis of structure, regulation, specificity and function of DLC1 protein in the presence of the modulatory protein 14-3-3γ and inhibitory protein p120RasGAP. Planned studies will be achieved by the established techniques of protein chemistry, biochemistry as well as molecular, structural and cellular biology. Addressing these issues of fundamental importance will ultimately advance our knowledge in the field of signal transduction and disclose molecular details of dysregulated signaling pathways.