As critical regulators of vesicular trafficking, Rab proteins comprise the largest GTPase family, with thirty-seven functionally distinct members and another twenty isoforms in the human genome. Like other GTPases, Rab proteins cycle between inactive (GDP bound) and active (GTP bound) states. Activation and inactivation are regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), respectively. Once activated, Rab GTPases interact with effector proteins involved in vesicle budding, cargo sorting, transport, tethering, docking and fusion. The specificity of Rab interactions with effectors and regulatory factors is hypothesized to play a central role with respect to the fidelity of membrane trafficking. However, an increasing number of effectors, GEFs and GAPs are known to interact with two or more Rab GTPases. Moreover, the determinants for recognition of Rab GTPases are elaborate, involving both direct and indirect structural mechanisms. The long range objective of this proposal is to understand the structural bases and mechanisms underlying the network of protein-protein interactions with Rab GTPases and how structurally diverse regulatory factors and effectors recognize Rab GTPases. Capitalizing on the information available from genomic sequencing projects and building on extensive preliminary data, the aims of this proposal address the recognition problem from a global, family wide perspective. Specifically, we will:
(Aim 1) determine high resolution crystal structures of as many Rab GTPases as possible in the active and inactive conformations;
(Aim 2) determine the specificity of selected effectors and regulatory factors for the entire Rab GTPase family;
and (Aim 3) determine the structural bases for interaction of effectors and regulatory factors with Rab GTPases that function in the endocytic and recycling pathways. The combined results will provide novel, family wide insight regarding the determinants for recognition of Rab GTPases. Defects in Rab GTPases and their accessory factors have been implicated in several genetically linked disorders and likely contribute to the pathology of complex disease states including diabetes and cancer. Consequently, Rab accessory and regulatory factors are potential targets for mechanism based pharmacological interdiction.
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