Functional analysis of the interaction of the cytohesin 2 GTP-exchange factor with MAGUIN-1

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The ADP-ribosylation factor (ARF)6 small GTPase act as a GDP/GTP-regulated switch in the pathways that stimulate actin reorganisation and membrane ruffling, which are essential for cell growth and development. The formation of active ARF-GTP is stimulated by guanine nucleotide exchange factors (GEFs) such as cytohesin 2. The localisation of active ARF6 to the plasma membrane is essential for biological activity and therefore cytohesin 2 must be at the plasma membrane for ARF6 activation. Cytohesin 2 translocates to the plasma membrane in agonist-stimulated cells by binding the lipid second messenger PIP3 through its PH domain. However, cytohesin 2 is found in the membrane fraction of some cell lines under non-stimulation conditions, suggesting that localisation of this protein may be determined by the properties of binding partner proteins. We have recently obtained membrane-associated guanylate kinase-interactor 1(MAGUIN-1) as a cytohesin 2-interactor by yeast two-hybrid screening. Importantly, we have also shown an interaction between endogenous MAGUIN-1 and cytohesin 2 by co-immunoprecipitation. MAGUIN-1, through its C-terminus, interacts with the coiled-coil domain of cytohesin 2 and recruits cytohesin 2 to the plasma membrane. These preliminary results provide support for the HYPOTHESIS of this proposal, that MAGUIN-1 regulates ARF6-associated cellular functions by constitutively recruiting cytohesin 2 to the plasma membrane and activating it. The specific aims described in the proposal will address this hypothesis.