Membrane localization of RasGRP1 is controlled by an EF-hand, and by the GEF domain

RasGRP1 is an exchange factor for membrane-localized Ras GTPases. Activation of RasGRP1 requires its translocation to membranes, which can be directly mediated by either its PT or C1 domains. RasGRP1 also has a pair of EF-hands which have been proposed to regulate RasGRP1 by sensing receptor-induced calcium fluxes. We determined that one of these EF-hands, EF1, is required for receptor-induced translocation of RasGRP1 to the plasma membrane in B cell lines. EF1 enables plasma membrane targeting of RasGRP1 by counteracting the SuPT domain, a negative regulator of the PT domain. Contrary to expectations, EF1-mediated translocation of RasGRP1 does not involve antigen receptor-induced intracellular calcium flux. Instead, alternative splicing affecting EF1 serves to modulate RasGRP1 localization. Excision of an exon encoding part of EF1 selectively disables PT domain-mediated plasma membrane targeting of RasGRP1, without affecting C1 domain-mediated localization to endomembranes. While EF1 specifically controls PT-mediated plasma membrane targeting, the Ras binding site in the catalytic GEF domain of RasGRP1 is required for both PT-mediated plasma membrane targeting and C1-mediated localization to endomembranes. Positive feedback between its GEF domain and membrane-binding domains could be important for full activation of RasGRP1, with occupation of the Ras binding sites in the GEF domain resulting in functional liberation of the PT and C1 domains, and membrane binding by these domains serving to maintain the Ras-GEF interaction.