Phosphoinositide 3-kinase regulates plasma membrane targeting of the Ras-specific exchange factor RasGRP1.

Receptor-induced targeting of exchange factors to specific cellular membranes is the predominant mechanism for initiating and compartmentalizing signal transduction by Ras GTPases. The exchange factor RasGRP1 has a C1 domain that binds the lipid diacylglycerol and thus can potentially mediate membrane localization in response to receptors that are coupled to diacylglycerol-generating phospholipase Cs. However, the C1 domain is insufficient for targeting RasGRP1 to the plasma membrane. We found that a basic/hydrophobic cluster of amino acids within the plasma membrane-targeting domain of RasGRP1 is instead responsible for plasma membrane targeting. This basic/hydrophobic cluster binds directly to phospholipid vesicles containing phosphoinositides via electrostatic interactions with polyanionic phosphoinositide headgroups and insertion of a tryptophan into the lipid bilayer. B cell antigen receptor ligation and other stimuli induce plasma membrane targeting of RasGRP1 by activating the phosphoinositide 3-kinase signaling pathway, which generates phosphoinositides within the plasma membrane. Direct detection of phosphoinositides by the basic/hydrophobic cluster of RasGRP1 provides a novel mechanism for coupling and co-compartmentalizing phosphoinositide 3-kinase and Ras signaling and, in coordination with diacylglycerol detection by the C1 domain, gives RasGRP1 the potential to serve as an integrator of converging signals from the phosphoinositide 3-kinase and phospholipase C pathways.

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.

Regulation of RasGRP1 by B cell antigen receptor requires cooperativity between three domains controlling translocation to the plasma membrane.

RasGRP1 is a Ras-activating exchange factor that is positively regulated by translocation to membranes. RasGRP1 contains a diacylglycerol-binding C1 domain, and it has been assumed that this domain is entirely responsible for RasGRP1 translocation. We found that the C1 domain can contribute to plasma membrane-targeted translocation of RasGRP1 induced by ligation of the B cell antigen receptor (BCR). However, this reflects cooperativity of the C1 domain with the previously unrecognized Plasma membrane Targeter (PT) domain, which is sufficient and essential for plasma membrane targeting of RasGRP1. The adjacent suppressor of PT (SuPT) domain attenuates the plasma membrane-targeting activity of the PT domain, thus preventing constitutive plasma membrane localization of RasGRP1. By binding to diacylglycerol generated by BCR-coupled phospholipase Cgamma2, the C1 domain counteracts the SuPT domain and enables efficient RasGRP1 translocation to the plasma membrane. In fibroblasts, the PT domain is inactive as a plasma membrane targeter, and the C1 domain specifies constitutive targeting of RasGRP1 to internal membranes where it can be activated and trigger oncogenic transformation. Selective use of the C1, PT, and SuPT domains may contribute to the differential targeting of RasGRP1 to the plasma membrane versus internal membranes, which has been observed in lymphocytes and other cell types.

Differential membrane binding and diacylglycerol recognition by C1 domains of RasGRPs.

RasGRPs (guanine-nucleotide-releasing proteins) are exchange factors for membrane-bound GTPases. All RasGRP family members contain C1 domains which, in other proteins, bind DAG (diacylglycerol) and thus mediate the proximal signal-transduction events induced by this lipid second messenger. The presence of C1 domains suggests that all RasGRPs could be regulated by membrane translocation driven by C1-DAG interactions. This has been demonstrated for RasGRP1 and RasGRP3, but has not been tested directly for RasGRP2, RasGRP4alpha and RasGRP4beta. Sequence alignments indicate that all RasGRP C1 domains have the potential to bind DAG. In cells, the isolated C1 domains of RasGRP1, RasGRP3 and RasGRP4alpha co-localize with membranes and relocalize in response to DAG, whereas the C1 domains of RasGRP2 and RasGRP4beta do not. Only the C1 domains of RasGRP1, RasGRP3 and RasGRP4alpha recognize DAG as a ligand within phospholipid vesicles and do so with differential affinities. Other lipid second messengers were screened as ligands for RasGRP C1 domains, but none was found to serve as an alternative to DAG. All of the RasGRP C1 domains bound to vesicles which contained a high concentration of anionic phospholipids, indicating that this could provide a DAG-independent mechanism for membrane binding by C1 domains. This concept was supported by demonstrating that the C1 domain of RasGRP2 could functionally replace the membrane-binding role of the C1 domain within RasGRP1, despite the inability of the RasGRP2 C1 domain to bind DAG. The RasGRP4beta C1 domain was non-functional when inserted into either RasGRP1 or RasGRP4, implying that the alternative splicing which produces this C1 domain eliminates its contribution to membrane binding.