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 membranetargeting activity of the PT domain, thus preventing constitutive plasma membrane localization of RasGRP1. By binding to diacylglycerol generated by BCR-coupled phospholipase C␥2, 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. INTRODUCTIONRasGRP1 is a guanine nucleotide exchange factor that couples antigen receptors to the activation of Ras GTPases (Dower et al., 2000;Ebinu et al., 2000;Priatel et al., 2002;Bivona et al., 2003;Caloca et al., 2003b;Layer et al., 2003;Norment et al., 2003;Oh-hora et al., 2003;Guilbault and Kay, 2004;Perez de Castro et al., 2004;Reynolds et al., 2004;Zugaza et al., 2004;Coughlin et al., 2005;Roose et al., 2005). Deletion of the RasGRP1 gene perturbs the immunological selection and activation of lymphocytes (Dower et al., 2000;Priatel et al., 2002Priatel et al., , 2006Layer et al., 2003) and mast cells (Liu et al., 2007), whereas aberrant expression of RasGRP1 initiates oncogenic transformation of lymphocytes (Li et al., 1999;Mikkers et al., 2002;Suzuki et al., 2002;Kim et al., 2003;Dupuy et al., 2005;Klinger et al., 2005), fibroblasts (Ebinu et al., 1998;Tognon et al., 1998) and keratinocytes (Oki-Idouchi and Lorenzo, 2007).To be active as an exchange factor, RasGRP1 must be localized to cell membranes where Ras GTPases reside. This requirement provides an opportunity for positive or negative regulation. RasGRP1 contains a C1 domain that binds the lipid second messenger diacylglycerol (DAG), or DAGmimetic phorbol esters (Ebinu et al., 1998;Lorenzo et al., 2000;Shao et al., 2001;Rong et al., 2002;Carrasco and Merida, 2004;Madani et al., 2004). Treatment of cells with DAG or phorbol esters results in the translocation of RasGRP1 to membranes (Ebinu et al., 1998;Tognon et al., 1998;Bivona et al., 2003;Rambaratsingh et al., 2003;Caloca et al., 2004;Stone et al., 2004), and it stimulates Ras activation via RasGRP1 (Ebinu et al., 1998;...
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.Ras GTPases are membrane-bound signal-transducing proteins that control a wide range of cellular responses to external stimuli. Their attachment to specific cellular membranes allows Ras GTPases to confine, concentrate, and organize networks of signal detectors and transmitters (1). Guanine nucleotide exchange factors directly activate Ras GTPases by promoting their conversion to the active GTP-bound state (2). Additionally, exchange factors regulate Ras activation by detecting and integrating inputs from signal transducers coupled to cell-surface receptors. Conversion of receptor-initiated signals into Ras activation is often achieved by spatial regulation of an exchange factor (2, 3). In the absence of signal detection, the exchange factor is sequestered away from its Ras GTPase substrates, whereas the activating signal induces membrane binding of the exchange factor and thus co-localizes it with Ras GTPase substrates. RasGRP1 is a Ras-activating exchange factor that is expressed in many cell types and can be activated by diverse receptors (2, 4). In lymphocytes, initiation of Ras signaling by RasGRP1 controls progression through developmental and immunoselection checkpoints, whereas misregulation of RasGRP1 can trigger autoimmunity or oncogenic transformation (5-10). Depending on the quality and intensity of signaling from receptors, RasGRP1 can be specifically targeted to the plasma membrane (11)(12)(13)(14)(15) or to endomembranes such as Golgi or the endoplasmic reticulum (11, 13, 16 -19). The site of RasGRP1 localization can h...
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.
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