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.