Differential membrane binding and diacylglycerol recognition by C1 domains of RasGRPs

Abstract

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.

The kinetics of αIIbβ3 activation determines the size and stability of thrombi in mice: implications for antiplatelet therapy

“…Previous studies in cell lines demonstrated that the C1 domain in CalDAG-GEFI is atypical and thus binds DAG with very low affinity. 16,41,42 C1 domains, however, are also known as protein interaction modules. 43 Their binding partners include adapter proteins such as 14-3-3 44 and Ras guanosine triphosphtases, 45 both of which are important in integrin inside-out activation.…”

“…[13][14][15] In addition to the GEF domain, CalDAG-GEFI contains a pair of Ca 2ϩ binding EF hand domains and a C1-like domain with hitherto unknown function. 16 DAG is a well-established activator of protein kinase C (PKC) and therefore granule release in platelets. 17,18 PKC-dependent integrin activation depends on positive feedback by the Gi-coupled P2Y12 receptor, 19 which again leads to the activation of Rap1.…”

Crystal Structure of a Mammalian CTP: Phosphocholine Cytidylyltransferase Catalytic Domain Reveals Novel Active Site Residues within a Highly Conserved Nucleotidyltransferase Fold

“…coli Expression and Purification of Selenomethionine-incorporated CCT236-The cDNA encoding CCT236 was transferred from the baculovirus shuttle vector into pGEX6p1 via NcoI and BamHI cloning sites, to create a fusion protein with a PreScission protease-cleavable glutathione S-transferase tag. The pGEX6p1-CCT236 plasmid was transformed into the BL21-derived Rosetta cell strain (Novagen) for protein expression (18). The overexpression protocol for selenomethionineincorporated protein was based on the method of Doublié (19).…”

“…The H168A,Y173A double mutant was created by introducing the Y173A mutation into the H168A template plasmid, using QuickChange and the same mutagenic primer detailed above. The GST-CCT mutant proteins were expressed in BL21 Rosetta cells (18), purified via glutathione affinity chromatography, and cleaved with PreScission protease, as described above. The protein concentrations in the pooled elution fractions were measured by the Bradford assay (37).…”

Ras guanyl nucleotide releasing protein 2 affects cell viability and cell–matrix adhesion in ECV304 endothelial cells

“…1,2 Because RasGRP2 cannot bind DAG, it fails to translocate to membranes in vivo in response to DAG analogs. 3 RasGRP2, also called CalDAG-GEFI, plays a role in controlling T-cell and platelet adhesion through Rap1 activation, 4,5 and is involved in integrin-independent neutrophil chemotaxis. 6 RasGRP2 has been reported to be associated with Huntington disease and immune-mediated thrombocytopenia and thrombosis.…”