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.
Johnson
Joanne E
JE
Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada, V5A 1S6.
Goulding
Rebecca E
RE
Ding
Ziwei
Z
Partovi
Amir
A
Anthony
Kira V
KV
Beaulieu
Nadine
N
Tazmini
Ghazaleh
G
Cornell
Rosemary B
RB
Kay
Robert J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D000838:Anions / Q000737:chemistry; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004075:Diglycerides / Q000737:chemistry* / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010703:Phorbol Esters; D010743:Phospholipids / Q000737:chemistry; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D016415:Sequence Alignment; D017385:Sequence Homology; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
D000838:Anions; D004268:DNA-Binding Proteins; D004075:Diglycerides; D020662:Guanine Nucleotide Exchange Factors; D010703:Phorbol Esters; D010743:Phospholipids; D018631:ras Proteins