title: The mechanism of cell membrane ruffling relies on a phospholipase D2 (PLD2), Grb2 and Rac2 association.
abstract: Membrane ruffling is the formation of actin rich membrane protrusions, essential for cell motility. The exact mechanism of ruffling is not fully known. Using YFP and CFP fluorescent chimeras, we show for the first time a co-localization of Phospholipase D2 (PLD2) and Growth factor Receptor Bound protein-2 (Grb2) with actin-rich membrane protrusions of macrophages. Grb2 cooperates with PLD2 in enhancing membrane ruffling, whether in resting cells or in cells stimulated with the growth factor M-CSF, although in the latter an increase in dorsal ruffles was observed, consistent with receptor-ligand internalization. Cells transfected with PLD2 mutated in the PH domain (Y169F) or with Grb2 mutated in the SH2 site (R86K) negate this effect, indicating an association PLD2(Y169)-SH2-Grb2 that was confirmed by immunoprecipitation and Western blotting. The association results in enhanced PLD activity, but the lipase activity can only partially explain the formation of membrane ruffles in vivo. A third component involves the Rho-GTPase Rac2 and it is only when Rac2 is overexpressed along with PLD2 and Rac2 that a full biological effect, including actin polymerization in vivo, is obtained. The mechanism involved is, then, as follows: PLD enzymatic action, after having been increased due to the binding to Grb2-SH2 via Y169, cooperates with Rac2, and the three molecules stimulate actin polymerization and consequently, membrane ruffle formation. Since membrane ruffling precedes cell migration, the results herein provide a novel mechanism for control of membrane dynamics, crucial for the physiology of leukocytes.
authors.affiliation: Dept. Biochemistry & Molecular Biology, Wright State University School Medicine, Dayton, OH 45435, USA.
authors.forename: Madhu
authors.initials: M
authors.lastname: Mahankali
authors.forename: Hong-Juan
authors.initials: HJ
authors.lastname: Peng
authors.forename: Dianne
authors.initials: D
authors.lastname: Cox
authors.forename: Julian
authors.initials: J
authors.lastname: Gomez-Cambronero
mesh_terms: D000199:Actins / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism*; D002465:Cell Movement; D022081:Cell Surface Extensions / Q000201:enzymology / Q000378:metabolism; D002522:Chlorocebus aethiops; D051380:GRB2 Adaptor Protein / Q000378:metabolism*; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D051379:Mice; D009154:Mutation; D010739:Phospholipase D / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D020744:rac GTP-Binding Proteins / Q000378:metabolism*; D000097766:RAC2 GTP-Binding Protein
chemical_list: D000199:Actins; D051380:GRB2 Adaptor Protein; D016173:Macrophage Colony-Stimulating Factor; D010739:Phospholipase D; D020744:rac GTP-Binding Proteins; D000097766:RAC2 GTP-Binding Protein; C105606:phospholipase D2