ARF6 is required for growth factor- and Rac-mediated membrane ruffling in macrophages at a stage distal to Rac membrane targeting

Activation of Rad, a member of the Rho family of GTPases, is associated wit h multiple cellular responses, including membrane ruffling and focal comple x formation. The mechanisms by which Rad is coupled to these functional res ponses are not well understood. It was recently shown that ARF6, a GTPase i mplicated in cytoskeletal alterations and a membrane recycling pathway, is required for Rad-dependent phagocytosis in macrophages (Q. Zhang et al., J. Biol. Chem. 273:19977-19981, 1998). To determine whether ARF6 is required for Rad-dependent cytoskeletal responses in macrophages, we expressed wild- type (WT) or guanine nucleotide binding-deficient alleles (T27N) of ARF6 in macrophages coexpressing activated alleles of Rad (Q61L) or Cdc42 (Q61L) o r stimulated with colony-stimulating factor I (CSF-1). Expression of ARF6 T 27N but not ARF6 WT inhibited ruffles mediated by Rad Q61L or CSF-1. In con trast, expression of ARF6 T27N did not inhibit Rad Q61L-mediated focal comp lex formation and did not impair Cdc42 Q61L-mediated filopodial formation. Cryoimmunogold electron microscopy demonstrated the presence of ARF6 in mem brane ruffles induced by either CSF-1 or Rad Q61L. Addition of CSF-1 to mac rophages led to the redistribution of ARF6 from the interior of the cell to the plasma membrane, suggesting that this growth factor triggers ARF6 acti vation. Direct targeting of Rad to the plasma membrane did not bypass the b lockade in ruffling induced by ARF6 T27N, indicating that ARF6 regulates a pathway leading to membrane ruffling that occurs after the activation and m embrane association of Rac. These data demonstrate that intact ARF6 functio n is required for coupling activated Rac to one of several effector pathway s and suggest that a principal function of ARF6 is to coordinate Rac activa tion with plasma membrane-based protrusive events.