INHIBITION OF NADPH OXIDASE ACTIVATION BY SYNTHETIC PEPTIDES MAPPING WITHIN THE CARBOXYL-TERMINAL DOMAIN OF SMALL GTP-BINDING PROTEINS - LACK OF AMINO-ACID-SEQUENCE SPECIFICITY AND IMPORTANCE OF POLYBASIC MOTIF

The small GTP-binding protein (G protein) Rac1 is an obligatory partic ipant in the assembly of the superoxide (O-2(radical anion))-generatin g NADPH oxidase complex of macrophages. We investigated the effect of synthetic peptides, mapping within the near carboxyl-terminal domains of Rac1 and of related G proteins, on the activity of NADPH oxidase in a cell-free system consisting of solubilized guinea pig macrophage me mbrane, a cytosolic fraction enriched in p47(phox) and p67(phox) (or t otal cytosol), highly purified Rac1-GDP dissociation inhibitor for Rho (Rho GDI) complex, and the activating amphiphile, lithium dodecyl sul fate. Peptides Rac1-(178-188) and Rac1-(178-191), but not Rac2-(178-18 8), inhibited NADPH oxidase activity in a Rac1-dependent system when a dded prior to or simultaneously with the initiation of activation. How ever, undecapeptides corresponding to the near carboxyl-terminal domai ns of RhoA and RhoC and, most notably, a peptide containing the same a mino acids as Rac1-(178-188), but in reversed orientation, were also i nhibitory. Surprisingly, O-2(radical anion) production in a Rac2-depen dent cell-free system was inhibited by Rac1-(178-188) but not by Rac2- (178-188). Finally, basic polyamino acids containing lysine, histidine , or arginine, also inhibited NADPH oxidase activation. We conclude th at inhibition of NADPH oxidase activation by synthetic peptides mappin g within the carboxyl-terminal domain of certain small G proteins is n ot amino acid sequence-specific but related to the presence of a polyb asic motif. It has been proposed that such a motif serves as a plasma membrane targeting signal for a number of small G proteins (Hancock, J .F., Paterson, H., and Marshall, C.J. (1990) Cell 63, 133-139).