FC RECEPTOR-MEDIATED PHAGOCYTOSIS REQUIRES CDC42 AND RAC1

At the surface of phagocytes, antibody-opsonized particles are recogni zed by surface receptors for the Fc portion of immunoglobulins (FcRs) that mediate their capture by an actin-driven process called phagocyto sis which is poorly defined. We have analyzed the function of the Rho proteins Rad and CDC42 in the high affinity receptor for IgE (Fc epsil on RI)-mediated phagocytosis using transfected rat basophil leukemia ( RBL-2H3) mast cells expressing dominant inhibitory forms of CDC42 and Rad. Binding of opsonized particles to untransfected RBL-2H3 cells led to the accumulation of F-actin at the site of contact with the partic les and further, to particle internalization. This process was inhibit ed by Clostridium difficile toxin B, a general inhibitor of Rho GTP-bi nding proteins, Dominant inhibition of Rad or CDC42 function severely inhibited particle internalization but not F-actin accumulation. Inhib ition of CDC42 function resulted in the appearance of pedestal-like st ructures with particles at their tips, while particles bound at the su rface of the Rad mutant cell line were enclosed within thin membrane p rotrusions that did not fuse. These phenotypic differences indicate th at Rac1 and CDC42 have distinct functions and may act cooperatively in the assembly of the phagocytic cup. Inhibition of phagocytosis in the mutant cell lines was accompanied by the persistence of tyrosine-phos phorylated proteins around bound particles. Phagocytic cup closure and particle internalization were also blocked when phosphotyrosine depho sphorylation was inhibited by treatment of RBL-2H3 cells with phenylar sine oxide, an inhibitor of protein phosphotyrosine phosphatases. Alto gether, our data show that Rad and CDC42 are required to coordinate ac tin filament organization and membrane extension to form phagocytic cu ps and to allow particle internalization during FcR-mediated phagocyto sis, Our data also suggest that Rac1 and CDC42 are involved in phospho tyrosine dephosphorylation required for particle internalization.