TRANSLOCATION OF MICROFILAMENT-ASSOCIATED INHIBITORY GUANINE-NUCLEOTIDE-BINDING PROTEINS TO THE PLASMA-MEMBRANE IN MYELOID DIFFERENTIATED HUMAN LEUKEMIA (HL-60) CELLS

The cytoskeletal localization of inhibitory guanine-nucleotide-binding (Gi) proteins and the coupling of these proteins to formyl peptide re ceptors were studied in myeloid differentiated human leukemia (HL-60) cells. Treatment of HL-60 cells with cytochalasin B or botulinum C2 to xin, which leads to the disruption of microfilaments, increased the bi nding of the stable GTP analogue guanosine 5'-[gamma-thio]triphosphate (GTP[SI) to permeabilized cells by about 30%. In contrast, the microt ubule-disrupting agents colchicine and vinblastine. and cytochalasin B treatment of isolated HL-60 membranes did not affect GTP[S] binding. The stimulatory effect of cytochalasin B treatment was concentration a nd time dependent, with maximal increases observed at 5 mu g/ml cytoch alasin B and an incubation time of 10 min, and was counteracted by the F-actin-stabilizing toxin phalloidin. Cytochalasin B treatment increa sed the amount of G proteins activated by chemoattractant receptors by about 25%. Furthermore, the number of G(i)-protein-coupled receptors for the chemoattractant, N-formyl-Met-Leu-Phe, was increased by about 25% upon cytochalasin B treatment. Based on these functional data, whi ch suggest an association of G proteins with actin filaments, the Trit on X-100 (1%)-insoluble cytoskeleton was analyzed for the presence of G proteins. G(i) alpha subunits were detected in the cytoskeleton prep arations, both by specific antisera and by pertussis-toxin-catalyzed A DP-ribosylation. Cytochalasin B pretreatment depleted the cytoskeleton in G(i) alpha, with an approximately 20% concomitant increase in memb rane G(i) alpha content. In conclusion, evidence is presented that par t of the cellular G(i) alpha is localized at actin filaments in HL-60 cells. After filament disruption, these G(i) alpha subunits seem to be translocated to the plasma membrane, where they can productively inte ract with chemoattractant receptors.