Abstract
The cytoskeletal localization of inhibitory guanine-nucleotide-binding (Gi) proteins and the coupling of these proteins to formyl peptide receptors were studied in myeloid differentiated human leukemia (HL-60) cells. Treatment of HL-60 cells with cytochalasin B or botulinum C2 toxin, which leads to the disruption of microfilaments, increased the binding of the stable GTP analogue guanosine 5'[gamma-thio]triphosphate (GTPS[S]) to permeabilized cells by about 30%. In contrast, the microtubule-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 and time dependent, with maximal increases observed at 5 micrograms/ml cytochalasin B and an incubation time of 10 min, and was counteracted by the F-actin-stabilizing toxin phalloidin. Cytochalasin B treatment increased the amount of G proteins activated by chemoattractant receptors by about 25%. Furthermore, the number of Gi-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, which suggest an association of G proteins with actin filaments, the Triton X-100 (1%)-insoluble cytoskeleton was analyzed for the presence of G proteins. Gia subunits were detected in the cytoskeleton preparations, both by specific antisera and by pertussis-toxin -catalyzed ADP-ribosylation. Cytochalasin B pretreatment depleted the cytoskeleton in Gialpha, with an approximately 20% concomitant increase in membrane Gialpha content. In conclusion, evidence is presented that part of the cellular Gia is localized at actin filaments in HL-60 cells. After filament disruption, these Gia subunits seem to be translocated to the plasma membrance, where they can productively interact with chemoattractant receptors.
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