Receptor-induced translocation of activated guanine-nucleotide-binding protein alpha(i) subunits to the cytoskeleton in myeloid differentiated human leukemia (HL-60) cells

The regulation of the cytoskeletal localization of guanine-nucleotide-binding protein alpha(i) subunits by formyl peptide receptors was studied in myeloid differentiated human leukemia (HL-60) cells. Stimulation of formyl peptide receptors with N-formyl-Met-Leu-Phe (fMet-Leu-Phe) transiently increased the amount of alpha(1) subunits in the Triton X-100-insoluble cytoskeleton, Similar to tire biphasic regulation of the actin content, fMet-Leu-Phe (greater than or equal to 10 nM) rapidly increased the cytoskeletal alpha(i) content (about threefold at 30 s), which was followed by a rapid reversal to control levels. The formyl peptide receptor increased the cytoskeletal content of both alpha(i) subtypes, alpha(i2) and alpha(i3), present in HL-60 cells. In cells permeabilized with Staphylococcus aureus alpha-toxin, fMet-Leu-Phe increased binding of the stable GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), to cytoskeletal proteins in a pertussis-toxin-sensitive manner, which was completely abolished by the F-actin-disrupting agent, cytochalasin B. Using the photoreactive CTP analogue, m-acetylanilido-GTP, the formyl peptide receptor-regulated GTP binding sires at the cytoskeleton were identified as 40-kDa proteins, the molecular size of alpha(i) subunits. Cytoskeleton prepared from stimulated cells did not exhibit increased GTP[S] binding, which suggests that activated alpha(i) subunits are translocated to the cytoskeleton. Finally in alpha-toxin-permeabilized HL-60 cells, fMet-Leu-Phe and GTP[S] cooperatively stimulated actin polymerization. In conclusion, evidence is provided that chemoattractant receptors cause translocation of activated alpha(i) subunits to the cytoskeleton coincidentally with F-actin formation. The data therefore argue for a potential role of translocated alpha(i) subunits in the process of receptor-induced actin polymerization.