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

Abstract

The regulation of the cytoskeletal localization of guanine-nucleotide-binding protein α(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 (fMel-Leu-Phe) transiently increased the amount of α, subunits in the Triton X-100-insoluble cytoskeleton. Similar to the biphasic regulation of the actin content, fMet-Leu-Phe (≤10 nM) rapidly increased the cytoskeletal α(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 α(i) subtypes, α(i2) and α(i3), present in HL-60 cells. In cells permeabilized with Staphylococcus aureus α-toxin, fMet-Leu-Phe increased binding of the stable GTP-analogue, guanosine 5'-[γ-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 GTP analogue, m-acetylanilido-GTP, the formyl peptide receptor-regulated GTP binding sites at the cytoskeleton were identified as 40-kDa proteins, the molecular size of α(i) subunits. Cytoskeleton prepared from stimulated cells did not exhibit increased GTP[S] binding, which suggests that activated α(i) subunits are trans located to the cytoskeleton. Finally, in α-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 α(i) subunits to the cytoskeleton coincidentally with F-actin formation. The data therefore argue for a potential role of translocated α(i) subunits in the process of receptor-induced actin polymerization.