Signal amplification in HL-60 granulocytes. Evidence that the chemotactic peptide receptor catalytically activates guanine-nucleotide-binding regulatory proteins in native plasma membranes

Receptors for the chemotactic peptide fMet-Leu-Phe (fMet, N-formylmethionine) are present in membranes of myeloid differentiated human leukemia (HL-60) cells and stimulate phospholipase C via a pertussis-toxin-sensitive guanine-nucleotide-binding regulatory protein(s) G-protein(s)]. We have developed methods for the assessment of formyl-peptide-receptor-stimulated binding of radiolabeled guanosine 5'-gamma-thio]triphosphate (35S]GTPS]) to native HL-60 membranes. Agonist stimulation of 35S]GTPS] association with the membrane was minimal (less than or equal to 20%) when GTPS] was the sole nucleotide present in the incubation medium. In contrast, receptor activation led to a marked (up to sixfold) stimulation of 35S]GTPS] binding when GDP or GTP were present in high (greater than 100-fold) excess of 35S]GTPS]. The increase in 35S]GTPS] binding caused by the chemotactic agonist was strictly dependent on the presence of Mg2+ and was significantly increased by Na+. Agonist-independent binding of 35S]GTPS] and the increase due to the chemotactic agonist were markedly attenuated by both pertussis and cholera toxin. Comparison of the number of chemotactic-peptide-sensitive 35S]GTPS]-binding sites to the number of chemotactic peptide receptors present in HL-60 membranes provided direct evidence that a single formyl-peptide receptor is capable of catalyzing the binding of 35S]GTPS] to, and thus the activation of, multiple (up to 20) G-proteins in native plasma membranes.