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 ([S-35]GTP[S]) to native HL-60 membranes. Agonist stimulation of [S-35]GTP[S] association with the membrane was minimal (less-than-or-equal-to 20%) when GTP[S] was the sole nucleotide present in the incubation medium. In contrast, receptor activation led to a marked (up to sixfold) stimulation of [S-35]GTP[S] binding when GDP or GTP were present in high (> 100-fold) excess of [S-35]GTP[S]. The increase in [S-35]GTP[S] binding caused by the chemotactic agonist was strictly dependent on the presence of Mg2+ and was significantly increased by Na+. Agonist-independent binding of [S-35]GTP[S] 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 [S-35]GTP[S]-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 [S-35]GTP[S] to, and thus the activation of, multiple (up to 20) G-proteins in native plasma membranes.