Signal amplification in HL‐60 granulocytes

Open Access
Publisher Website
Google Scholar
Abstract
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′‐[γ‐thio]triphosphate ([35S]GTP[S]) to native HL‐60 membranes. Agonist stimulation of [35S]GTP[S] association with the membrane was minimal ( 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 [35S]GTP[S] binding when GDP or GTP were present in high (> 100‐fold) excess of [35S]GTP[S]. The increase in [35S]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 [35S]GTP[S] and the increase due to the chemotactic agonist were markedly attenuated by both pertussis and cholera toxin. Comparision of the number of chemotactic‐peptide‐sensitive [35S]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 [35S]GTP[S] to, and thus the activation of, multiple (up to 20) G‐proteins in native plasma membranes.

This publication has 69 references indexed in Scilit: