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]GTP[S]) to native HL-60 membranes. Agonist stimulation of [35S]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 [35S]GTP[S] binding when GDP or GTP were present in high (greater than 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. Comparison 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.
Gierschik
P
P
Pharmakologisches Institut, Universität Heidelberg, Federal Republic of Germany.
Moghtader
R
R
Straub
C
C
Dieterich
K
K
Jakobs
K H
KH
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006098:Granulocytes / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000502:physiology*; D012965:Sodium Chloride / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D006153:Guanosine Diphosphate; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012965:Sodium Chloride; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019204:GTP-Binding Proteins; D008274:Magnesium