Chemotactic peptide receptor-supported ADP-ribosylation of a pertussis toxin substrate GTP-binding protein by cholera toxin in neutrophil-type HL-60 cells.
A 40-kDa protein, in addition to the alpha-subunits of Gs (a GTP-binding protein involved in adenylate cyclase stimulation), was [32P]ADP-ribosylated by cholera toxin (CT) in the membranes of neutrophil-like HL-60 cells, only if formyl Met-Leu-Phe (fMLP) was added to the ADP-ribosylation mixture. The 40-kDa protein proved to be the alpha-subunit of Gi serving as the substrate of pertussis toxin, islet-activating protein (IAP). No radioactivity was incorporated into this protein in membranes isolated from HL-60 cells that had been exposed to IAP. Gi-alpha purified from bovine brain and reconstituted into IAP-treated cell membranes was ADP-ribosylated by CT plus fMLP. Gi-alpha was ADP-ribosylated by IAP, but not by CT plus fMLP, in membranes from cells that had been pretreated with CT plus fMLP. When membrane Gi-alpha [32P]ADP-ribosylated by CT plus fMLP or IAP was digested with trypsin, the radiolabeled fragments arising from the two proteins were different from each other. These results suggest that CT ADP-ribosylates Gi-alpha in intact cells when coupled fMLP receptors are stimulated and that the sites modified by two toxins are not identical. CT-induced and fMLP-supported ADP-ribosylation of Gi-alpha was favored by Mg2+ and allow concentrations of GTP or its analogues but suppressed by GDP. The ADP-ribosylation did not occur at all, even in the presence of ADP-ribosylation factor that supported CT-induced modification of Gs, in phospholipid vesicles containing crude membrane extract in which Gi was functionally coupled to stimulated fMLP receptors. Thus, Gi activated via coupled receptors is the real substrate of CT-catalyzed ADP-ribosylation. This reaction may depend on additional factor(s) that are too labile to survive the process of membrane extraction.
Iiri
T
T
Department of Life Science, Faculty of Science, Tokyo Institute of Technology Yokohama, Japan.
Tohkin
M
M
Morishima
N
N
Ohoka
Y
Y
Ui
M
M
Katada
T
T
D020727:ADP-Ribosylation Factors; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D037361:Adenylate Cyclase Toxin; D000818:Animals; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002417:Cattle; D002454:Cell Differentiation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002772:Cholera Toxin / Q000494:pharmacology*; D003600:Cytosol / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000378:metabolism*; D037342:Pertussis Toxin; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000502:physiology*; D010566:Virulence Factors, Bordetella / Q000378:metabolism* / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D002352:Carrier Proteins; D006150:Guanine Nucleotides; D008565:Membrane Proteins; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D009240:N-Formylmethionine Leucyl-Phenylalanine; D002772:Cholera Toxin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors