Abstract
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.
Highlights
A 40-kDa protein, in addition to the a-subuofniGts, brane-associated GTP-binding proteins (G proteins)’ are (a GTP-binding protein involved in adenylate cyclase present in a variety of vertebrate cellsw, as j3*P]ADP-ribosylated bycholera review)
This occurs in intact HL-60 cells as well as in HL-60 membrane preparations only if Gi is activated by stimulationof coupled fMLP receptors
Cholera Toxin-catalyzAedDP-ribosylation of IAP Substrate G Protein is uncoupling of the IAP-modified Gi.' from fMLP receptors
Summary
NAD, 2.5 mM MgC12, 10 p~ fMLP, and theindicated concentrations of guanine nucleotides. A marked difference between the two toxin- incubation of the membranes isolated therefrom with the catalyzed ADP-ribosylations was observed for the effect of same toxin and [32P]NAD(compare lanes 2 with 1 in Fig. GTPyS thereon (Fig. 4B). Induced ADP-ribosylationat any concentration employed; it Fig. 5A shows that cholera toxin-induced and fMLP-supcaused gradualinhibition as theconcentration was increased ported ADP-ribosylation of Gi-a in membranes was inbeyond 0.1 p ~ A.n additional disparity between IAP- and hibited markedly by prior exposure of the membrane-donor cholera toxin-induced ADP-ribosylations of Gi-a was their HL-60 cells to thesame toxin and chemotactic peptide The inhibition was much less when fMLP was Cholera Toxin-catalyzAedDP-ribosylation of IASPubstrate G Protein
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