Pertussis toxin-catalyzed ADP-ribosylation of Gi-2 and Gi-3 in CHO cells is modulated by inhibitors of intracellular trafficking.
In previous studies, an in vitro ADP-ribosylation assay was developed to quantitatively evaluate the in vivo ADP-ribosylation of eukaryotic target proteins in intact Chinese hamster ovary (CHO) cells by pertussis toxin (PT). Immunoblot analysis identified the two PT-sensitive target proteins in CHO cells as Gi-2 and Gi-3. In this in vitro ADP-ribosylation assay, the ability of PT and ADP-ribosylate Gi-2 and Gi-3 intact CHO cells was not inhibited by cytochalasin D but was inhibited by chloroquine, monensin, and nocodazole. These data implicated the involvement of a cytochalasin D-independent endocytic mechanism, a pH-sensitive step, and microtubules in the ADP-ribosylation of Gi-2 and Gi-3 by PT in intact CHO cells. Preincubation of CHO cells with cycloheximide, at concentrations that reduced protein synthesis by > 95%, did not inhibit the ability of PT to ADP-ribosylate Gi-2 and Gi-3. Control experiments showed that these agents did not affect either the ability of PT to directly ADP-ribosylate the heterotrimeric G protein, Gt, or the binding of PT to CHO cells, except that monensin slightly inhibited the binding of PT to CHO cells. These results are consistent with a model in which PT is internalized by receptor-mediated endocytosis, probably via a cytochalasin D-independent pathway, which involves intracellular trafficking through late endosomes and the Golgi apparatus. An alternative model predicts the presence of a eukaryotic factor that traffics within cells via this pathway and is required by PT to ADP-ribosylate Gi proteins.
Xu
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Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226, USA.
Barbieri
J T
JT
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002738:Chloroquine / Q000494:pharmacology; D006224:Cricetinae; D015638:Cytochalasin D / Q000494:pharmacology; D004705:Endocytosis; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008985:Monensin / Q000494:pharmacology; D015739:Nocodazole / Q000494:pharmacology; D037342:Pertussis Toxin; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D015638:Cytochalasin D; D002738:Chloroquine; D008985:Monensin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D015739:Nocodazole