Involvement of ADP-ribosylation factor 1 in cholera toxin-induced morphological changes of Chinese hamster ovary cells.
ADP-ribosylation factor 1 (ARF1) was originally found as a cofactor in CT-catalyzed ADP-ribosylation of Galpha(s) but is now known to participate in vesicle trafficking. We asked whether ARF1 function in vesicular trafficking is necessary for CT-induced morphological changes in Chinese hamster ovary (CHO) cells, which result from increased intracellular cAMP. Brefeldin A treatment of cells suppressed CT action, confirming a requirement for Golgi integrity. Overexpression of a GFP-ARF1 fusion protein did not affect the morphological changes induced by CT, but changes were reduced in cells overexpressing guanine nucleotide exchange-defective ARF1(T31N) or GTP hydrolysis-deficient ARF1(Q71L) mutants. In cells expressing these mutants, 8-bromo-cAMP induced changes similar to those seen in cells transfected with ARF1 or vector. Inhibition of CT action was specific for mutants of ARF1 and not reproduced by analogous mutants of ARF5 or ARF6. ARF1(Q71L) was mostly colocalized with betaCOP, but ARF5(Q71L) less so. ARF6(Q67L) did not colocalize with betaCOP and was partially associated with the plasma membrane. These data are consistent with the conclusion that ARF1 influenced CT action in cells by its specific function in the vesicular transport pathway used by CT to travel from plasma membrane to Golgi to ER.
Morinaga
N
N
Second Department of Microbiology, Chiba University School of Medicine, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan. moringa@med.m.chiba-u.ac.jp
Kaihou
Y
Y
Vitale
N
N
Moss
J
J
Noda
M
M
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D020823:ADP-Ribosylation Factor 1 / Q000502:physiology*; D000818:Animals; D001483:Base Sequence; D020126:Brefeldin A / Q000494:pharmacology; D016466:CHO Cells; D002772:Cholera Toxin / Q000494:pharmacology*; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers; D005260:Female; D010053:Ovary / Q000166:cytology / Q000187:drug effects*; D021381:Protein Transport
D017931:DNA Primers; D020126:Brefeldin A; D015124:8-Bromo Cyclic Adenosine Monophosphate; D002772:Cholera Toxin; D020823:ADP-Ribosylation Factor 1