title: Cholera toxin differentially decreases membrane levels of alpha and beta subunits of G proteins in NG108-15 cells.
abstract: Treatment of NG108-15 neuroblastoma x glioma cells (24 h) with cholera toxin (0.1-10 micrograms/ml) resulted in a concentration-dependent reduction of the membrane levels of subunits of GTP-binding regulatory proteins (G proteins), as determined by quantitative immunoblot procedures. The extent of reduction differed for different types of subunits: the levels of Go alpha and G beta 1 were reduced by 40-50%, whereas those of G alpha common immunoreactivity and Gi2 alpha were only reduced by 10-20% following treatment with 10 micrograms/ml cholera toxin. This effect of the toxin could not be mimicked by incubation with the resolved B oligomer of cholera toxin, nor by exposure of cells to agents able to raise the intracellular levels of cAMP. Basal adenylate cyclase was stimulated in a biphasic manner by cholera toxin, being stimulated at low concentrations (0.01-10 ng/ml) and then decreased at high (0.1-10 micrograms/ml) concentrations. Thus, the down regulation of G-protein subunits produced by cholera toxin requires its (ADP-ribosyl)transferase activity but does not result from a cAMP-mediated mechanism. The toxin-mediated decrease of Go alpha in the membrane was correlated with a diminution of opioid-receptor-mediated stimulation of high-affinity GTPase activity, suggesting that opioid receptors interact with Go in native membranes of NG108-15 cells. Northern-blot analysis of cytoplasmic RNA prepared from cells treated with cholera toxin showed that the levels of mRNA coding for G beta 1 did not change. Thus, the cholera-toxin-induced decrease of G-protein subunits may not result from an alteration in mRNA levels, but may involve a direct effect of the toxin on the process of insertion and/or clearance of G proteins into and/or from the membrane. These data indicate that cholera toxin, besides catalyzing the ADP-ribosylation of Gs and Gi/Go types of G proteins, can also reduce the steady state levels of Go alpha and G beta 1 subunits in the membrane and thus alter by an additional mechanism the function of inhibitory receptor systems.
authors.affiliation: Department of Neuropharmacology, Max-Planck-Institut für Psychiatric, Planegg-Martinsried, Federal Republic of Germany.
authors.forename: F J
authors.initials: FJ
authors.lastname: Klinz
authors.forename: T
authors.initials: T
authors.lastname: Costa
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000302:isolation & purification; D037361:Adenylate Cyclase Toxin; D000818:Animals; D015152:Blotting, Northern; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D002772:Cholera Toxin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000494:pharmacology; D004247:DNA / Q000302:isolation & purification; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification*; D005910:Glioma / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification*; D009447:Neuroblastoma; D037342:Pertussis Toxin; D012333:RNA, Messenger / Q000302:isolation & purification; D013963:Thyroid Hormones / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D010566:Virulence Factors, Bordetella
chemical_list: D037361:Adenylate Cyclase Toxin; D008565:Membrane Proteins; D012333:RNA, Messenger; D013963:Thyroid Hormones; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D004247:DNA; D002772:Cholera Toxin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins