Pertussis toxin treatment in vivo is associated with a decline in G-protein beta-subunits.
The effects of pertussis toxin on the steady-state levels of G-protein alpha- and beta-subunits were investigated both in vitro and in vivo. The steady-state level Go alpha, a major substrate for pertussis toxin-catalyzed ADP-ribosylation, was unaltered by pertussis toxin treatment for periods up to 100 h for 3T3-L1 cells in culture or up to 3 days in vivo. In 3T3-L1 cells pertussis toxin treatment did not alter levels of Gs alpha-subunits; in S49 cells the level of Gs alpha-subunits declined moderately following by pertussis toxin treatment. The steady-state levels of G beta-subunits, in contrast, were found to decline to less than 50% of the normal cellular complement following pertussis toxin treatment in vitro and in vivo. Inhibitory control of adenylate cyclase, pertussis toxin-catalyzed ADP-ribosylation of Gi alpha and Go alpha, and the GTP-dependent shift in agonist-specific binding to beta-adrenergic receptors were attenuated or abolished within 5 h of pertussis toxin treatment, representing "early" effects of the toxin. Stimulatory regulation of adenylate cyclase, in contrast, displayed a progressive enhancement that was first observed 4 h after pertussis toxin treatment, increasing thereafter up until 100 h, the last time point measured. This progressive enhancement of the stimulatory pathway of adenylate cyclase was not manifest at the level of stimulatory receptors, since the Kd and Bmax for one such receptor, the beta-adrenergic receptor, were shown to be unaltered in toxin-treated cells. Furthermore, the potentiation of stimulation of adenylate cyclase was observed in cells stimulated by the beta-adrenergic agonist isoproterenol and PGE1 alike. The progressive enhancement of the stimulatory pathway correlated best with the decline in G beta-subunit levels that occurs following pertussis intoxication. The changes in both of these parameters occur "late" (12-48 h), as compared to the early events that occur within 5 h. Pertussis toxin action appears to be composed of two, temporally distinct, groups of effects. Pertussis toxin-catalyzed ADP-ribosylation of G alpha-subunits, attenuation of the inhibitory regulation of adenylate cyclase, and attenuation of the ability of GTP to induce an agonist-specific shift in receptor affinity are members of the early group of effects. The second group of late effects includes the decline in G beta-subunit levels and the progressive enhancement of the stimulatory pathway of adenylate cyclase. This enhanced stimulatory control at these later times cannot be explained by the attenuation of the inhibitory pathway occurring early, but rather appears as G beta-subunit levels decline.
Watkins
D C
DC
Department of Pharmacological Sciences, State University of New York, Stony Brook 11794-8651.
Northup
J K
JK
Malbon
C C
CC
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D005459:Fluorides / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology; D006801:Humans; D008114:Liver Neoplasms, Experimental; D046911:Macromolecular Substances; D051379:Mice; D037342:Pertussis Toxin; D010566:Virulence Factors, Bordetella / Q000633:toxicity*
D037361:Adenylate Cyclase Toxin; D006150:Guanine Nucleotides; D046911:Macromolecular Substances; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D005459:Fluorides