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  • Title: In vivo pertussis toxin administration: effects on the function and levels of Gi alpha proteins and their messenger ribonucleic acids.
    Author: Ramkumar V, Stiles GL.
    Journal: Endocrinology; 1990 Feb; 126(2):1295-304. PubMed ID: 2105204.
    Abstract:
    Pertussis toxin ADP-ribosylates and functionally inactivates a number of guanine nucleotide-binding proteins (G-proteins), including the inhibitory G-protein, Gi. Once ADP-ribosylated, the subsequent duration of inactivation and fate of these proteins in cells are unknown. In the present study, therefore, we have attempted to answer some of these questions in an in vivo rat adipocyte model. Our results indicate a rather prolonged (greater than 2 weeks) inactivation of Gi, as evidenced by loss of R-phenylisopropyladenosine-mediated inhibition of adenylate cyclase activity in rat adipocyte membranes after administration of the toxin in vivo. This was associated with a complete loss of pertussis toxin ADP-ribosylatable substrates on days 3 and 15, with only partial recovery by day 28 after initiation of treatment. Specific antibodies used to quantitate the levels of alpha i1- and alpha 12-subunits of alpha i indicate a 2-fold increase in the levels of these proteins by day 3, followed by a progressive decrease and subsequent recovery in these subunits to 53%, 47%, and 87% of the control value on days 15, 20, and 28, respectively. No change in the levels of alpha s was observed at all time points tested. The levels of mRNA encoding the alpha i proteins were measured using specific oligonucleotide probes to determine whether changes in the levels of these transcripts could explain the observed regulation of alpha i. At the early time point (day 3), there was no alteration in the mRNA for alpha 3, alpha i1, alpha i2, and alpha i3 in fat pads from control and treated rats. However, the levels of the alpha i1 transcript decreased slightly in the treated groups on day 15-20 by a maximum of 33% and partially recovered by day 28. No changes in the alpha i2, alpha i3, alpha s, or tubulin transcripts were observed over the same treatment period. These results provide the first evidence that ADP ribosylation of the alpha i proteins by pertussis toxin leads to significant alterations in their levels. Furthermore, alterations in the levels of alpha i proteins (except alpha i1) do not appear to result from transcriptional regulation.
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