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VICKRAM RAMKUMAR, GARY L. STILES, In Vivo Pertussis Toxin Administration: Effects on the Function and Levels of Giα Proteins and Their Messenger Ribonucleic Acids, Endocrinology, Volume 126, Issue 2, 1 February 1990, Pages 1295–1304, https://doi.org/10.1210/endo-126-2-1295
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Pertussis toxin ADP-ribosylates and functionally inactivates a number of guanine nucleotide-binding proteins (G-proteins), including the inhibitory G-protein, Gi. Once ADPribosylated, 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 (>2 weeks) inactivation of Gi, as evidenced by loss of Rphenylisopropyladenosine- 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 αi1- and αi2- subunits of αi-, 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 α8 was observed at all time points tested. The levels of mRNA encoding the αi proteins were measured using specific oligonucleotide probes to determine whether changes in the levels of these transcripts could explain the observed regulation of αi. At the early time point (day 3), there was no alteration in the mRNA for α8, αi1,αi2, and αi3 in fat pads from control and treated rats. However, the levels of the αi1 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 αi2, αi3, α8, or tubulin transcripts were observed over the same treatment period. These results provide the first evidence that ADP ribosylation of the αi1, proteins by pertussis toxin leads to significant alterations in their levels. Furthermore, alterations in the levels of αi proteins (except αi1) do not appear to result from transcriptional regulation. (Endocrinology126: 1295–1304, 1990)
