grantor:
University of TorontoThis study examined whether the stimulatory G protein à subunit, Ãs, undergoes ADP-ribosylation in postmortem human brain, and whether disturbances in this pathway contribute to the elevated à s levels reported in bipolar affective disorder (BD). Endogenous, and cholera toxin (CTX)-catalyzed [32P]ADP-ribosylated à s were characterized in postmortem temporal cortex by immunoprecipitation, and overlay comparisons of autoradiograms and Western blots of the [ 32P]ADP-ribosylated Ãs isoforms. Endogenous and CTX-catalyzed [32P]ADP-ribosylated Ãs in temporal, occipital and cerebellar cortices of BD, and age/postmortem delay-matched controls were then separated by SDS-PAGE and autoradiograms quantified by densitometry. à s protein levels were determined by Western blotting. Two major endogenous [32P]ADP-ribosylated products (48 kDa and 45 kDa) were identified as Ãs-L and à s-S, respectively. Immunoprecipitation with Ãs specific antibody revealed a third endogenous [32P]ADP-ribosylated protein (39 kDa). Resolution by SDS-PAGE and limited protease digestion supported that this product corresponded to an Ãs-like protein, possibly a previously reported N-terminal truncated Ãs splice variant. Two major CTX-catalyzed [32P]ADP-ribosylated products were also identified as Ãs-L (52 kDa) and Ãs-S (45 kDa). No differences were observed in either endogenous, and CTX-catalyzed [32P]ADP-ribosylation of Ãs-L in BD temporal cortex. However, Ãs-L immunolabeling was significantly elevated and correlated inversely with endogenously [32P]ADP-ribosylated à s-L in this brain region suggesting reduced clearance of à s-L through the ADP-ribosylation pathway in BD temporal cortex. Reduced CTX-catalyzed [32P]ADP-ribosylation of Ãs-S in BD temporal cortex also supports this interpretation. Moreover, lack of differences in endogenous [32P]ADP-ribosylation of myelin basic protein (MBP) in temporal cortex of BD compared with controls suggests that elevations of Ãs are not due to underlying disturbances in ADP-ribosyltransferase activity but rather may reflect changes in disposition or availability that are specific to Ãs. Endogenous [32P]ADP-ribosylation of the 39 kDa à s-like protein was reduced only in BD temporal cortex. This decrement correlated with lithium concentrations suggesting that lithium may modify the activity of specific ADP-ribosyltransferases in this region. While the results of this study demonstrate that Ãs is a substrate for ADP-ribosylation and that a 39 kDa Ãs-like protein is also expressed in human brain, the findings do not support the hypothesis that alterations in ADP-ribosyltransferase activity per se are responsible for the higher Ãs levels seen in BD cerebral cortex regions.Ph.D
