Characterization of αs-immunoreactive ADP-ribosylated proteins in postmortem human brain

Abstract

ADP-ribosylation of the stimulatory G protein alpha subunit, alpha(s), has been demonstrated in a number of different mammalian tissues. However, little is known about the occurrence and role of this process in modifying alpha(s) levels/function in human brain. In the present study, endogenous and cholera toxin (CTX)-catalyzed [32P]ADP-ribosylated products were characterized in postmortem human temporal cortex by (1) immunoprecipitation with alpha(s) antisera (RM/1), (2) comparisons of immunoblots and autoradiograms of the [32P]ADP-ribosylated products, and (3) limited protease digestion. Of the three major endogenous [32P]ADP-ribosylated products (48, 45, and 39 kDa) in postmortem brain, the 48-kDa and 45-kDa bands were clearly identified as alpha(s-L) (long isoform) and alpha(s-S) (short isoform), respectively. RM/1 immunoprecipitated the 39-kDa [32P]ADP-ribosylated protein, and overlays of immunoblots and autoradiograms showed that this product corresponded to an alpha(s)-like-immunoreactive protein. Furthermore, limited protease digestion of the 39-kDa endogenous [32P]ADP-ribosylated band generated peptide fragments similar to both endogenous and CTX-catalyzed [32P]ADP-ribosylated alpha(s-S). Two major CTX-catalyzed [32P]ADP-ribosylated products were also identified as alpha(s-L) (52 kDa) and alpha(s-S) (45 kDa). These findings clearly demonstrate that alpha(s) is a substrate for endogenous and CTX-catalyzed [32P]ADP-ribosylation in postmortem human brain. Furthermore, a lower molecular weight alpha(s)-like immunoreactive protein is also expressed in human brain and is a substrate for endogenous but not CTX-catalyzed [32P]ADP-ribosylation.

Altered CTX‐catalyzed and endogenous [32P]ADP‐ribosylation of stimulatory G protein αs isoforms in postmortem bipolar affective disorder temporal cortex

“…Details of patient histories, including cause of death and antemortem drug treatment, were obtained from available medical records as described previously (Rahman et al, 1997). Postmortem brain was dissected as detailed elsewhere (Andreopoulos et al, 1999). Brain regions assayed included temporal (middle temporal gyrus, Brodmann area 21, inferior temporal gyrus, Brodmann area 20), and occipital (lips of the calcarine sulcus, Brodmann area 17) cortex and cerebellum.…”

“…Endogenous [ 32 P]ADP‐ribosylation was carried out on postmortem human brain tissues as described previously (Andreopoulos et al, 1999). Briefly, after thawing, tissues (20–25 mg) were homogenized in 2 vol of ice‐cold buffer containing 20 mM Tris (pH 7.0), 1 mM EDTA, 0.5 mM BZ and 0.001% SBTII.…”

“…Samples were then incubated with 10 μl 100 mM NEM for 15 min at 21 °C and then 100 μl of gel‐loading buffer (250 mM Tris‐HCl [pH 6.8], 4% SDS, 5 mM EGTA, 3 mM EDTA, 10% mercaptoethanol, and 10% glycerol) were added and the samples heated for 3 min at 90°C. The denatured sample proteins were separated electrophoretically by SDS‐polyacrylamide gel electrophoresis (PAGE) on 10% gels that were dried subsequently for 2 hr at 80°C under vacuum (99.9 kPa) and autoradiographed at −70°C (18–72 hr exposures) (Andreopoulos et al, 1999).…”

“…Samples were then incubated at 30°C for 60 min and reactions terminated with 3 μl TCA. After washing, samples were processed subsequently for SDS‐PAGE and autoradiography as described previously (Andreopoulos et al, 1999). Autoradiographs were quantified by densitometric image analysis (MCID Image Analysis System, St. Catherines, ON).…”

“…We tested this hypothesis by comparing the extent to which α s isoforms can be [ 32 P]ADP‐ribosylated in postmortem temporal and occipital cortex (two regions reported previously to show elevated α s levels) and cerebellum from BD and nonpsychiatric comparison subjects. Endogenous and CTX‐catalyzed [ 32 P]ADP‐ribosylation of α s have been characterized recently in cerebral cortex of postmortem human brain (Andreopoulos et al, 1999). CTX catalyzed the ADP‐ribosylation of both α s‐L (52 kDa) and α s‐S (45 kDa) whereas three major endogenously ADP‐ribosylated α s products were detected, corresponding to the long and short forms of α s , and a novel α s subtype likely representing a N‐terminal truncated α s spliced variant described previously (Ishikawa et al, 1990).…”