Characterization of the distribution of G alpha o in rat striatal synaptosomes and its colocalization with tyrosine hydroxylase.
Dopaminergic striatal synaptosomes can be detected and isolated with a fluorescence-activated cell sorter (FACS). In the present study, two antigens were detected simultaneously with primary antisera raised in different species and species-specific fluorescent secondary antibodies with different emission spectra. Double-label FACS analysis was used to determine whether tyrosine hydroxylase (TH) and the alpha subunit of Go (G alpha o) are colocalized in striatal synaptosomes. Rabbit antibodies generated against a synthetic fragment of G alpha o (corresponding to amino acids 22-35) combined with fluorescein-conjugated secondary antibodies were used to detect G alpha o-containing striatal synaptosomes. Preadsorption of G alpha o antiserum with the synthetic peptide antigen reduced labeling to the level obtained with preimmune serum. Approximately 65-75% of striatal synaptosomes were specifically labeled by G alpha o antiserum. Tyrosine hydroxylase-containing synaptosomes were detected with a mouse monoclonal antibody to TH and R-phycoerythrin-conjugated secondary antibody. They comprised 15-17% of total striatal synaptosomes. Double-label studies indicated that at least 50% of TH-containing synaptosomes also contained G alpha o. These findings suggest that G alpha o may not be a protein component of all striatal nerve terminals, and provide a basis for a role for G alpha o in signal transduction within subpopulations of intrinsic and afferent nerve terminals, including those of nigrostriatal dopamine neurons.
Center for Cell Biology, Sinai Research Institute, Detroit, Michigan 48235.
M E
ME
Wolf
J G
JG
Granneman
G
G
Kapatos
D000818:Animals; D015153:Blotting, Western; D003342:Corpus Striatum / Q000166:cytology / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D019204:GTP-Binding Proteins / Q000032:analysis*; D007106:Immune Sera; D008297:Male; D051381:Rats; D013574:Synaptosomes / Q000378:metabolism* / Q000648:ultrastructure; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis*
D007106:Immune Sera; D014446:Tyrosine 3-Monooxygenase; D019204:GTP-Binding Proteins