Pertussis toxin-sensitive G protein alpha-subunits: production of monoclonal antibodies and detection of differential increases on differentiation of PC12 and LA-N-5 cells.
Monoclonal antibodies were produced that are specific for the three major pertussis toxin-sensitive G protein alpha-subunits present in mammalian brain--alpha o, alpha i1, and alpha i2--using purified bovine brain G proteins, purified rat brain G proteins, and purified recombinant alpha i2, respectively. These monoclonal antibodies were used to monitor changes in the concentrations of the three G protein alpha-subunits during differentiation of PC12 cells and human neuroblastoma LA-N-5 cells. In PC12 cells, levels of alpha i1 but not alpha i2 increased during nerve growth factor-induced differentiation. In contrast, alpha i2 but not alpha i1 increased when LA-N-5 cells were differentiated with retinoic acid. The concentration of alpha o increased in both cell lines during differentiation. Electrophoretic resolution of alpha o subtypes revealed that although alpha o2 was the major subtype in undifferentiated cells, only the concentration of alpha o1 increased during differentiation of both PC12 and LA-N-5 cells. The level of 43-kDa growth-associated protein, a protein known to associate with alpha o, increased similarly to that of alpha o1. ADP-ribosylation of alpha o, alpha i1, and alpha i2 with pertussis toxin did not alter the reactivities of the monoclonal antibodies, but toxin treatment of cells reduced the concentrations of each protein after 24 h. There was no change in the concentration of alpha q, which is not ADP-ribosylated by pertussis toxin. Thus, these new monoclonal antibodies enabled the detection of differential increases in subtypes of alpha i and alpha o associated with neuronal differentiation.
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Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham 35294-0017.
Mumby
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SM
Greenwood
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Jope
R S
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D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002454:Cell Differentiation; D019922:GAP-43 Protein; D019204:GTP-Binding Proteins / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000378:metabolism; D009414:Nerve Growth Factors / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009447:Neuroblastoma; D016716:PC12 Cells; D037342:Pertussis Toxin; D051381:Rats; D013574:Synaptosomes / Q000378:metabolism; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins