Pertussis Toxin-Sensitive G Protein α-Subunits: Production of Monoclonal Antibodies and Detection of Differential Increases on Differentiation of PC12 and LA-N-5 Cells

Abstract: Monoclonal antibodies were produced that are specific for the three major pertussis toxin-sensitive G protein α-subunits present in mammalian brain—αo, αi1, and αi2—using purified bovine brain G proteins, purified rat brain G proteins, and purified recombinant αi2, respectively. These monoclonal antibodies were used to monitor changes in the concentrations of the three G protein α-subunits during differentiation of PC12 cells and human neuroblastoma LA-N-5 cells. In PC12 cells, levels of αi1 but not αi2 increased during nerve growth factor-induced differentiation. In contrast, αi2 but not αi1 increased when LA-N-5 cells were differentiated with retinoic acid. The concentration of αo increased in both cell lines during differentiation. Electrophoretic resolution of αo subtypes revealed that although αo2 was the major subtype in undifferentiated cells, only the concentration of αo1 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 αo, increased similarly to that of αo1. ADP-ribosylation of αo, αi1, and αi2 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 αq, which is not ADP-ribosylated by pertussis toxin. Thus, these new monoclonal antibodies enabled the detection of differential increases in subtypes of αi and αo associated with neuronal differentiation.