ABSTRACT
The heterotrimeric G protein Go is highly enriched in the growth cones of neuronal cells and makes up 10% of the membrane protein of growth cones from neonatal rat brain. We have used PC12 cells, a cell line that differentiates to a neuron-like phenotype, as a model with which to study the mechanism of G protein localization. First, the role of the βγ-subunit was investigated. The attachment of the βγ-subunit to the membrane depends on the isoprenylation of the γ-subunit. The drug lovastatin blocks isoprenylation by inhibiting a key enzyme in the biosynthetic pathway. After treatment of PC12 cells with 10 μM lovastatin for 48 hours 50% of the βγ-subunits were cytosolic compared with 100% membrane bound βγ in control cells, as determined by cell fractionation, gel electrophoresis and western blot. Addition of 200 μM mevalonic acid reverses this effect. However, lovastatin affects neither the membrane attachment of αo nor its localization to the growth cones as determined by immunohistochemistry. This suggests that the localization and retention of αo are independent of the membrane attachment of the full complement of βγ-subunits. Second, pertussis toxin was used to block the interaction between αo and receptors. PC12 cells were treated with 0.1 μg/ml pertussis toxin prior to and during nerve growth factor-induced differentiation. In vitro [32P]ADP-ribosylation confirmed that αo and αi were completely ADP-ribosylated by this treatment. The ADP-ribosylation by pertussis toxin did not interfere with neurite outgrowth. The localization of αo to the growth cones was indistinguishable from that in untreated cells. We conclude that G protein-receptor interaction is not necessary for the distribution of αo to growth cones.
