Localization of G alpha 0 to growth cones in PC12 cells: role of G alpha 0 association with receptors and G beta gamma

Abstract

The heterotrimeric G protein G0 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 beta gamma-subunit was investigated. The attachment of the beta gamma-subunit to the membrane depends on the isoprenylation of the gamma-subunit. The drug lovastatin blocks isoprenylation by inhibiting a key enzyme in the biosynthetic pathway. After treatment of PC12 cells with 10 microM lovastatin for 48 hours 50% of the beta gamma-subunits were cytosolic compared with 100% membrane bound beta gamma in control cells, as determined by cell fractionation, gel electrophoresis and western blot. Addition of 200 microM mevalonic acid reverses this effect. However, lovastatin affects neither the membrane attachment of alpha 0 nor its localization to the growth cones as determined by immunohistochemistry. This suggests that the localization and retention of alpha 0 are independent of the membrane attachment of the full complement of beta gamma-subunits. Second, pertussis toxin was used to block the interaction between alpha 0 and receptors. PC12 cells were treated with 0.1 microgram/ml pertussis toxin prior to and during nerve growth factor-induced differentiation. In vitro [32P]ADP-ribosylation confirmed that alpha 0 and alpha i were completely ADP-ribosylated by this treatment. The ADP-ribosylation by pertussis toxin did not interfere with neurite outgrowth. The localization of alpha 0 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 alpha 0 to growth cones.