HIGH-LEVEL EXPRESSION OF TRANSFECTED G-PROTEIN ALPHA-I3 SUBUNIT IS REQUIRED FOR PLASMA-MEMBRANE TARGETING AND ADENYLYL CYCLASE INHIBITION IN NIH 3T3 FIBROBLASTS

The alpha subunits of pertussis toxin-sensitive G proteins G(i1), G(i2) and G(i3) have been shown to inhibit adenylyl cyclase in transfected cells. However, G(i3) has recently been associated with protein transport and localized to the Golgi apparatus in a number of cell lines, rather than to the plasma membrane. We studied NIH 3T3 clones stably expressing different levels of a constitutively activated mutant of the alpha subunit of G(i3) (alpha(i3)-Q204L). Transfected alpha(i3) subunits were localized to the Golgi apparatus in all NIH 3T3 clones. In clones expressing alpha(i3)-Q204L at high levels, alpha(i3) subunits were also localized to the plasma membrane. Those clones which demonstrated expression of alpha(i3) at the plasma membrane showed a 40% to 60% inhibition of forskolin-induced cAMP accumulation. Transfected NIH 3T3 clones in which plasma membrane alpha(i3) was undetectable, did not show inhibition of forskolin-induced cAMP accumulation. These data suggest that, unless high expression is achieved in transfected cells, alpha(i3) is targeted predominantly to the Golgi, not to the plasma membrane, and does not control adenylyl cyclase activity in NIH 3T3 fibroblasts.