High level expression of transfected G protein α_i3 subunit is required for plasma membrane targeting and adenylyl cyclase inhibition in NIH 3T3 fibroblasts

The α 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 α subunit of G_i3 (α_i3-Q204L). Transfected α_i3 subunits were localized to the Golgi apparatus in all NIH 3T3 clones. In clones expressing α_i3-Q204L at high levels, α_i3 subunits were also localized to the plasma membrane. Those clones which demonstrated expression of α_i3 at the plasma membrane showed a 40% to 60% inhibition of forskolin-induced cAMP accumulation. Transfected NIH 3T3 clones in which plasma membrane α_i3 was undetectable, did not show inhibition of forskolin-induced cAMP accumulation. These data suggest that, unless high expression is achieved in transfected cells, α_i3 is targeted predominantly to the Golgi, not to the plasma membrane, and does not control adenylyl cyclase activity in NIH 3T3 fibroblasts.