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 Gi1, Gi2 and Gi3 have been shown to inhibit adenylyl cyclase in transfected cells. However, Gi3 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 Gi3 (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.
Hermouet
S
S
Molecular Pathophysiology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
de Mazancourt
P
P
Spiegel
A M
AM
Farquhar
M G
MG
Wilson
B S
BS
D016475:3T3 Cells; D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D014162:Transfection
D000067956:Adenylyl Cyclase Inhibitors; D000242:Cyclic AMP; D019204:GTP-Binding Proteins