Dynamic Interactions Between The G Protein, G Alpha S, Lipid Rafts And Adenylyl Cyclase Are Altered By Chronic Antidepressants, Cholesterol Removal, Cytoskeletal Modification And Acylation Or Prenylation

Numerous studies suggest that chronic, but not acute antidepressant treatment increases physical coupling between Gαs and adenylyl cyclase resulting in increased cAMP signaling. Biochemical studies have demonstrated that this is due, at least in part, to the translocation of Gαs from lipid rafts into non‐raft membrane fractions where it is more effectively coupled to adenylyl cyclase. Although this effect is highly reproducible, 4° C lipid raft extractions require millions of cells and are criticized because they may not reflect the same physiology as living cells (whether or not detergents are used to extract lipid raft fractions). Here we describe a novel monomeric variant of our previously developed GFP‐Gαs fusion protein. The monomeric variant has allowed stable transfection of Gαs‐GFP into C6 glioma cells from which clones have been selected. Gαs‐GFP not only relocalizes out of lipid rafts similar to endogenous Gαs in response to antidepressant treatment, but can be used to study individual cells under physiologic conditions. Using Fluorescent Recovery After Photobleaching (FRAP), we observed that treatment with antidepressants, which decrease GFP‐Gαs raft association, but increase Gαs association with adenylyl cyclase, also increase recovery time. This appears due to the very slow recovery of adenylyl cyclase. Mutations to Gαs that render it cytosolic (palmitoylation deficient) shorten recovery time and mutations that augment membrane association retard recovery further. Disruption of lipid rafts or microtubules have a similar effect, but actin disruption does not alter Gαs FRAP. It is suggested that both rafts and adenylyl cyclase anchor Gαs, and removal from the former facilitates the latter. Grant Funding Source : VA BX001149 and NIH MH067631