Upon binding hormones or drugs, many G protein-coupled receptors are internalized, leading to receptor recycling, receptor desensitization, and down-regulation. Much less understood is whether heterotrimeric G proteins also undergo agonist-induced endocytosis. To investigate the intracellular trafficking of G␣s, we developed a functional G␣s-green fluorescent protein (GFP) fusion protein that can be visualized in living cells during signal transduction. C6 and MCF-7 cells expressing G␣s-GFP were treated with 10 M isoproterenol, and trafficking was assessed with fluorescence microscopy.

Recent in vivo and in vitro studies have demonstrated that Gs␣ migrates from a Triton X-100 (TX-100)-insoluble membrane domain (lipid raft) to a TX-100-soluble nonraft membrane domain in response to chronic, but not acute, treatment with tricyclic or selective serotonin reuptake inhibitor antidepressants. This migration resulted in a more facile association with adenylyl cyclase. Our hypothesis is that Gs␣ may be ensconced, to a greater extent, in lipid rafts during depression, and that one action of chronic antidepressant treatment is to reverse this. In this postmortem study, we examined Gs␣ membrane localization in the cerebellum and prefrontal cortex of brains from nonpsychiatric control subjects and suicide cases with confirmed unipolar depression. Sequential TX-100 and TX-114 detergent extractions were performed on the brain tissue. In the cerebellum, the ratio of TX-100/TX-114-soluble Gs␣ is ϳ2:1 for control versus depressed suicides. Results with prefrontal cortex samples from each group demonstrate a similar trend. These data suggest that depression localizes Gs␣ to a membrane domain (lipid rafts) where it is less likely to couple to adenylyl cyclase and that antidepressants may upregulate Gs␣ signaling via disruption of membrane microenvironments. Raft localization of Gs␣ in human peripheral tissue may thus serve as a biomarker for depression and as a harbinger of antidepressant responsiveness.

Previous studies demonstrated that Gsa migrates from a Triton X-100 (TTX-100) insoluble membrane domain to a TTX-100 soluble membrane domain in response to chronic treatment with the antidepressants desipramine and fluoxetine. Antidepressant treatment also causes a Gsa redistribution in cells as seen by confocal microscopy. The current studies have focused on examining the possibility that the association between Gsa and the plasma membrane and/or cytoskeleton is altered in response to antidepressant treatment, and that this is relevant to both Gsa redistribution and the increased coupling between Gsa and adenylyl cyclase seen after chronic antidepressant treatment. Chronic treatment of C6 cells with two fuctionally and structurally distinct antidepressants, desipramine and fluoxetine, decreased the Gsa content of TTX-100 insoluble membrane domains by as much as 60%, while the inactive fluoxetine analog LY368514 had no effect. Disruption of these membrane domains with the cholesterol chelator methyl-b-cyclodextrin altered the localization of many proteins involved in the cAMP signaling cascade, but only Gsa localization was altered by antidepressant treatment. In addition, microtubule disruption with colchicine elicited the movement of Gsa out of detergent-resistant membrane domains in a manner identical to that seen with antidepressant treatment. The data presented here further substantiate the role of Gsa as a major player in antidepressant-induced modification of neuronal signaling and also raise the possibility that an interaction between Gsa and the cytoskeleton is involved in this process.

Abstract:Results from previous studies suggested that chronic treatment of rats or C6 glioma cells with antidepressants augments the coupling between G s and adenylyl cyclase. As these effects on C6 glioma cells are seen in the absence of presynaptic input, several antidepressant drugs may have a direct "postsynaptic" effect on their target cells. It was hypothesized that the target of antidepressant action was some membrane protein that may regulate coupling between G proteins and adenylyl cyclase. To test this, C6 glioma cells were treated with amitriptyline, desipramine, iprindole, or fluoxetine for 3 days. Chlorpromazine served as a control for these treatments. Membrane proteins were extracted sequentially with Triton X-100 and Triton X-114 from C6 glioma cells. Triton X-100 extracted more G s␣ in membranes prepared from antidepressant-treated C6 glioma cells than from control groups. In addition, cell fractionation studies revealed that the amount of G s␣ in caveolin-enriched domains was reduced after antidepressant treatment and that adenylyl cyclase comigrated with G s␣ in the gradients. These data suggest that some postsynaptic component that increases availability of G s to activate effector molecules, such as adenylyl cyclase, might be a target of antidepressant treatment.