Transience of antidepressant‐induced Gαs signaling potentiation revealed by high‐throughput real‐time cAMP accumulation

Abstract

Gαs is found at high concentrations in cholesterol rich membrane microdomains known as lipid rafts. These domains suppress coupling between Gαs and effectors such as adenylyl cyclase (AC), leading to decreased cAMP. Antidepressant drugs with diverse mechanisms of action have reliably shown the ability to promote the translocation of Gαs out of lipid rafts in cellular and animal models, leading to increased cAMP, pCREB and BDNF. In cells from depressed subjects Gαs is found at higher concentrations in lipid rafts. Importantly, this difference is eliminated in individuals who respond positively to antidepressant drug treatment, while in non‐responders the increased raft localization of Gαs is maintained. This differential localization may allow Gαs translocation out of lipid rafts to serve as a novel biomarker to screen for effective antidepressants.Although antidepressant drugs have been shown to gradually accumulate in lipid rafts, the relevant binding site, or sites, remain unknown. Notably, the S‐stereoisomer of the selective serotonin reuptake inhibitor citalopram binds to these raft regions, even in cells lacking the serotonin transporter, while the non‐antidepressant R‐stereoisomer does not. This suggests a novel, stereospecific protein target for antidepressant drugs may exist within lipid rafts. Little is known about the reversibility of this process, or whether antidepressant drug withdrawal itself produces unique signaling effects.In this study we use a combination of techniques including live cell cAMP accumulation analysis, fluorescence recovery after photobleaching (FRAP) of GFP tagged Gαs, and detection of antidepressant drug concentration via mass spectrometry. This allows determination of both the effects of extended antidepressant treatment on Gαs localization and signaling, as well as the effect of antidepressant withdrawal. Agents that increase cAMP concentrations, such as forskolin, produce greater effects following antidepressant treatment due to Gαs translocation out of lipid rafts and into closer association with AC. Gαs mobility within the membrane is also inhibited following antidepressant treatment due to formation of this Gαs/AC complex, and this reduction in mobility is observed as an increased FRAP half‐time with GFP tagged Gαs. The localization of the antidepressant drugs themselves are also quantified via mass spectrometry following separation of raft vs. non‐raft membrane regions.Analyzing these effects both during antidepressant treatment and following removal of the drug provides insight not only into the ways these drugs regulate cellular processes, but also how the cell responds to their withdrawal. It appears that antidepressant drug removal not only reverses the effects seen with extended treatment, but can also produce a lasting suppression of these processes. This work provides the next steps towards determining a binding site for antidepressants within lipid rafts, and will lead to the development of a more realistic screen for antidepressant drug development.Support or Funding InformationFunding: NIH R01AT009169; VA BX001149This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.