Dynamic Interactions Between G Proteins and Plasma Membrane Nanodomains: Effects of Antidepressants and PUFAs

Cellular neurotransmission is a complex array of processes vital for transforming chemical signals into behavior. Aside from ion channels, no other class of receptors is more prominent to this process than G-protein coupled receptors. These receptors transmit signals in cell throughout the body, constitute the largest family of genes and are a target for a plurality of drugs. Most GPCRs, however, would be at least partially neutralized without their coupled G-proteins for which their name derives. This dissertation focuses on the complex regulation of the G alpha stimulatory subunit. In particular, lipids are vital mediators of Gαs signal transduction. In a simple sense, lipids are necessary for Gαs function since GPCRs and adenylyl cyclase are both transmembrane proteins. Delving deeper however, membranes are not simple environments – hundreds of lipid species exist in complex patterns that cell biologists are only beginning to understand. Among these exists the concept of lipid rafts, signaling centers organized by hydrophobic saturated phosopholipids, cholesterol and cytoskeleton corrals that concentrate or segregate signaling pathways. In the case of Gαs, lipid rafts are thought to dampen coupling to adenylyl cyclase. The nature of Gαs raft association is still poorly understood. Part of this stems from the controversial existence of lipid rafts themselves. Most studies of protein regulation by rafts, including those of Gαs, rely of lipid raft extractions. This thesis complements this single approach with GFP-Gαs diffusion studies to better understand the relationship between Gαs and lipid raft association. First, a monomeric variant of the previously characterized eGFP-Gαs was developed. Unlike eGFP-Gαs, monomeric GFP-Gαs properly localizes to lipid rafts similar to endogenous Gαs. Next this tool was used in a fluorescent recovery after photobleaching (FRAP) assay under conditions that altered Gαs raft localization. In particular, antidepressant treatment decreases Gαs localization to rafts. FRAP reveals after chronic, but not acute antidepressant treatment, GFP-Gαs diffuses significantly slower. Results also suggest this decrease in diffusion could be attributed to increased scaffolding to slow diffusing transmembrane proteins.