Abstract
<b>Abstract ID 25402</b> <b>Poster Board 371</b> <b>Background:</b> The trimeric G-proteins, composed of a Gα and a dimeric Gβg subunit, function to relay extracellular stimuli from G-protein-coupled-receptors (GPCRs) and transduce intracellular signaling cascades by directly interacting with their cognate coupling partners in confined membrane microdomains. Chronic intermittent ethanol (CIE) exposure alters the function of many GPCRs, although the molecular mechanism is largely unknown. Our preliminary data indicate that CIE exposure increases membrane cholesterol content in the brain. Membrane cholesterol critically regulates the compartmentalization of membrane proteins and the assembly of signaling complexes. Thus, this project aimed to test the hypothesis that CIE exposure may result in spatial alterations of membrane localization of G-proteins and their coupling partners within lipid raft and non-raft microdomains, disrupting GPCR-stimulated G-protein and downstream signaling. <b>Method:</b>Ethanol Vapor Exposure: Male Sprague Dawley rats were exposed to either ethanol vapor (CIE) or room air (AIR) during the light cycle (12 hr/day) for seven consecutive days. Following 24 hr withdrawal from the last ethanol exposure, all animals were euthanized, and prefrontal cortex (PFC) tissue was dissected. Sucrose Density Gradient Ultracentrifugation: PFC tissue was homogenized and then fractionated by discontinuous sucrose density (5%/30%/40%) ultracentrifugation. Fifteen fractions were collected along the sucrose density gradient for each sample. Western blotting was performed to determine the localization of G-protein subunits and their interacting proteins within lipid raft and non-lipid raft microdomains. <b>Results:</b> We found that CIE induced differential changes in the localization of G-protein subunits within lipid rafts and non-raft regions. In CIE-exposed animals, Gαi and Gαo subunits, but not Gαs and Gαq subunits, translocated from lipid rafts toward non-rafts, while Gβγ subunits translocated from non-rafts toward lipid rafts when compared to AIR-exposed control animals. Interestingly, CIE exposure did not alter the compartmentalization of the protein kinases adenylyl cyclase type 1 and phospholipase Cβ1, which are direct G-protein interacting proteins. Further, CIE exposure induced an increase in lipid raft localization of mGluR2, a Gαi/o-interacting protein, and Kv1.2, a Gβγ-interacting protein, but had no effect on Gαq-coupled mGluR1 localization. Finally, CIE exposure significantly increased rat PFC membrane cholesterol content. <b>Conclusions:</b> Our data suggest that spatial localization of G-proteins and their interacting proteins is susceptible to regulation by CIE. Importantly, the present study highlights a potential role of lipid metabolism in GPCR dysregulation and may open a new avenue for targeting cholesterol metabolism as a treatment for alcohol use disorder. Support/Funding Information: T32-AA007565 R21DA056857 R01DA042862
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