Cholesterol Enrichment Alters Compartmentalization of G-Proteins Into Lipid Raft Microdomains

Abstract ID 130365 Poster Board 141 Background: The function of many of G-protein coupled receptors (GPCRs) is dysregulated in neurodegenerative diseases. Interestingly, disruption of cholesterol metabolism in the brain is also commonly found in neurodegenerative diseases. For example, increased cholesterol content is observed in the brain of patients with Alzheimer's disease. Cholesterol is not evenly distributed along the plasma membrane. Cholesterol is mostly localized within microdomains throughout the plasma membrane referred to as lipid rafts, which are tightly packed with cholesterol molecules, sphingolipids, and membrane-bound proteins. As cholesterol on the plasma membrane regulates the localization and function of membrane proteins, it is possible that altered cholesterol content contributes to GPCR dysregulation. Therefore, the purpose of this study was to investigate the effect of increased cholesterol content on the membrane localization of GPCRs and G-protein subtypes and determine GPCR function. Method: Cholesterol Addition Mouse neuroblastoma 2A (N2A) cells stably expressing mGluR2 were treated with water-soluble cholesterol (0.05, 0.5, or 5 mM) for 30 min at 37°C prior to experimentation. An Amplex Red cholesterol assay kit was used to determine the content of membrane cholesterol after cholesterol treatment. Discontinuous sucrose density gradient ultracentrifugation N2A cells treated with cholesterol were homogenized and fractionated by discontinuous sucrose density (5%/30%/40%) ultracentrifugation. Fifteen fractions were collected from the sucrose density gradient for each sample. Western blotting was performed to determine the localization of G-protein subunits (G αq, G αi2, G αs, and Gβγ) as well as metabotropic glutamate receptor 2 (mGluR2) within lipid raft and non-raft microdomains. cAMP assay A cAMP accumulation assay was performed to determine the effect cholesterol addition has on mGluR2, a Gi/o coupled GPCR. Cells were pre-incubated with LY379268, a mGluR2 agonist, before forskolin treatment. The accumulation of cAMP was determined using a cAMP Gs HiRange assay kit from Cisbio. Results: We found cholesterol induced trafficking of G-protein subunits and mGluR2 from non-raft microdomains towards lipid raft microdomains in a concentration-dependent manner, although the localization of flotillin and transferrin, markers for lipid raft and non-raft microdomains, were not altered. We also observed a functional alteration in mGluR2 inhibition of cAMP levels in cells treated with cholesterol. Conclusions: Enhanced cholesterol content significantly increased the localization of G-proteins and mGluR2 in lipid rafts, which in turn effects the functionality of mGluR2. This study indicates a direct causal relationship between cholesterol content and GPCR localization/function. The outcome of our study may be informative for understanding neurological diseases that show impaired cholesterol metabolism and dysregulated GPCR function. This work was supported by the National Institute on Drug Abuse R21DA056857 and R01AA030676.