Disruption of Caveolae Lipid Rafts and the Effects on Melanin-concentrating Hormone Receptor-1 Localization: A Pharmacological Study

Abstract

Melanin-concentrating hormone (MCH) is integral to the regulation of human appetite. MCH targets G protein-coupled receptors in the brain and peripheral tissues. When MCH receptor 1 binds MCH on the surface of cells, it activates multiple signaling pathways, then desensitizes. Internalization of MCH-bound MCHR1 is only thought to be partially responsible for the loss of MCH signaling capacity of cells. Previous research has shown that MCH receptors are enriched in caveolae and specifically complex with caveolin-1. Caveolin-1 is a key structural component of caveolae, which are cholesterol-based lipid rafts that are known for concentrating signaling molecules and clathrin-independent endocytosis. This study aims to investigate how MCH signaling would be affected if MCH receptors weren’t enriched in these regions. Pharmacological treatments were used to achieve this goal as caveolae formation was disrupted with the antibiotic nystatin, a cholesterol inhibitor. It has been shown that sodium carbonate-based extraction procedure followed by flotation on sucrose density centrifugation isolates caveolae from other cell contents. Caveolae-isolation procedures to detect caveloin-1 were undertaken to indicate that untreated BHK-570 cells contained caveolin-1 in fractions 4 and 5 of the sucrose gradients while a gradient shift of caveolin-1 to fractions 7-10 in nystatin-treated cells occurred. Such shift confirmed that there was a partial disruption of caveolae within the treated cells. Future experiments will test whether other pharmacological inhibitors such as filipin and methyl-?-cyclodextrin as well as caveolin-1 RNAi are better able to deplete caveolae from cells as well as their impact on MCH signaling.