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. We have previously 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. We are interested in investigating the role of MCH localization to caveolae on MCH signaling. We hypothesize that MCH signaling would be disrupted if MCH receptors werenít enriched in these regions and our first approach is pharmacological; we are disrupting caveolae with nystatin, a cholesterol inhibitor. We have previously utilized a sodium-carbonate based extraction procedure followed by flotation on sucrose density centrifugation to isolate caveolae from other cell contents. To confirm whether nystatin does indeed deplete caveolae in BHK-570 cells we performed our caveolae isolation procedure on cells pre-treated with or without nystatin. Caveolin-1 can usually be detected by Western Blot in Fractions 4 and 5 of our gradients. We observed a gradient shift of caveolin-1 to Fractions 7-10 in nystatin-treated cells confirming that we at least partially disrupted caveolae. 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.
|Presenter:||Colin M. King (Undergraduate Student)|
|Location:||Lobby of Edwards|
|Time:||1:15 pm (Session III)
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