Caveolae are free cholesterol (FC) and sphingolipid -rich microdomains abundant at the surface of vascular cells. Caveolae take part in signal transduction from the plasma membrane as scaffolds binding intermediates of the MAP kinase and PK-A pathways. They have also been implicated in FC homeostasis, and potentially, as sensors of cell surface FC levels. Caveolin, a major structural protein of caveolae, is a FC- binding protein. In this project the molecular basis of FC binding to caveolin, and the mechanism of its effects on FC transport and signaling, will be analyzed. The hypothesis to be tested is that FC efflux to a physiological acceptor (apolipoprotein A-1/phospholipid complex) is coupled to dissociation of FC the caveola, and changes in the conformation of residual FC and SPH within the organelle. In initial studies, photoactivable analogs of FC and sphingomyelin (SPH) will be used to define lipid-protein binding sites within the caveolae of living cells. The location of crosslinks within the protein sequence will be determined after protease digestion by HPLC/MS. Subsequent studies deal with the relationship between FC efflux and signal transduction. Vanadate increases caveolin phosphorylation, and under the same conditions downregulates expression of cell surface caveolae and caveolin and inhibits FC efflux (>80%) from vascular cells. This Project will determine if FC dissociation from caveolin initiates signal transduction to the nucleus to provide the missing link between membrane lipid composition and transcriptional regulation in FC sensing. A second inhibitor of FC efflux, 7-ketocholesterol, at concentrations similar to those present in human atherosclerotic plaques, also downregulates caveolae and caveolin expression and FC efflux. The hypothesis will be tested that oxysterols can displace FC into the cell from the caveolin sterol binding site, leading to inappropriate FC storage and downregulation of caveolin expression. The studies in this Project explore fundamental issues in how normal vascular cells sense and regulate their FC content. The results obtained will have significance for understanding normal FC homeostasis, as well as for the consequences of cholesterol loading under pathophysiologic conditions.
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