Fellowship: The Role of the Cytoplasmic Tails in SR-BI Recycling and Cholesterol Trafficking

The Scavenger Receptor class B type I (SR-BI) has a clear anti-atherogenic effect as it is involved in reverse cholesterol transport, a major route of the body to dispose of excess cholesterol. During this process, cholesterol from peripheral tissues is transferred into HDL particles, subsequently taken up by the liver and partially secreted into bile. SR-BI is responsible for HDL cholesteryl ester uptake at the basolateral side of the hepatocyte, via a selective uptake process with limited degradation of HDL proteins. SR-BI is also involved in subsequent biliary secretion of cholesterol at the apical side. The exact mechanism of these processes is not known. The current model proposes uptake of the HDL particle at the basolateral membrane, followed by differential sorting of HDL protein and lipid, resulting in apical secretion of cholesterol and basolateral resecretion of proteins. It is not known how SR-BI internalization and selective sorting is regulated. The observation that an alternative splicing variant of SR-BI (designated SR-BII) with a different cytoplasmic C-terminus displays significantly reduced selective uptake capacity leads us to hypothesize that the cytoplasmic "tails" of SR-BI are involved in its uptake and intracellular cycling and sorting, perhaps through interaction with other proteins. At present only one protein, PDZK1, is known to interact with SR-BI, by binding to a C-terminal sequence that is absent in SR-BII. PDZK1 indeed appears to affect SR-BI cycling and functioning. No protein is known to bind SR-BII. We propose to investigate the role of SR-BI's cytoplasmic tails in uptake, recycling, and selective sorting of SR-BI and HDL-derived proteins and lipids. We will employ non-polarized CHO cells and primary hepatocytes, as well as polarized hepatocyte couplets and MDCK cells, all expressing various artificial SR-BI mutants. Those mutants will either lack the N- or C- terminus, or both, or will contain N- or C- termini of CD36, a closely related scavenger receptor with greatly reduced selective uptake capacity. SR-BII and CD36 will also be compared. Furthermore, we propose to search for proteins that interact with the cytoplasmic tails of SR-BI and SR-BII, by employing yeast-2-hybrid analyses and affinity chromatography.