Abstract 169: BiFC-FRET: A Novel Tool for Visualizing SR-BI Oligomerization in Vivo

Abstract

Scavenger receptor BI (SR-BI) is a cell surface receptor that binds high-density lipoprotein (HDL) and mediates the selective uptake of HDL-cholesteryl esters (CE) into the liver for excretion. As cholesterol flux from peripheral tissues to the liver is a critical step for cholesterol disposal via reverse cholesterol transport, a better understanding of the SR-BI/HDL complex and mechanisms that enhance this interaction will aid in developing strategies for preventing cardiovascular diseases such as atherosclerosis. Previous fluorescence resonance energy transfer (FRET) data from our laboratory revealed that SR-BI exists as a dimer in live cells. Although dimers and higher order oligomers are detected in vitro, live cell FRET analysis is limited to interactions between two molecules only. Our in vitro data also reveals several mutant receptors that are unable to mediate selective uptake of HDL-CE are also unable to form higher order oligomers, suggesting that a functional SR-BI complex consists of greater than two molecules. Therefore, we hypothesize that SR-BI function correlates with higher order oligomer formation (i.e. a hydrophobic channel). To test this hypothesis, we have developed an assay that couples bimolecular fluorescence complementation (BiFC) with FRET, which will help us visualize, for the first time, higher order oligomer formation in vivo. Wild-type SR-BI, Cys-less-SR-BI, and ΔLeuZip-SR-BI were cloned into pBiFC vectors containing either the N-terminal half of Venus (VN), the C-terminal half of Venus (VC) [fluorescence acceptor halves], or full-length Cerulean [fluorescence donor]. Total cell expression and cell surface expression of each construct was confirmed by protein immunoblotting and flow cytometry, respectively. To confirm that addition of the BiFC tags did not alter protein function, fusion proteins were transiently expressed in COS7 cells and assayed for selective uptake efficiency. All wild-type tagged SR-BI constructs displayed similar levels of selective uptake efficiency as untagged SR-BI. To confirm the presence of dimers and validate the BiFC methodology, COS7 cells were transiently transfected with (i) SRBI-VN alone, (ii) SR-BI-VC alone, or (iii) both SR-BI-VN and SR-BI-VC, and we measured Venus fluorescence two days post-transfection. Co-expression of SR-BI-VN and SR-BI-VC in COS7 cells resulted in reconstitution of Venus fluorescence and a positive BiFC signal following excitation at 514 nm, while single transfections of either SR-BI-VN or SR-BI-VC displayed no detectable fluorescence as expected. BiFC was also observed for Cys-less-SR-BI and ΔLeuZip-SR-BI, confirming that these mutants are still able to dimerize, despite being unable to mediate selective uptake. Therefore, we have successfully validated the use of BiFC to confirm the presence of SR-BI dimers in cells. As we have confirmed proper cell expression of Cerulean-tagged SR-BI, we now have all the necessary tools to couple BiFC with FRET methodologies to provide the first evidence for the existence of SR-BI oligomers in vivo.