Scavenger receptor class B, type I (SR-BI) homo-dimerizes via its C-terminal region: Fluorescence resonance energy transfer analysis

Autor: Yolanda F. Darlington, Daisy Sahoo, Yinan Peng, Margery A. Connelly, Jeffery R. Smith
Rok vydání: 2007
Předmět:
Zdroj: Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 1771:818-829
ISSN: 1388-1981
Popis: Expression of the scavenger receptor class B, type I (SR-BI) receptor facilitates high density lipoprotein cholesterol transport and correlates with protection against atherosclerosis. Studies have shown that SR-BI self-associates, but many of the techniques used to characterize SR-BI homo-oligomerization were wrought with the prospect of producing artifacts. Therefore, we employed fluorescence resonance energy transfer (FRET) to visualize SR-BI homo-oligomerization with the benefit of gaining information about its quaternary structure in the absence of typical membrane receptor artifacts. To this end, SR-BI was tagged at the N- or C-termini with either cyan (CFP) or yellow (YFP) fluorescent protein. To test whether SR-BI subunits oligomerize through N-N, N-C or C-C terminal interactions, we co-expressed the appropriate SR-BI fusion protein combinations in COS-7 cells and measured live-cell FRET following acceptor photobleaching. We did not observe FRET with co-transfection of SR-BI with CFP and YFP at the N-termini nor at the N- and C-termini, suggesting that the N-termini are not proximal to each other or to the C-termini. However, FRET was observed with co-transfection of SR-BI with CFP and YFP at the C-termini, suggesting that the C-terminal ends are within 10 nm of each other, consistent with SR-BI dimerization via its C-terminal region.
Databáze: OpenAIRE
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