Scavenger receptor class B, type I (SR-BI) homo-dimerizes via its C-terminal region: fluorescence resonance energy transfer analysis.
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.
Sahoo
Daisy
D
Department of Pharmacological Sciences, University Medical Center, Stony Brook University, Stony Brook, NY 11794-8651, USA. sahoo@pharm.sunysb.edu
Peng
Yinan
Y
Smith
Jeffery R
JR
Darlington
Yolanda F
YF
Connelly
Margery A
MA
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D038761:Photobleaching; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051122:Scavenger Receptors, Class B / Q000737:chemistry* / Q000378:metabolism*
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D051122:Scavenger Receptors, Class B