Cholesterol binding, efflux, and a PDZ-interacting domain of scavenger receptor–BI mediate HDL-initiated signaling
J. Clin. Invest. Chatchawin Assanasen, et al. 115:969 doi:10.1172/JCI23858 [Go to this article.]

Figure 7
SR-BI C-terminal cytoplasmic domain is not sufficient for eNOS activation, and the C-terminal transmembrane domain, which binds cholesterol, is also required. (A) COS-M6 cells were transfected with eNOS cDNA and either wild-type SR-BI, CD36, CD/SRCT, or CD/SRCTMT cDNA. For detection of eNOS phosphorylation (top), 24 hours after transfection, the cells were starved in serum-free DMEM for another 24 hours and incubated with HDL (50 μg/ml) for 0 or 10 minutes, and cell lysates were analyzed by Western blotting using anti–phospho-eNOS–specific (Ser1179-specific) antibody and anti-eNOS monoclonal antibody. For detection of eNOS activation (bottom), parallel sets of transfected cells were used, and [³H]L-arginine conversion to [³H]L-citrulline was measured during 15-minute incubations with control buffer or HDL (50 μg/ml). Values (mean ± SEM) are expressed as percent of HDL-stimulated activity above basal activity; n = 4. *P < 0.05 versus wild-type SR-BI. (B) HEK 293 cells were transfected and labeled with [³H]photocholesterol; receptors were immunoprecipitated with antibody to the SR-BI C-terminal tail; and the [³H]photocholesterol associated with the receptor was assessed by autoradiography (top). The abundance of immunoprecipitated receptor was assessed by Western blotting (bottom), and the relative amount of [³H]photocholesterol-bound receptor versus total receptor was calculated.