Abstract 1275: Mutations in the C-terminal Transmembrane Domain of Scavenger Receptor Class B Type I (SR-BI) Selectively Inhibit the Cholesterol Sensing that is Required for HDL-initiated Intracellular Signaling
HDL binding to scavenger receptor class B type I (SR-BI) induces intracellular signaling, which in endothelium activates endothelial NO synthase (eNOS) to generate the anti-atherogenic molecule NO. HDL-SR-BI signaling also promotes NO-independent endothelial cell migration. SR-BI has intracellular N- and C-terminal domains, N- and C-terminal transmembrane domains (NTTM, CTTM), and an extracellular loop that binds HDL. Our prior studies showed that HDL-SR-BI signaling requires the binding of cell cholesterol to the receptor, cholesterol efflux, and C-terminal domains of SR-BI. The CTTM of SR-BI uniquely binds cell cholesterol, whereas the CTTM of the related class B scavenger receptor, CD36, does not. We have also shown that HDL-SR-BI signaling requires interaction with the adaptor protein PDZK1. To further understand the molecular basis of HDL-initiated SR-BI signaling, we created mutant forms of SR-BI in which highly conserved residues of the CTTM of SR-BI were mutated to those of CD36. One mutant designated MT1 contains 5 such mutations (H442G, Y443M, Q445A, Y446M, L455M), and another contains only the single substitution Q445A. When expressed in COS M6 cells, both MT1 and Q445A displayed HDL binding that was comparable to the binding to wild-type SR-BI, both mutants mediated normal cholesterol efflux to HDL, both displayed normal binding to cell cholesterol, and both interacted normally with PDZK1. However, in cells coexpressing eNOS, the mutants were incapable of mediating HDL-induced eNOS Ser1179 phosphorylation or enzyme activation. They also failed to activate c-Src kinase, which is the most proximal signaling event induced by HDL-SR-BI. We further found that brief treatment with methyl-β-cyclodextrin, which causes cholesterol efflux independent of SR-BI, activated signaling in cells expressing wild-type SR-BI but not in cells expressing the receptor mutants. Thus, we have identified conserved amino acids in the SR-BI CTTM that are required for the unique capacity of the receptor to function as a plasma membrane cholesterol sensor. In doing so, we have shown that cholesterol sensing by SR-BI is required for the HDL-initiated intracellular signaling that underlies its cardiovascular protective actions in endothelium.
This research has received full or partial funding support from the American Heart Association, South Central Affiliate (Arkansas, New Mexico, Oklahoma & Texas).