Binding of high density lipoprotein (HDL) and discoidal reconstituted HDL to the HDL receptor scavenger receptor class B type I - Effect of lipid association and APOA-I mutations on receptor binding

The binding of apoA-I-containing ligands to the HDL receptor scavenger rece ptor class B type I (SR-BI) was characterized using two different assays. T he first employed conventional binding or competition assays with I-125-lab eled ligands. The second is a new nonradioactive ligand binding assay, in w hich the receptor-associated ligand is detected by quantitative immunoblott ing ("immunoreceptor assay"). Using both methods, we observed that the K-d value for spherical HDL (density = 1.1-1.13 g/ml) was similar to 16 mu g of protein/ml, while the values for discoidal reconstituted HDL (rHDL) contai ning proapoA-I or plasma apoA-I were substantially lower (similar to 0.4-5 mu g of protein/ml). We also observed reduced affinity and/or competition f or spherical I-125-HDL cell association by higher relative to lower density HDL and very poor competition by lipid-free apoA-I and pre-beta-1 HDL. Del etion of either 58 carboxyl-terminal or 59 amino-terminal residues from apo A-I, relative to full-length control apoA-I, resulted in little or no chang e in the affinity of corresponding rHDL particles. However, rHDL particles containing a double mutant lacking both terminal domains competed poorly wi th spherical I-125-HDL for binding to SR-BI. These findings suggest an impo rtant role for apoA-I and its conformation/organization within particles in mediating HDL binding to SR-BI and indicate that the NH2 and COOH termini of apoA-I directly or indirectly contribute independently to binding to SR- BI.