The binding of apoA-I-containing ligands to the HDL receptor scavenger receptor class B type I (SR-BI) was characterized using two different assays. The first employed conventional binding or competition assays with 125 I-labeled ligands. The second is a new nonradioactive ligand binding assay, in which the receptor-associated ligand is detected by quantitative immunoblotting ("immunoreceptor assay"). Using both methods, we observed that the K d value for spherical HDL (density ‫؍‬ 1.1-1.13 g/ml) was ϳ16 g of protein/ml, while the values for discoidal reconstituted HDL (rHDL) containing proapoA-I or plasma apoA-I were substantially lower (ϳ0.4 -5 g of protein/ml). We also observed reduced affinity and/or competition for spherical 125 I-HDL cell association by higher relative to lower density HDL and very poor competition by lipid-free apoA-I and pre-␤-1 HDL. Deletion of either 58 carboxyl-terminal or 59 amino-terminal residues from apoA-I, relative to full-length control apoA-I, resulted in little or no change in the affinity of corresponding rHDL particles. However, rHDL particles containing a double mutant lacking both terminal domains competed poorly with spherical 125 I-HDL for binding to SR-BI. These findings suggest an important role for apoA-I and its conformation/organization within particles in mediating HDL binding to SR-BI and indicate that the NH 2 and COOH termini of apoA-I directly or indirectly contribute independently to binding to SR-BI.

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