Abstract 1183: Evidence for a Tandem Two-binding Site Model for ABCA1-Mediated Nascent HDL Formation
It is well accepted that both apoA-I and ABCA1 play crucial roles in HDL biogenesis and in the human atheroprotective system. However, the nature and specifics of apoA-I/ABCA1 interactions remain poorly understood. To investigate the structural characteristics of cellular apoA-I binding sites and their relationship to nascent HDL formation, we developed a rapid and efficient cross-linking assay in which 125I-apoA-I was cross-linked to ABCA1, and then the complex was co-immunoprecipitated with anti-ABCA1 antibody. The amount of 125I-apoA-I associated with ABCA1 or with other binding sites was quantitatively determined in BHK cells stably overexpressing ABCA1. Transferrin a ligand that is known to specifically interact with transferrin receptor was used to verify the efficiency of the cross-linking reaction. Here is presented evidence for a new binding site characterized by a 10-fold higher capacity to binds apoA-I compared to ABCA1 site (Bmax = 0.40 ± 0.02 vs. 0.039 ± 0.01 ng apoA-I/μg cell protein). This new cellular apoA-I binding site was designated “high capacity binding site” (HCBS). Glyburide drastically reduced 125I-apoA-I binding to the HCBS, whereas 125I-apoA-I showed no significant binding to the HCBS in Tangier disease fibroblasts. Interestingly, reconstituted HDL particles exhibited reduced affinity for the HCBS compared to lipid-free apoA-I. Deletion in the C-terminal region of apoA-I (delta 187–243) drastically reduced the binding of apoA-I to the HCBS. The majority of the HCBS was localized to plasma membrane and was not associated with membrane raft domains. Importantly, treatment of cells with phosphatidylcholine-specific phospholipase C (PC-PLC), but not sphingomyelinase, concomitantly reduced the binding of 125I-apoA-I to the HCBS, apoA-I-mediated cholesterol efflux and the formation of nascent apoA-I-containing particles. Altogether these data suggest that a functional ABCA1 leads to the formation of a phosphatidylcholine-containing site for the binding and the lipidation of apoA-I at the plasma membrane. Our results provide a biochemical basis for HDL biogenesis pathway that involves both ABCA1 and the HCBS, supporting a novel two-binding site model for ABCA1-mediated nascent HDL genesis.