Abstract 452: Detailed Characterization of the Role of the Epidermal Growth Factor Precursor Homology Domain A of the Low-density Lipoprotein Receptor in Proprotein Convertase Subtilisin/kexin-type 9 Binding

Abstract

Proprotein convertase subtilisin/kexin-type 9 (PCSK9) promotes the LDL receptor (LDLR) degradation, thereby preventing clearance of LDL-C levels and playing a central regulatory role in cholesterol homeostasis. PCSK9 interacts with the LDLR at the cell surface and binds the receptor with a much higher affinity at the acidic environment of the endosome. Consequently, the receptor traffics from the endosome to the lysosome for degradation, rather than being recycled. Previously, we have shown that the epidermal growth factor precursor homology domain A (EGF-A) of the LDLR is critical for PCSK9 binding at the cell surface (pH7.4) and that leucine at the position 318 in the EGF-A is critical for PCSK9 binding. Here, we further characterized the role of EGF-A in the binding of PCSK9 to the LDLR in more details. We found that mutation of Asp299 to Val or Arg329 to Pro in the EGF-A significantly reduced PCSK9 binding at pH 7.4. In addition, we observed that deletion of EGF-A significantly reduced PCSK9 binding at the acidic endosomal environment (pH 6.0) and that PCSK9 bound to purified recombinant EGF-A in a pH-dependent manner with a stronger binding at pH6.0. However, mutation of Leu318 to Arg or His306 to Tyr that dramatically enhanced PCSK9 binding at pH7.4 had no significant effect on the binding of PCSK9 to the LDLR at pH6.0. Thus, our findings demonstrate that EGF-A of the LDLR plays an important role in PCSK9 binding at the cell surface (pH 7.4) and the endosomal environment (pH6.0). Our data also indicate that different amino acid residues in EGF-A are involved in PCSK9 binding to the LDLR at the cell surface and in the endosome.