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Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the epidermal growth factor homology domain repeat A of the low-density lipoprotein receptor (LDLR) at the cell surface and disrupts recycling of the internalized LDLR. As a consequence, the LDLR is rerouted to the lysosomes for degradation. Although PCSK9 may bind to an LDLR lacking the ligand-binding domain, at least three ligand-binding repeats of the ligand-binding domain are required for PCSK9 to reroute the LDLR to the lysosomes. In this study, we have studied the binding of PCSK9 to an LDLR with or without the ligand-binding domain at increasingly acidic conditions in order to mimic the milieu of the LDLR:PCSK9 complex as it translocates from the cell membrane to the sorting endosomes. These studies have shown that PCSK9 is rapidly released from an LDLR lacking the ligand-binding domain at pH in the range of 6.9-6.1. A similar pattern of release at acidic pH was also observed for the binding to the normal LDLR of mutant PCSK9 lacking the C-terminal domain. Together these data indicate that an interaction between the negatively charged ligand-binding domain of the LDLR and the positively charged C-terminal domain of PCSK9 is required for PCSK9 to remain bound to the LDLR during the early phase of endosomal acidification as the LDLR translocates from the cell membrane to the sorting endosome.

The proprotein convertase subtilisin/kexin type 9 (PCSK9) gene is involved in the post-transcriptional regulation of the low-density lipoprotein (LDL) receptors (LDLR). Mutations in the PCSK9 gene have been associated with both hypocholesterolemia and hypercholesterolemia through 'loss-of-function' and 'gain-of-function' mechanisms, respectively. We have studied the effect of the four loss-of-function mutations R46L, G106R, N157K and R237W and the two gain-of-function mutations S127R and D374Y on the autocatalytic activity of PCSK9, as well as on the amount of the cell surface LDLR and internalization of LDL in transiently transfected HepG2 cells. The two groups of mutations did not differ with respect to autocatalytic activity of PCSK9, but they did differ with respect to the amount of cell surface LDLR and internalization of LDL. The four loss-of-function mutations had a 16% increased level of cell surface LDLR and a 35% increased level of internalization of LDL as compared with WT-PCSK9. The two gain-of-function mutations had a 23% decreased level of cell surface LDLR and a 38% decreased level of internalization of LDL as compared with WT-PCSK9. Our studies have also shown that transfer of media from transiently transfected HepG2 cells to untransfected HepG2 cells, reduces the amount of cell surface LDLR and internalization of LDL in the untransfected cells within 20 min of media transfer. Thus, PCSK9 or a factor acted upon by PCSK9, is secreted from the transfected cells and degrades LDLR both in transfected and untransfected cells.