No abstract

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.

PCSK9 (proprotein convertase subtilisin/kexin type 9) binds to the LDLR (low-density lipoprotein receptor) at the cell surface and disrupts recycling of the LDLR. However, PCSK9 also interacts with the LDLR in the ER (endoplasmic reticulum). In the present study we have investigated the role of PCSK9 for the transport of the LDLR from the ER to the cell membrane. A truncated LDLR consisting of the ectodomain (ED-LDLR) was used for these studies to avoid PCSK9-mediated degradation of the LDLR. The amount of secreted ED-LDLR was used as a measure of the amount of ED-LDLR transported from the ER. From co-transfection experiments of various PCSK9 and ED-LDLR plasmids, PCSK9 increased the amount of WT (wild-type) ED-LDLR in the medium, but not of an ED-LDLR lacking the EGF (epidermal growth factor)-A repeat or of a Class 2a mutant ED-LDLR which fails to exit the ER. Mutant PCSK9s which failed to undergo autocatalytic cleavage or failed to exit the ER, failed to increase the amount of WT-ED-LDLR in the medium. These mutants also reduced the amount of WT-ED-LDLR intracellularly, which could partly be prevented by the proteasome inhibitor lactacystine. WT-ED-LDLR promoted autocatalytic cleavage of pro-PCSK9. The findings of the present study indicate that the binding of WT-ED-LDLR to pro-PCSK9 in the ER promotes autocatalytic cleavage of PCSK9, and autocatalytically cleaved PCSK9 acts as a chaperone to promote the exit of WT-ED-LDLR from the ER.

HighlightsMutation G805R is in the transmembrane domain of the LDLR.A polar residue in the transmembrane domain induced metalloproteinase cleavage.Mutation G805R caused reduced amounts of the precursor LDLR.Reduced amounts of precursor LDLR led to reduced amounts of the mature LDLR.Mutation G805R prevented γ-secretase cleavage within the transmembrane domain.

No abstract