The Proprotein Convertase PCSK9 Induces the Degradation of Low Density Lipoprotein Receptor (LDLR) and Its Closest Family Members VLDLR and ApoER2

Abstract

The proprotein convertase PCSK9 gene is the third locus implicated in familial hypercholesterolemia, emphasizing its role in cardiovascular diseases. Loss of function mutations and gene disruption of PCSK9 resulted in a higher clearance of plasma low density lipoprotein cholesterol, likely due to a reduced degradation of the liver low density lipoprotein receptor (LDLR). In this study, we show that two of the closest family members to LDLR are also PCSK9 targets. These include the very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) implicated in neuronal development and lipid metabolism. Our results show that wild type PCSK9 and more so its natural gain of function mutant D374Y can efficiently degrade the LDLR, VLDLR, and ApoER2 either following cellular co-expression or re-internalization of secreted human PCSK9. Such PCSK9-induced degradation does not require its catalytic activity. Membrane-bound PCSK9 chimeras enhanced the intracellular targeting of PCSK9 to late endosomes/lysosomes and resulted in a much more efficient degradation of the three receptors. We also demonstrate that the activity of PCSK9 and its binding affinity on VLDLR and ApoER2 does not depend on the presence of LDLR. Finally, in situ hybridization show close localization of PCSK9 mRNA expression to that of VLDLR in mouse postnatal day 1 cerebellum. Thus, this study demonstrates a more general effect of PCSK9 on the degradation of the LDLR family that emphasizes its major role in cholesterol and lipid homeostasis as well as brain development.Familial hypercholesterolemia is mainly characterized by elevated plasma LDL 2 cholesterol that is highly correlated with cardiovascular diseases (1). The main player in regulating the circulating cholesterol is the low density lipoprotein receptor (LDLR), which is expressed mostly in the liver. Recently, natural mutations in the proprotein convertase PCSK9 (2, 3) have been identified and associated with the third locus implicated in familial hypercholesterolemia (4 -6). The major function of PCSK9 seems to be an enhancement of the degradation of the LDLR (7, 8) in acidic subcellular compartments (3), likely endosomes/lysosomes (9, 10). This can occur either via an extracellular endocytotic route (11), or possibly by a direct cellular circuit not involving cell surface endocytosis of the LDLR (12). The gain of function PCSK9 mutations D374Y (13, 14) or D374H (15) have the highest impact on the development of hypercholesterolemia (16), likely through enhanced binding (17) and degradation of the LDLR (18, 19). The major binding site of LDLR to PCSK9 seems to reside within its first epidermal growth factor-like repeat namely EGF-A (20). Finally, it was recently suggested that the PCSK9-induced degradation of the cell surface LDLR does not require its proteolytic activity (21). One of the unanswered questions is the target specificity of PCSK9, and it is not known, nor obvious, whether other members of the LDLR family are also affected by PCSK9. This family consists of str...

Dissection of the Endogenous Cellular Pathways of PCSK9-induced Low Density Lipoprotein Receptor Degradation

“…In addition, we showed that LDLR and PCSK9 interact early in the secretory pathway (10) and questioned whether some PCSK9⅐LDLR complex or PCSK9 alone can be targeted directly from the transGolgi network to LE/L, without cycling through the cell surface. Aside from the extracellular pathway, the existence of an intracellular one is supported by the ability of PCSK9 to degrade the LDLR in vivo in the absence of ARH (9) and on the capability of PCSK9-Lamp1 chimeras, which directly traffic to LE/L, to enhance the degradation of the LDLR, VLDLR and apo-ER2 efficiently (26). In liver, ARH seems to be critical for the endocytosis of the extracellular PCSK9⅐LDLR complex (16) but does not seem to play an important role in regulating total levels of endogenous LDLR (Fig.…”

Annexin A2 Is a C-terminal PCSK9-binding Protein That Regulates Endogenous Low Density Lipoprotein Receptor Levels

“…Furthermore, the identification of the R1 domain of AnxA2 that mediates binding to PCSK9 provides a potential new peptidederived tool to regulate PCSK9 function. Derivatives or mimics of this segment could be used to inhibit PCSK9 function on LDLR and possibly on other targets, such as VLDLR or ApoER2 (19). The peptide could also be used to derive a co-crystal structure to better define the CHRD residues, including Gln 554 , seemingly implicated in this interaction (Fig.…”

“…PCSK9 is the sole PC that is secreted as a catalytically inhibited prosegment⅐PC complex (1, 3,18). Accordingly, the enhanced degradation of the LDLR (3,4,16), very low density lipoprotein receptor (VLDLR), and ApoER2 (19) in endosomes/ lysosomes (14) induced by PCSK9 does not seem to require its catalytic activity (18,20). This intriguing twist in the function of this convertase is supported by the crystal structure of PCSK9, which revealed an extended tight binding complex of the enzyme and its inhibitory prosegment (21).…”

Pcsk9

“…Thus, PCSK9 was first found to enhance the extracellular and intracellular degradation of the closest LDLR family members, namely the VLDLR and ApoER2 (LRP8). 42,43 However, although the interaction of the catalytic domain of PCSK9 with the EGF-A-like domains of these receptors was confirmed, the specific aa in each protein implicated in such interactions are not the same as for the LDLR. For example, in contrast to the LDLR, the GOF D374Y PCSK9 does not degrade these other receptors more efficiently than wild-type PCSK9.…”

“…For example, in contrast to the LDLR, the GOF D374Y PCSK9 does not degrade these other receptors more efficiently than wild-type PCSK9. 42 The receptors LDLR, VLDLR, and ApoER2 have been confirmed as PCSK9 target proteins in mice 25,26,65,66 and the LDLR in monkeys 67 and human. 68 Recently, we showed that PCSK9 can enhance the degradation of LRP1 in various cells 44 although proof of this activity in vivo is still lacking.…”