title: Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis.
abstract: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR.
authors: Lilly Research Laboratories, Eli Lilly & Company, Indianapolis, IN 46285, USA.
authors: Yue-Wei
authors: YW
authors: Qian
authors: Robert J
authors: RJ
authors: Schmidt
authors: Youyan
authors: Y
authors: Zhang
authors: Shaoyou
authors: S
authors: Chu
authors: Aimin
authors: A
authors: Lin
authors: He
authors: H
authors: Wang
authors: Xiliang
authors: X
authors: Wang
authors: Thomas P
authors: TP
authors: Beyer
authors: William R
authors: WR
authors: Bensch
authors: Weiming
authors: W
authors: Li
authors: Mariam E
authors: ME
authors: Ehsani
authors: Deshun
authors: D
authors: Lu
authors: Robert J
authors: RJ
authors: Konrad
authors: Patrick I
authors: PI
authors: Eacho
authors: David E
authors: DE
authors: Moller
authors: Sotirios K
authors: SK
authors: Karathanasis
authors: Guoqing
authors: G
authors: Cao
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000502:physiology; D000818:Animals; D002460:Cell Line; D008078:Cholesterol, LDL / Q000097:blood; D015536:Down-Regulation; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D008954:Models, Biological; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D017434:Protein Structure, Tertiary; D011973:Receptors, LDL / Q000096:biosynthesis* / Q000502:physiology; D012697:Serine Endopeptidases / Q000378:metabolism / Q000502:physiology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D008078:Cholesterol, LDL; C500241:Ldlrap1 protein, mouse; D011973:Receptors, LDL; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases