title: Effects of pH and low density lipoprotein (LDL) on PCSK9-dependent LDL receptor regulation.
abstract: Mutations within PCSK9 (proprotein convertase subtilisin/kexin type 9) are associated with dominant forms of familial hyper- and hypocholesterolemia. Although PCSK9 controls low density lipoprotein (LDL) receptor (LDLR) levels post-transcriptionally, several questions concerning its mode of action remain unanswered. We show that purified PCSK9 protein added to the medium of human endothelial kidney 293, HepG2, and Chinese hamster ovary cell lines decreases cellular LDL uptake in a dose-dependent manner. Using this cell-based assay of PCSK9 activity, we found that the relative potencies of several PCSK9 missense mutants (S127R and D374Y, associated with hypercholesterolemia, and R46L, associated with hypocholesterolemia) correlate with LDL cholesterol levels in humans carrying such mutations. Notably, we found that in vitro wild-type PCSK9 binds LDLR with an approximately 150-fold higher affinity at an acidic endosomal pH (K(D) = 4.19 nm) compared with a neutral pH (K(D) = 628 nm). We also demonstrate that wild-type PCSK9 and mutants S127R and R46L are internalized by cells to similar levels, whereas D374Y is more efficiently internalized, consistent with their affinities for LDLR at neutral pH. Finally, we show that LDL diminishes PCSK9 binding to LDLR in vitro and partially inhibits the effects of secreted PCSK9 on LDLR degradation in cell culture. Together, the results of our biochemical and cell-based experiments suggest a model in which secreted PCSK9 binds to LDLR and directs the trafficking of LDLR to the lysosomes for degradation.
authors.affiliation: Department of Cardiovascular Diseases, Merck Research Laboratories, Rahway, NJ 07065, USA.
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Fisher
authors.forename: Paola
authors.initials: P
authors.lastname: Lo Surdo
authors.forename: Shilpa
authors.initials: S
authors.lastname: Pandit
authors.forename: Marco
authors.initials: M
authors.lastname: Mattu
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Santoro
authors.forename: Doug
authors.initials: D
authors.lastname: Wisniewski
authors.forename: Richard T
authors.initials: RT
authors.lastname: Cummings
authors.forename: Alessandra
authors.initials: A
authors.lastname: Calzetta
authors.forename: Rose M
authors.initials: RM
authors.lastname: Cubbon
authors.forename: Paul A
authors.initials: PA
authors.lastname: Fischer
authors.forename: Anil
authors.initials: A
authors.lastname: Tarachandani
authors.forename: Raffaele
authors.initials: R
authors.lastname: De Francesco
authors.forename: Samuel D
authors.initials: SD
authors.lastname: Wright
authors.forename: Carl P
authors.initials: CP
authors.lastname: Sparrow
authors.forename: Andrea
authors.initials: A
authors.lastname: Carfi
authors.forename: Ayesha
authors.initials: A
authors.lastname: Sitlani
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005799:Genes, Dominant; D030342:Genetic Diseases, Inborn / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006937:Hypercholesterolemia / Q000235:genetics / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D020125:Mutation, Missense; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D011485:Protein Binding / Q000235:genetics; D011973:Receptors, LDL / Q000819:agonists / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D008077:Lipoproteins, LDL; D011973:Receptors, LDL; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases