title: A chimeric LDL receptor containing the cytoplasmic domain of the transferrin receptor is degraded by PCSK9.
abstract: Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the extracellular domain of the low density lipoprotein receptor (LDLR) at the cell surface, and disrupts the normal recycling of the LDLR. However, the exact mechanism by which the LDLR is re-routed for lysosomal degradation remains to be determined. To clarify the role of the cytoplasmic domain of the LDLR for re-routing to the lysosomes, we have studied the ability of PCSK9 to degrade a chimeric receptor which contains the extracellular and transmembrane domains of the LDLR and the cytoplasmic domain of the transferrin receptor. These studies were performed in CHO T-REx cells stably transfected with a plasmid encoding the chimeric receptor and a novel assay was developed to study the effect of PCSK9 on the LDLR in these cells. Localization, function and stability of the chimeric receptor were similar to that of the wild-type LDLR. The addition of purified gain-of-function mutant D374Y-PCSK9 to the culture medium of stably transfected CHO T-REx cells showed that the chimeric receptor was degraded, albeit to a lower extent than the wild-type LDLR. In addition, a mutant LDLR, which has the three lysines in the intracellular domain substituted with arginines, was also degraded by D374Y-PCSK9. Thus, the mechanism for the PCSK9-mediated degradation of the LDLR does not appear to involve an interaction between the endosomal sorting machinery and LDLR-specific motifs in the cytoplasmic domain. Moreover, ubiquitination of lysines in the cytoplasmic domain does not appear to play a critical role in the PCSK9-mediated degradation of the LDLR.
authors.affiliation: Medical Genetics Laboratory, Department of Medical Genetics, Oslo University Hospital Rikshospitalet, NO-0027 Oslo, Norway.
authors.forename: Øystein L
authors.initials: ØL
authors.lastname: Holla
authors.forename: Thea Bismo
authors.initials: TB
authors.lastname: Strøm
authors.forename: Jamie
authors.initials: J
authors.lastname: Cameron
authors.forename: Knut Erik
authors.initials: KE
authors.lastname: Berge
authors.forename: Trond P
authors.initials: TP
authors.lastname: Leren
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis; D057075:Enzyme Assays; D006801:Humans; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011973:Receptors, LDL / Q000737:chemistry / Q000378:metabolism*; D011990:Receptors, Transferrin / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D014162:Transfection
chemical_list: D050505:Mutant Proteins; D011973:Receptors, LDL; D011990:Receptors, Transferrin; D011994:Recombinant Proteins; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases