Internalized PCSK9 dissociates from recycling LDL receptors in PCSK9-resistant SV-589 fibroblasts

J Lipid Res. 2014 Feb;55(2):266-75. doi: 10.1194/jlr.M044156. Epub 2013 Dec 2.


Secreted PCSK9 binds to cell surface LDL receptor (LDLR) and directs the receptor for lysosomal degradation. PCSK9 is potent at inducing LDLR degradation in cultured liver-derived cells, but it is considerably less active in immortalized fibroblasts. We examined PCSK9 trafficking in SV-589 human skin fibroblasts incubated with purified recombinant wild-type PCSK9 or gain-of-function mutant PCSK9-D374Y with increased LDLR binding affinity. Despite LDLR-dependent PCSK9 uptake, cell surface LDLR levels in SV-589 fibroblasts were only modestly reduced by wild-type PCSK9, even at high nonphysiological concentrations (20 µg/ml). Internalized (125)I-labeled wild-type PCSK9 underwent lysosomal degradation at high levels, indicating its dissociation from recycling LDLRs. PCSK9-D374Y (2 µg/ml) reduced cell surface LDLRs by approximately 50%, but this effect was still blunted compared with HepG2 hepatoma cells. Radioiodinated PCSK9-D374Y was degraded less efficiently in SV-589 fibroblasts, and Alexa488-labeled PCSK9-D374Y trafficked to both lysosomes and endocytic recycling compartments. Endocytic recycling assays showed that more than 50% of internalized PCSK9-D374Y recycled to the cell surface compared with less than 10% for wild-type PCSK9. These data support that wild-type PCSK9 readily dissociates from the LDLR within early endosomes of SV-589 fibroblasts, contributing to PCSK9-resistance. Although a large proportion of gain-of-function PCSK9-D374Y remains bound to LDLR in these cells, degradative activity is still diminished.

Keywords: degradation; endosome; ligand dissociation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Endocytosis*
  • Fibroblasts / cytology*
  • Fibroblasts / metabolism
  • Humans
  • Intracellular Space / metabolism
  • Lysosomes / metabolism
  • Mutation
  • Proprotein Convertase 9
  • Proprotein Convertases / genetics
  • Proprotein Convertases / metabolism*
  • Protein Transport
  • Proteolysis
  • Receptors, LDL / metabolism*
  • Serine Endopeptidases / genetics
  • Serine Endopeptidases / metabolism*
  • Skin / cytology
  • Sterols / metabolism


  • Receptors, LDL
  • Sterols
  • PCSK9 protein, human
  • Proprotein Convertase 9
  • Proprotein Convertases
  • Serine Endopeptidases