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.