Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that 
regulates the number of cell surface low-density lipoprotein receptors (LDLRs) 
and the levels of low-density lipoprotein cholesterol in plasma. Intact cells 
have not previously been used to determine the characteristics of binding of 
PCSK9 to LDLR. Using PCSK9 iodinated by the tyramine cellobiose (TC) method 
([(125)I]TC-PCSK9), we measured the affinity and kinetics of binding of PCSK9 to 
LDLR on HepG2 cells at 4 °C. The extent of [(125)I]TC-PCSK9 binding increased as 
cell surface LDLR density increased. Unlabeled wild-type and two 
gain-of-function mutants of PCSK9 reduced binding of [(125)I]TC-PCSK9. The 
Scatchard plot of the binding-inhibition curve was curvilinear, indicative of 
high-affinity and low-affinity sites for PCSK9 binding on HepG2 cells. Nonlinear 
regression analysis of the binding data also indicated that a two-site model 
better fitted the data. The time course of [(125)I]TC-PCSK9 binding showed two 
phases in the association kinetics. Dissociation of [(125)I]TC-PCSK9 also 
occurred in two phases. Unlabeled PCSK9 accelerated the dissociation of 
[(125)I]TC-PCSK9. At low pH, only one phase of dissociation was apparent. 
Furthermore, the dissociation of [(125)I]TC-PCSK9 under pre-equilibrium 
conditions was faster than under equilibrium conditions. Overall, the data 
suggest that PCSK9 binding to cell surface LDLR cannot be described by a simple 
bimolecular reaction. Possible interpretations that can account for these 
observations are discussed.