FIGURE 5.

PCSK9 displays similar binding to the cell surface LDLR-WT and LDLR-RS. A and B, HEK293 cells transfected with LDLR-WT (WT) or LDLR-R410S (RS) were incubated for 4 h at 4 °C with conditioned media from HEK293 cells: no PCSK9 control media (Cnt) or media containing PCSK9-WT. PCSK9 was non-tagged (PC9-WT) ∼1.3 μg/ml (A), or mCherry-tagged (PC9-mCherry), ∼1 μg/ml (B). PCSK9 binding to cell surface receptor was quantified by FACS (A) or fluorescence spectroscopy (B). Bars represent averages ± S.D. from three independent experiments. C, HEK293 cells overexpressing LDLR-WT (WT) or LDLR-RS (RS) were incubated with purified recombinant human PCSK9 for 4 h at 4 °C. The curve through the data is the fit to a rectangular hyperbola and is representative of two independent experiments. The average PCSK9 binding constants determined from the two experiments are 1.8 ± 0.8 μm (WT) and 1.3 ± 0.1 μm (RS).