PCSK9 binds to multiple receptors and can be functionally inhibited by an
LiXin Shana,1, Ling Panga,1, Rumin Zhangb, Nicholas J. Murgolob, Hong Lana, Joseph A. Hedricka,*
aDepartment of Cardiovascular and Metabolic Disease Research, Schering-Plough Research Institute, 2015 Galloping Hill Road, K-15-1/1945, Kenilworth, NJ 07033, USA
bDepartment of Discovery Technologies, Schering-Plough Research Institute, 2015 Galloping Hill Road, K-15-1/1945, Kenilworth, NJ 07033, USA
a r t i c l ei n f o
Received 22 July 2008
Available online 31 July 2008
Low density lipoprotein receptor
LDL receptor associated protein
a b s t r a c t
Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to low density lipoprotein receptor (LDLR)
and induces its internalization and degradation. PCSK9 binding to LDLR is mediated through the LDLR
epidermal growth factor-like repeat A (EGF-A) domain. We show for the first time that an EGF-A peptide
inhibits PCSK9-mediated degradation of LDLR in HepG2 cells. In addition to LDLR, we show that PCSK9
also binds directly to ApoER2 and mouse VLDLR. Importantly, binding of PCSK9 to either LDLR or mouse
VLDLR was effectively inhibited by EGF-A while binding to ApoER2 was less affected. In contrast, LDL
receptor-associated protein (RAP), which interacts with LDL receptor repeat type A (LA) domains, inhib-
ited PCSK9 binding to ApoER2 with greater efficacy than either LDLR or mVLDLR. These data demonstrate
that while PCSK9 binds several receptors via its EGF-A binding domain, additional contacts with other
receptor domains are also involved.
? 2008 Elsevier Inc. All rights reserved.
PCSK9 was originally cloned as neural apoptosis-regulated
convertase 1 . Although some reports have suggested PCSK9
is involved in neuronal development [1–3], more attention has
been focused on its role in regulating LDLR expression and sub-
sequently serum LDL levels [4–9]. The ability of PCSK9 to influ-
reduction in liver LDLR protein [10–12]. The reduction of LDLR
is also independent of PCSK9 enzymatic activity, though auto-
catalytic activity is required for secretion [13,14]. PCSK9 appears
to alter the fate of LDLR, either preventing its transport to the
surface or interfering with recycling of internalized receptor
. Poirier et al. have raised the possibility that, in addition
to LDLR, PCSK9 may also interact with VLDLR and ApoER2 .
These authors showed that PCSK9 could associate with cells that
had been transfected with either of these receptors and that
PCSK9 could mediate their degradation in transfected cells. The
structural basis by which PCSK9 could bind to ApoER2 and
VLDLR has not been explored, nor has binding to either of these
proteins been directly demonstrated.
Recently, Zhang et al. revealed that the EGF-A domain of LDLR
is important for PCSK9 binding . As is the case with LDLR
, PCSK9 binding to EGF-A is pH and calcium dependent
. The interaction between PCSK9 and LDLR has been further
illuminated by the co-crystal structure of PCSK9 and the LDLR
EGF-A domain . This structure shows that PCSK9 and EGF-
A interact across a relatively small and flat 530 Å2contact patch
Here we report that PCSK9 directly and specifically binds to
ApoER2 and mVLDLR in addition to LDLR. We also report that a
synthetic EGF-A domain peptide blocks the interaction of PCSK9
with LDLR. EGF-A also diminished PCSK9-mediated degradation
of LDLR and maintained LDL uptake in HepG2 cells. This is the first
demonstration that the effect of PCSK9 on LDLR can be blocked. In
EGF-A inhibited PCSK9 binding to mVLDLR and to a lesser extent
with ApoER2. In contrast, recombinant RAP inhibited PCSK9 bind-
ing to ApoER2 more effectively than binding to LDLR and VLDLR,
suggesting the involvement of additional receptor domains in
Materials and methods
Recombinant proteins and antibodies. Polyhistidine-tagged hu-
man LDLR, human ApoER2, mouse VLDLR and mouse reelin were
purchased from R&D Systems as were antibodies to LDLR and
mouse VLDLR. Polyhistidine-tagged human JNK2a2 and mouse
endostatin were purchased from Calbiochem Inc. Recombinant
rat RAP was obtained from PROSPEC Protein Specialists and human
RAP from Molecular Innovations. Polyclonal rabbit anti-LDLR used
for Western blotting and in-cell Western was obtained from PRO-
GEN Biotechnik. Mouse monoclonal anti-b-actin and M2 anti-FLAG
antibodies were obtained from Sigma.
0006-291X/$ - see front matter ? 2008 Elsevier Inc. All rights reserved.
* Corresponding author. Fax: +1 908 740 7101.
E-mail address: email@example.com (J.A. Hedrick).
1These authors contributed equally to this work.
Biochemical and Biophysical Research Communications 375 (2008) 69–73
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Expression and purification of PCSK9. Human PCSK9 (GenBank
NM_174936.2) was amplified from liver cDNA using a pair of prim-
ers (N-terminal primer, 50-GCCGCCACCATGGGCACCGTCAGCTCCAG
GCG-30and C-terminal primer, 50-TCACTTGTCATCGTCGTCCTTGTA
GTCCTGGAGCTCCTGGGAGGCCTGCGCCAG-30) designed to insert a
C-terminal FLAG epitope tag. The product was cloned into pCDNA
3.1 and sequence confirmed. Human PCSK9-FLAG was purified
from supernatants of stably transfected HEK293 cells essentially
as described by Lagace et al. .
Synthetic LDLR EGF-A domain peptide. The human LDLR EGF-A
domain peptide GTNECLDNNGGCSHVCNDLKIGYECLCPDGFQLVAQ
RRCEDI-NH2 (S:S = C1–C3, C2–C4, C5–C6) was synthesized by CS
Bio Inc. Sequence and purity of the product were confirmed by
HPLC and mass spectrophotometry. The dissociation constant
(Kd) between PCSK9 and EGF-A was estimated by temperature-
dependent fluorescence (TdF). Briefly, 20 ll of 1 lM of PCSK9 and
2.5–20 lM of EGF-A peptide were mixed in buffer containing Sypro
Orange (5?) in a strip-sealed 96-well PCR plate. For pH 5.2 deter-
minations, the buffer was 25 mM citric–phosphate, pH 5.2,
150 mM NaCl, ±10 mM CaCl2; pH 7.4 buffer was 25 mM HEPES,
pH 7.4, 150 mM NaCl, ±10 mM CaCl2. The plate was placed in a real
time thermocycler (Chromo4, Bio-Rad) for melting in 45 min from
room temperature to 90 ?C in increments of 0.5 ?C. The measured
fluorescent intensities were fitted to generate melting point data.
The concentration-dependent melting point shifts were subjected
to nonlinear regression analysis to derive TdF Kdvalues using cus-
tom algorithms based on previous literature [20–22] and were
based on an assumed average enthalpy change of binding at
7000 cal/mol with an assigned uncertainty of approximately 50%.
Alphascreen binding assays. An Amplified Luminescent Proximity
Homogeneous Assay (ALPHA, Perkin-Elmer) capable of directly
determining the interaction between PCSK9-FLAG and a putative
binding partner was established. This technique requires that
‘‘donor” and ‘‘acceptor” beads be brought into proximity via pro-
tein–protein interaction, resulting in increased luminescence
. Receptor binding to PCSK9 was determined as follows: 5 ll
of recombinant receptor at the appropriate concentrations was
incubated with 2.5 ll PCSK9-FLAG (1.4 lg/ml, 30 min). About
2.5 ll of biotinylated anti-Flag-M2 antibody (1.8 lg/ml) was added
and the mixture incubated for 1 h. Afterward 5 ll of streptavidin
donor bead and nickel chelate acceptor bead (1:1 mixture) was
added and the assay incubated overnight. AlphaScreen signal
(counts per second) was analyzed using an EnVision microplate
reader (Perkin-Elmer). All data points were determined in tripli-
cate. Assays were carried out at 23 ?C in buffer containing 25 mM
HEPES, 0.1 M NaCl, pH 7.4, 0.1% BSA.
The inhibition assays were determined similarly with slight
adjustments to assay volumes and protein concentrations. Briefly,
5 ll of 1.25 lg/ml of PCSK9-Flag and 1.25 lg/ml of His-tagged
receptor was incubated with 2.5 ll of inhibitor at the appropriate
concentrations for 30 min followed by the addition of 2.5 ll of
anti-Flag-BioM2 (1.8 lg/ml) and a 1 h incubation.
LDLR degradation assays. LDLR degradation was determined by
Western blotting of whole-cell lysate from HepG2 cells (ATCC,
HB-8065). HepG2 cells were treated with media or media contain-
ing PCSK9 (100 nM), EGF-A (at concentration indicated) or both for
6 and 18 h. Total cell protein (35 lg/lane) was run on a reducing
10% SDS–PAGE and protein transferred to PVDF membrane. LDLR
Fig. 1. PCSK9 specifically interacts with LDLR, ApoER2, and mVLDLR. (A) Serial dilutions of the His-tagged LDLR, ApoER2, mVLDLR were incubated with PCSK9-Flag and the
interaction detected by Alphascreen as described in experimental procedures. His-tagged mEndostatin was used as an unrelated protein control. (B) Polyclonal antibodies to
LDLR or mVLDR were used to block interaction of PCSK9 with LDLR. (C) Polyclonal antibody inhibition of PCSK9 binding to ApoER2. (D) Polyclonal antibody inhibition of
PCSK9 binding to mVLDLR. All assay points were determined in triplicate and EC50values were calculated using Graphpad Prism 4.0. Error bars (standard deviation) are
shown. Results shown are representative of at least three independent experiments.
L. Shan et al./Biochemical and Biophysical Research Communications 375 (2008) 69–73
was detected using rabbit anti-LDLR and IRDye 800CW goat anti-
rabbit (Li-Cor Biosciences). Bands were visualized with an Odyssey
infrared imaging system (Li-Cor Biosciences).
In addition to Western blotting, in-cell western was performed
to quantitate EGF-A inhibition of PCSK9-mediated LDLR degrada-
tion. HepG2 cells were seeded in 384-well collagen I coated plates
and treated with EGF-A (at the indicated concentrations) and/or
PCSK9 (100 nM) for 18 h. Detection of LDLR and b-actin was per-
formed according to the manufacturer’s protocol (Li-Cor Biosci-
ences) using the antibodies described above in conjunction with
IRDye 800CW goat anti-rabbit (Li-Cor) and IRDye 680 goat anti-
mouse (Li-Cor). The assay was read on an Odyssey infrared imaging
system (Li-Cor) and the signal for LDLR protein in each well nor-
malized to b-actin content.
LDL uptake assay. For LDL uptake determinations, HepG2 cells
were treated with appropriate dilutions of EGF-A made in HBSS
containing 1% BSA ± 100 nM PCSK9 and the mixture incubated
for 1 h, 23 ?C before addition to the cells. Following an 18 h incuba-
tion, treatment buffer was removed from the cells and diI-LDL
(9 lg/ml in HBSS, 1% BSA, Invitrogen) added for 90 min, 23 ?C. Cells
were then fixed and fluorescence intensity read out using an Ana-
lyst GT (Molecular Devices).
Results and discussion
Conflicting results have been reported in regard to the ability of
PCSK9 to interact with proteins other than LDLR. Zhang et al. re-
ported that no binding of PCSK9 to VLDLR could be detected in
transfected COS-M cells . In addition, these authors showed
that binding to VLDLR could be conferred by introducing the
EGF-A domain of LDLR into VLDLR. In contrast, Poirier et al. showed
enhanced PCKS9 association with CHO-A7 cells expressing either
ApoER2 or VLDLR after an overnight incubation . These authors
further demonstrated enhanced degradation of ApoER2 and VLDLR
when coexpressed with PCSK9 in a variety of cell lines. In order to
assess PCSK9’s interaction with these proteins more directly we
developed a cell-free binding assay as described above. We ob-
served a concentration-dependent binding of PCSK9 to LDLR,
ApoER2 and mVLDLR (Fig. 1A). The omission of either of the pro-
tein pair or substitution of His-tagged mouse endostatin resulted
in no increase in signal. Substitution of His-tagged mouse reelin
or JNK2a2 also resulted in no signal (data not shown).
Polyclonal antibodies raised against LDLR or mVLDLR selec-
tively inhibited the interaction with those proteins in a concentra-
tion-dependent manner (Fig. 1B–D). A
polyclonal IgG was also unable to block any of the interactions
(not shown). Commercial polyclonal antibodies capable of recog-
nizing the extracellular domain of ApoER2 were not available.
The EC50values obtained for receptor binding suggest that LDLR
may bind to PCSK9 with higher affinity than either mVLDLR or
As mentioned above, Zhang et al. have shown that the EGF-A
domain of LDLR is critical for PCSK9 interaction and the recently
reported crystal structure of PCSK9 complexed with the LDLR
EGF-A domain has provided critical insight into the nature of this
interaction [17,19]. In our analysis of the LDLR interface in the X-
ray structure coordinates (RCSB code 3BPS), there was no obvious
reason to why the corresponding EGF domains of ApoER2 and
VLDLR would not be able to interact with PCSK9, particularly as
the LDLR L318 difference with VLDLR, cited previously , com-
prises only a small portion of the interaction surface. We therefore
proceeded on the hypothesis that the PCSK9 domain shown to
Fig. 2. Interaction of PCSK9 with LDLR, ApoER2, and mVLDLR are inhibited by EGF-A peptide and recombinant RAP. (A) Serial dilutions of EGF-A (indicated) were used to
inhibit PCSK9 binding to LDLR in the presence or absence of 2 mM added calcium. (B and C) EGF-A was used at the indicated concentrations in the presence of 2 mM added
calcium to inhibit PCSK9 binding to mVLDLR and ApoER2. (D) IC50values for inhibition of PCSK9 binding to the indicated receptors using recombinant rat or human RAP as
indicated in the legend. The numerical IC50values obtained for the experiment shown are indicated. In all cases, the results shown are representative of at least two
L. Shan et al./Biochemical and Biophysical Research Communications 375 (2008) 69–73
interact with the LDLR EGF-A was also involved in binding ApoER2
and mVLDLR. In order to address this question we used a synthetic
LDLR EGF-A domain peptide in an attempt to block the PCSK9-
receptor interaction. The ability of the synthetic peptide to bind
to recombinant PCSK9 was confirmed by TdF. The binding of
EGF-A to PCSK9 as determined by this method was pH and calcium
sensitive with the highest affinity, Kd 0.3 lM, determined at pH 5.2
in the presence of added calcium versus a Kd of 1 lM at pH 7.4 in
the presence of added calcium or 10 lM with no added calcium.
These findings are consistent with previous reports showing bind-
ing of PCSK9 to either LDLR [18,24] or EGF-A . We could not
determine a TdF dissociation constant for PCSK9 in low pH in the
absence of calcium. The reason for this is unknown, but one plau-
sible hypothesis is that the calcium is absolutely required to main-
tain the EGF-A fold at low pH.
As expected, the synthetic EGF-A blocked the PCSK9-LDLR inter-
action with an IC50of 3.4 lM (12.3 lM in the absence of calcium,
Fig. 2A). EGF-A also inhibited the PCSK9-mVLDLR interaction with
an IC50of 4.7 lM (Fig. 2B). In contrast, the PCSK9-ApoER2 interac-
tion was poorly inhibited by the EGF-A peptide and we were un-
able to completely block binding even with 200 lM EGF-A
(Fig. 2C). Since PCSK9 binding to all three of these proteins was
inhibited by EGF-A it suggests the same PCSK9 domain is used to
engage each of them, however the binding of ApoER2 appeared
to be less reliant on this interaction.
In order to further explore PCSK9 interactions with these pro-
teins, we took advantage of another protein known to interact with
all three receptors, the LDL receptor-associated protein or RAP. RAP
has been shown to interact with the LA domains found in LDLR and
related receptors [25,26]. This interaction protects these receptors
from being bound by their ligands while in the endoplasmic retic-
ulum . The presence of either human or rat RAP inhibited bind-
ing of PCSK9 to each of the receptors tested, albeit with different
efficacies (Fig. 2D). This suggests that, in addition to the EGF-A do-
main, the LA domains of LDLR are also involved in binding PCSK9.
Interestingly, RAP was much better at blocking ApoER2 (IC50
0.4 nM for rRAP) than either LDLR or mVLDLR (IC50 of 18 and
61 nM with rRAP, respectively). Together with the data regarding
EGF-A inhibition of PCSK9 binding, this suggests a model wherein
PCSK9 interaction with ApoER2 may rely more on the LA domains
and less on the EGF-A domain than is the case with LDLR or
mVLDLR. Previous reports have shown that PCSK9 degrades LDLR
in a post-ER compartment [12,28]. It is possible that RAP protects
LDLR and the related receptors from being bound by PCSK9 while
still in the ER, shielding them from degradation at least until they
reach the Golgi.
We also examined whether the EGF-A peptide could inhibit
LDLR degradation and restore LDL uptake using HepG2 liver cells
as a model system. PCSK9 very effectively enhanced degradation
of mature LDLR in HepG2 cells incubated with recombinant protein
(Fig. 3A). The presence of EGF-A inhibited this effect and showed
increased LDLR protein in HepG2 cells incubated with PCSK9 for
6 or 18 h. This effect appeared minimal at 1.5 lM EGF-A, but
showed a clear increase at 15 lM. These concentrations of EGF-A
had no obvious effect on LDLR protein levels in the absence of
PCSK9. In order to make a quantitative determination of the effects
of EGF-A, we used in-cell protein detection to determine total LDLR
protein levels after 18 h exposure to PCSK9. EGF-A showed a clear
(Fig. 3B). In a parallel experiment EGF-A inhibition of PCSK9 func-
tion was reflected in a corresponding increase in HepG2 uptake of
diI-labeled LDL (Fig. 3C). Although we have not assessed the ability
to maintainLDLR protein
Fig. 3. EGF-A peptide inhibits PCSK9-mediated degradation of LDLR. (A) HepG2 cells were treated with culture media (control), PCSK9, PCSK9 + EGF-A at 1.5 lM or 15 lM
(P+E1.5, P+E15) or EGF-A alone (1.5 or 15 lM) as indicated for 6 and 18 h. Whole cell protein lysates were prepared and duplicate samples analyzed for LDLR protein by
Western blotting using an anti-LDLR polyclonal. The band corresponding to mature LDLR (?160 kD) is indicated. Results shown are from one of two experiments. (B) HepG2
cells were plated into a 384-well plate and treated with buffer, PCSK9 or PCSK9 + EGF-A as indicated for 18 h. The amount of total LDLR protein relative to the amount of b-
actin in each well was determined as described in experimental procedures. Results are expressed as a percent of LDLR protein relative to the buffer control and are the
average of five wells. Error bars shown are standard deviation. (C) Uptake of diI-labeled LDL was determined in HepG2 cells treated as indicated for 18 h. Where indicated,
**p < 0.01,***p < 0.001 as determined by one-way ANOVA and Bonferroni’s multiple comparison test.
L. Shan et al./Biochemical and Biophysical Research Communications 375 (2008) 69–73
of RAP to inhibit PCSK9-mediated degradation we did attempt to Download full-text
look at effects on LDL uptake. As might be predicted given its
known function, RAP proved to be very effective in blocking diI-la-
beled LDL uptake on its own, thus any effect of RAP on PCSK9 func-
tion could not be observed.
In conclusion, we have provided the first direct evidence for
PCSK9 binding to ApoER2 and mVLDLR as well as to LDLR. We have
shown that this binding can be inhibited by a synthetic LDLR EGF-A
domain peptide as well as by receptor-specific antibodies. We have
also provided data to suggest that, while it certainly binds to the
EGF-A domain of LDLR and likely to an EGF domain(s) of mVLDLR
and ApoER2, PCSK9 may also interact with the LA domains found in
LDLR, mVLDLR, and in particular ApoER2. Finally, we shown for the
first time that inhibition of PCSK9 binding to LDLR can prevent
LDLR degradation and maintain LDL uptake.
The Schering Plough Research Institute is funded entirely by
Schering-Plough Corporation. The authors would like to thank
Drs Harry Davis and Diane Hollenbaugh for valuable discussion
 N.G. Seidah, S. Benjannet, L. Wickham, J. Marcinkiewicz, S.B. Jasmin, S. Stifani,
A. Basak, A. Prat, M. Chretien, The secretory proprotein convertase neural
apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal
differentiation, Proc. Natl. Acad. Sci. USA 100 (2003) 928–933.
 S. Poirier, A. Prat, E. Marcinkiewicz, J. Paquin, B.P. Chitramuthu, D. Baranowski,
B. Cadieux, H.P. Bennett, N.G. Seidah, Implication of the proprotein convertase
NARC-1/PCSK9 in the development of the nervous system, J. Neurochem. 98
 B. Bingham, R. Shen, S. Kotnis, C.F. Lo, B.A. Ozenberger, N. Ghosh, J.D. Kennedy,
J.S. Jacobsen, J.M. Grenier, P.S. DiStefano, L.W. Chiang, A. Wood, Proapoptotic
effects of NARC 1 (= PCSK9), the gene encoding a novel serine proteinase,
Cytometry A 69 (2006) 1123–1131.
 M. Abifadel, M. Varret, J.P. Rabes, D. Allard, K. Ouguerram, M. Devillers, C.
Cruaud, S. Benjannet, L. Wickham, D. Erlich, A. Derre, L. Villeger, M. Farnier, I.
Beucler, E. Bruckert, J. Chambaz, B. Chanu, J.M. Lecerf, G. Luc, P. Moulin, J.
Weissenbach, A. Prat, M. Krempf, C. Junien, N.G. Seidah, C. Boileau, Mutations
in PCSK9 cause autosomal dominant hypercholesterolemia, Nat. Genet. 34
 J.J. Kastelein, S.W. Fouchier, J.C. Defesche, What promise does PCSK9 hold?, J
Am. Coll. Cardiol. 45 (2005) 1620–1621.
 M.S. Brown, J.L. Goldstein, Biomedicine. Lowering LDL — not only how low, but
how long?, Science 311 (2006) 1721–1723
 J. Cohen, A. Pertsemlidis, I.K. Kotowski, R. Graham, C.K. Garcia, H.H. Hobbs, Low
LDL cholesterol in individuals of African descent resulting from frequent
nonsense mutations in PCSK9, Nat. Genet. 37 (2005) 161–165.
 K.E. Berge, L. Ose, T.P. Leren, Missense mutations in the PCSK9 gene are
associated with hypocholesterolemia and possibly increased response to statin
therapy, Arterioscler. Thromb. Vasc. Biol. 26 (2006) 1094–1100.
 J.C. Cohen, E. Boerwinkle, T.H. Mosley Jr., H.H. Hobbs, Sequence variations in
PCSK9, low LDL, and protection against coronary heart disease, N. Engl. J. Med.
354 (2006) 1264–1272.
 K.N. Maxwell, J.L. Breslow, Adenoviral-mediated expression of Pcsk9 in mice
results in a low-density lipoprotein receptor knockout phenotype, Proc. Natl.
Acad. Sci. USA 101 (2004) 7100–7105.
 S.W. Park, Y.A. Moon, J.D. Horton, Post-transcriptional regulation of low
density lipoprotein receptor protein by proprotein convertase subtilisin/kexin
type 9a in mouse liver, J. Biol. Chem. 279 (2004) 50630–50638.
 K.N. Maxwell, E.A. Fisher, J.L. Breslow, Overexpression of PCSK9 accelerates the
degradation of the LDLR in a post-endoplasmic reticulum compartment, Proc.
Natl. Acad. Sci. USA 102 (2005) 2069–2074.
 J. Li, C. Tumanut, J.A. Gavigan, W.J. Huang, E.N. Hampton, R. Tumanut, K.F.
Suen, J.W. Trauger, G. Spraggon, S.A. Lesley, G. Liau, D. Yowe, J.L. Harris,
Secreted PCSK9 promotes LDL receptor degradation independently of
proteolytic activity, Biochem. J. 406 (2007) 203–207.
 M.C. McNutt, T.A. Lagace, J.D. Horton, Catalytic activity is not required for
secreted PCSK9 to reduce low density lipoprotein receptors in HepG2 cells, J.
Biol. Chem. 282 (2007) 20799–20803.
 T.A. Lagace, D.E. Curtis, R. Garuti, M.C. McNutt, S.W. Park, H.B. Prather, N.N.
Anderson, Y.K. Ho, R.E. Hammer, J.D. Horton, Secreted PCSK9 decreases the
number of LDL receptors in hepatocytes and in livers of parabiotic mice, J. Clin.
Invest. 116 (2006) 2995–3005.
 S. Poirier, G. Mayer, S. Benjannet, E. Bergeron, J. Marcinkiewicz, N. Nassoury, H.
Mayer, J. Nimpf, A. Prat, N.G. Seidah, The proprotein convertase PCSK9 induces
the degradation of low density lipoprotein receptor (LDLR) and its closest
family members VLDLR and ApoER2, J. Biol. Chem. 283 (2008) 2363–2372.
 D.W. Zhang, T.A. Lagace, R. Garuti, Z. Zhao, M. McDonald, J.D. Horton, J.C.
Cohen, H.H. Hobbs, Binding of proprotein convertase subtilisin/kexin type 9 to
epidermal growth factor-like repeat A of low density lipoprotein receptor
decreases receptor recycling and increases degradation, J. Biol. Chem. 282
 T.S. Fisher, P. Lo Surdo, S. Pandit, M. Mattu, J.C. Santoro, D. Wisniewski, R.T.
Cummings, A. Calzetta, R.M. Cubbon, P.A. Fischer, A. Tarachandani, R. De
Francesco, S.D. Wright, C.P. Sparrow, A. Carfi, A. Sitlani, Effects of pH and low
density lipoprotein (LDL) on PCSK9-dependent LDL receptor regulation, J. Biol.
Chem. 282 (2007) 20502–20512.
 H.J. Kwon, T.A. Lagace, M.C. McNutt, J.D. Horton, J. Deisenhofer, Molecular basis
for LDL receptor recognition by PCSK9, Proc. Natl. Acad. Sci. USA 105 (2008)
 T.W. Mayhood, W.T. Windsor, Ligand binding affinity determined by
inhibitors, Anal. Biochem. 345 (2005) 187–197.
 M.C. Lo, A. Aulabaugh, G. Jin, R. Cowling, J. Bard, M. Malamas, G. Ellestad,
Evaluation of fluorescence-based thermal shift assays for hit identification in
drug discovery, Anal. Biochem. 332 (2004) 153–159.
 D. Matulis, J.K. Kranz, F.R. Salemme, M.J. Todd, Thermodynamic stability of
carbonic anhydrase: measurements of binding affinity and stoichiometry
using ThermoFluor, Biochemistry 44 (2005) 5258–5266.
 E.F. Ullman, H. Kirakossian, S. Singh, Z.P. Wu, B.R. Irvin, J.S. Pease, A.C.
Switchenko, J.D. Irvine, A. Dafforn, C.N. Skold, et al., Luminescent oxygen
channeling immunoassay: measurement of particle binding kinetics by
chemiluminescence, Proc. Natl. Acad. Sci. USA 91 (1994) 5426–5430.
 D. Cunningham, D.E. Danley, K.F. Geoghegan, M.C. Griffor, J.L. Hawkins, T.A.
Subashi, A.H. Varghese, M.J. Ammirati, J.S. Culp, L.R. Hoth, M.N. Mansour, K.M.
McGrath, A.P. Seddon, S. Shenolikar, K.J. Stutzman-Engwall, L.C. Warren, D. Xia,
X. Qiu, Structural and biophysical studies of PCSK9 and its mutants linked to
familial hypercholesterolemia, Nat. Struct. Mol. Biol. 14 (2007) 413–419.
 C. Fisher, N. Beglova, S.C. Blacklow, Structure of an LDLR–RAP complex reveals
a general mode for ligand recognition by lipoprotein receptors, Mol. Cell 22
 D. Lee, J.D. Walsh, I. Mikhailenko, P. Yu, M. Migliorini, Y. Wu, S. Krueger, J.E.
Curtis, B. Harris, S. Lockett, S.C. Blacklow, D.K. Strickland, Y.X. Wang, RAP uses
a histidine switch to regulate its interaction with LRP in the ER and Golgi, Mol.
Cell 22 (2006) 423–430.
 T.E. Willnow, A. Rohlmann, J. Horton, H. Otani, J.R. Braun, R.E. Hammer, J. Herz,
RAP, a specialized chaperone, prevents ligand-induced ER retention and
degradation of LDL receptor-related endocytic receptors, EMBO J. 15 (1996)
 N. Nassoury, D.A. Blasiole, A. Tebon Oler, S. Benjannet, J. Hamelin, V. Poupon,
P.S. McPherson, A.D. Attie, A. Prat, N.G. Seidah, The cellular trafficking of the
secretory proprotein convertase PCSK9 and its dependence on the LDLR, Traffic
8 (2007) 718–732.
L. Shan et al./Biochemical and Biophysical Research Communications 375 (2008) 69–73