Ectodomains of the LDL Receptor-Related Proteins
LRP1b and LRP4 Have Anchorage Independent Functions
Martin F. Dietrich1., Louise van der Weyden2., Haydn M. Prosser3, Allan Bradley3, Joachim Herz1*,
David J. Adams2*
1Department of Molecular Genetics, UT Southwestern, Dallas, Texas, United States of America, 2Experimental Cancer Genetics, Wellcome Trust Sanger Institute, Hinxton,
Cambs, United Kingdom, 3Mouse Genomics, Wellcome Trust Sanger Institute, Hinxton, Cambs, United Kingdom
Background: The low-density lipoprotein (LDL) receptor gene family is a highly conserved group of membrane receptors
with diverse functions in developmental processes, lipoprotein trafficking, and cell signaling. The low-density lipoprotein
(LDL) receptor-related protein 1b (LRP1B) was reported to be deleted in several types of human malignancies, including
non-small cell lung cancer. Our group has previously reported that a distal extracellular truncation of murine Lrp1b that is
predicted to secrete the entire intact extracellular domain (ECD) is fully viable with no apparent phenotype.
Methods and Principal Findings: Here, we have used a gene targeting approach to create two mouse lines carrying
internally rearranged exons of Lrp1b that are predicted to truncate the protein closer to the N-terminus and to prevent
normal trafficking through the secretary pathway. Both mutations result in early embryonic lethality, but, as expected from
the restricted expression pattern of LRP1b in vivo, loss of Lrp1b does not cause cellular lethality as homozygous Lrp1b-
deficient blastocysts can be propagated normally in culture. This is similar to findings for another LDL receptor family
member, Lrp4. We provide in vitro evidence that Lrp4 undergoes regulated intramembraneous processing through
metalloproteases and c-secretase cleavage. We further demonstrate negative regulation of the Wnt signaling pathway by
the soluble extracellular domain.
Conclusions and Significance: Our results underline a crucial role for Lrp1b in development. The expression in mice of
truncated alleles of Lrp1b and Lrp4 with deletions of the transmembrane and intracellular domains leads to release of the
extracellular domain into the extracellular space, which is sufficient to confer viability. In contrast, null mutations are
embryonically (Lrp1b) or perinatally (Lrp4) lethal. These findings suggest that the extracellular domains of both proteins may
function as a scavenger for signaling ligands or signal modulators in the extracellular space, thereby preserving signaling
thresholds that are critical for embryonic development, as well as for the clear, but poorly understood role of LRP1b in
Citation: Dietrich MF, van der Weyden L, Prosser HM, Bradley A, Herz J, et al. (2010) Ectodomains of the LDL Receptor-Related Proteins LRP1b and LRP4 Have
Anchorage Independent Functions In Vivo. PLoS ONE 5(4): e9960. doi:10.1371/journal.pone.0009960
Editor: Pieter H. Reitsma, Leiden University Medical Center, Netherlands
Received October 8, 2009; Accepted March 2, 2010; Published April 7, 2010
Copyright: ? 2010 Dietrich et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: LvdW is supported by a fellowship from the Kay Kendall Leukaemia Foundation, and DJA is supported by Cancer Research UK and the Wellcome Trust.
MFD is supported by a Boehringer Ingelheim Fellowship, and JH is supported by the National Institutes of Health, the Perot Family Foundation, the American
Health Assistance Foundation and the Humboldt Foundation. HP and AB are supported by the Wellcome Trust. The funders have or had no influence on data
collection and sharing, experimental planning, data interpretation, decision to publish or time of publication.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (JH); email@example.com (DJA)
. These authors contributed equally to this work.
The LDL receptor gene family is a highly conserved class of cell
surface receptors involved in various functions, including cell
signaling, cargo transport, and gene regulation . LRP1b, initially
named LRP-DIT (Deleted in Tumors), was first described as a gene
that was frequently inactivated in non-small cell lung cancer . It
was subsequently also shown to be mutated in urothelial , head
and neck [4,5], esophageal tumors  and in B-cell lymphomas
. The specific deletion of LRP1b in certain tumors through
genetic and epigenetic silencing suggests a role as a tumor
suppressor. However, the exact mechanism by which LRP1b
functions in this manner remains elusive. LRP1 and LRP1b share
86 percent mRNA and 52 percent amino acid identity. Previously
reported mechanisms of action for LRP1b, including the regulation
of the urokinase (uPAR) and platelet-derived growth factor
(PDGF) receptor trafficking at the membrane level [8,9], overlap
with the functions of expressed and unmutated LRP1 in tumor
tissues. We have previously reported that mice that express of a
truncated allele lacking both the transmembrane and intracellular
domains of Lrp1b is viable .
Here, we extend our earlier findings by demonstrating
embryonic lethality of two lines of mice carrying null alleles of
Lrp1b. Interestingly, similar observations were made with the Lrp4
PLoS ONE | www.plosone.org1 April 2010 | Volume 5 | Issue 4 | e9960
knockout mice . While Lrp4 knockout mice fail to develop
neuromuscular junctions and succumbed to respiratory failure
post-natally , a truncated allele lacking the transmembrane
and intracellular domains displays a mitigated phenotype
compatible with postnatal survival [11,13]. The common feature
of the truncated Lrp1b and Lrp4 alleles is that they secrete an intact
and apparently physiologically functional extracellular domain.
All members of the LDL receptor gene family harbor at least
one structurally highly similar extracellular ligand binding domain
consisting of a series of negatively charged cysteine-rich Ca2+-
chelating repeat modules that bind numerous ligands . These
ligand binding domains have numerous and partially overlapping
functions in cell signaling and cargo transport . The ability of
the extracellular domain (ECD) to rescue embryonic or perinatal
lethality suggests a functional role for the isolated ECDs.
We therefore propose a model in which the ECDs of LRP1b
and LRP4 may modulate cellular signaling by scavenging and
neutralizing extracellular ligands, thereby preserving signaling
thresholds that are critical for proper embryonic development.
The same mechanisms could impact on the development and
progression of some malignancies.
Materials and Methods
Generation of Lrp1b-deficient mice
Mice carrying two different Lrp1b alleles were generated. These
lines were termed Lrp1btm1wtsiand Lrp1btm2wtsiand carry N-terminal
and C-terminal duplications of exons of Lrp1b, respectively.
Targeting vectors were obtained from the MICER collection
. Lrp1btm1wtsimice carry an internal duplication of exons 6–8 of
Lrp1b while mice with the Lrp1btm2wtsiallele carry an internal
duplication of exon 69 of Lrp1b (based on the Ensembl predicted
gene structure). Both alleles are predicted to cause frameshift
10 mg of the linearized targeting vectors were electroporated
into AB2.2 embryonic stem (ES) cells (from mouse strain 129S5/
SvEvBrd) . The ES cells were cultured on a lethally irradiated
SNL76/7 feeder layer and picked into 96-well plates after 7 days
of drug selection in G418 (180 mg/ml) . To check for
homologous recombination, genomic DNA was analyzed by
Southern blotting. For the Lrp1btm1wtsiallele, a 359 bp 39 external
probe was used (generated by PCR from AB2.2 genomic DNA,
using the primers: forward, 59- AAA AAA TCT TCC TTG AAG
GCT CTT GTA AG GTC -39 and reverse, 59- ATG CAT ATG
GAA TGC CAG GGG GAT GTT CAC AC -39) and hybridized
with EcoRV-digested DNA to identify restriction fragments of
18.2 kb for the wild-type and a 11.3 kb for the targeted allele. For
the Lrp1btm2wtsiallele, a 500 bp external probe was used (generated
by PCR from AB2.2 genomic DNA, using the primers: forward,
59-GAA AGT GAT CAA ATG AAC ATA TTC AAA TCC
TTC-39 and reverse, 59-CTT GAT CAC AGC TTT CTC TCA
ATG GAC TTT AC-39) on BamHI-digested DNA to identify a
10 kb wild-type and a 20 kb targeted allele.
Correctly targeted ES cell clones were injected into C57BL/6J
blastocysts and germline transmission of the targeted (mutant)
allele was demonstrated by Southern blot analysis of tail DNA.
Mice were maintained on a mixed 129/C57 background and
husbandry was in compliance with Home Office regulations
(United Kingdom). All animal work was conducted according to
the relevant national and international guidelines and in
accordance with the recommendations of the Weatherall report,
‘‘The use of non-human primates in research’’ (no primates were
used in this study). Animal experiments conducted in Dallas were
also reviewed and approved by the Institutional Committee on
Animal Use and Care (IACUC) at UT Southwestern Medical
Isolation and in vitro culture of mouse blastocysts
6–8 week old male and female heterozygous Lrp1b mice were
intercrossed (with each mouse carrying a different Lrp1b allele,
such that homozygote embryos could be detected as those carrying
both the Lrp1btm1wtsiand Lrp1btm2wtsialleles). The females were
inspected twice daily for signs of a plug (which was taken as
embryonic day 0.5), and three days later the females were
sacrificed and their uteri collected and flushed to harvest the
blastocysts (embryonic day 3.5). The blastocysts were then
cultured in Dulbecco’s modified Eagle medium (Invitrogen Ltd,
Paisley, UK) supplemented with 10% fetal calf serum, 1 mM L-
glutamine, 50 units penicillin/100 mg streptomycin per mL, 1%
non-essential amino acids, 0.1 mM b-mercaptoethanol and
overlaid with mineral oil in a humidified incubator containing
5% CO2at 37uC for up to 1 week. After culture, each embryo was
placed in 20 mL lysis buffer consisting of 50 mM KCl, 10 mM
Tris-HCl (pH 8.3), 2.5 mM MgCl2, 0.1 mg/mL gelatin, 0.45%
Tween-20, 0.45% NP-40 and 1 mg/mL proteinase K. The lysis
was carried out at 55uC for 5 hr, followed by 95uC for 15 min.
The lysate was then used to perform three separate PCR reactions:
to detect the Lrp1btm1wtsiallele (forward: 59-AAA CCG CCT CTC
CCC GCG CGT TGG C-39 and reverse: 59-CTA TAA GCC
AAT CTA ATA AAT TCC CAT CTC TCT-39), the Lrp1btm2wtsi
allele (forward: 59-TGT TTT CAG ACT AGA TAG GCA TTG
GGT CTA TA-39 and reverse: 59-GCG CCC AAT ACG CAA
ACC GCC TCT CCC CG-39) and an unrelated allele for quality
control of the lysate (forward: 59-GAA GAT GGC TTA GTC
GGC CAT CAT TGG GAA GA-39 and reverse: 59-GAT GAA
TAC ACT GGG TGT GAA ACA CAG CTA CC-39). The PCR
was performed in 50 mL reactions using 45 mL of Platinum PCR
Supermix (Invitrogen) and 100 ng of each primer pair with the
following PCR cycle profile: 1 cycle at 94uC for 2 min followed by
30 cycles at 94uC for 30 sec, 55uC for 1 min (or 65uC for the
Lrp1btm2wtsiallele primer pair), and 72uC for 30 sec with a final
cycle of 72uC for 10 min. The resulting PCR products were
visualized on an ethidium bromide-stained 2% agarose gel.
Lrp4 in vitro assays and Western Blotting
Subconfluent HEK293T cells were grown in 10% FCS/
DMEM High Glucose (Cellgro Mediatech Inc.). On day 1,
pcDNA3.1 constructs expressing either the extracellular domain
or full length murine Lrp4 were transfected into the cells using
FuGene6 (Roche Laboratories) according to the manufacturer’s
protocol. Briefly, 6 mg DNA and 2 uL FuGene6 reagent were
suspended (1:3 ratio of FuGene6:DNA) in a volume of 600 mL of
serum and incubated for 30 min at room temperature before
addition to the cell culture dish. The cells were incubated
overnight in 10% serum and then switched to serum free DMEM
High Glucose/0.2% bovine serum albumin for two days. Cells
were collected, washed three times in ice-cold PBS and lysed in
1% Triton-X lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl,
1 mM MgCl2, 1 mM CaCl2, 1% Triton X-100, with an EDTA-
free protease inhibitor cocktail (Complete Mini EDTA-free
Protease Inhibitor Cocktail, Roche Laboratories).
The supernatants were centrifuged at 4,000 rpm for 15 min to
remove detached or dead cells. Media was then concentrated
using 100 kDa size exclusion spin concentrators from Millipore
(Amicon Ultra Centrifuge Device 100.000 MWCO). 50 mg
cellular extract and 50 mL concentrated media (concentration
,200:1) were run on 4–15% gradient gels and subsequently
analyzed by immunoblotting for expression of both the intracel-
LRP Ectodomain Functions
PLoS ONE | www.plosone.org2 April 2010 | Volume 5 | Issue 4 | e9960
lular and extracellular domains of Lrp1b and Lrp4. All antibodies
were used at a 1:1,000 dilution in 5% milk/PBS-Tween, and were
generated as described below. The bands were visualised by
chemiluminescence (Thermo Scientific Pierce ECL Western
Blotting) according to the manufacturer’s instructions.
Accumulation of ICD was verified with c-secretase inhibitor
DAPT (10 mM, Sigma Aldrich). Treatment was initiated 24 hrs
after transfection of the Lrp4 full length construct (murine,
pcDNA3.1 vector) for 16 hrs overnight. Cells were then lysed in
1% Triton-X buffer as described previously and 20 mg per lane
subjected to Western blotting.
The intracellular domain (ICD) specific rabbit polyclonal
antibodies for Lrp1b  and Lrp4  have been described
previously. Extracellular domain (ECD) specific rabbit polyclonal
antibodies for Lrp1b and Lrp4 were generated by fusing the
murine full ligand binding domain (LBD 2 in case of Lrp1b) to
maltose binding protein (MBP) followed by bacterial expression of
the recombinant fusion protein. Briefly, DH5a E. coli cells were
transformed and induced with IPTG (0.5 mM) overnight for
16 hrs. Bacteria were then centrifuged and exposed to osmotic
shock to release the correctly folded fusion protein from the
periplasmic space. Proteins were then column-purified and
injected subcutaneously into 3 months old rabbits. The immuni-
zation was repeated every four weeks until high titer antibodies
HEK-293 cells were plated at 400 000 cells/well in 6-well plates
and grown to 50–80% confluency in 10% FBS/DMEM. Cells
were transfected using the TOP-Flash reporter assay system 
with the indicated expression plasmids for Wnt1, Dkk1, Lrp4
ECD, Lrp5 and Lrp6 in pcDNA3.1 backbones (0.5 mg/construct,
2.5 mg total). To account for the different numbers of transfected
plasmids, empty pcDNA 3.1 plasmid was added to a total of
2.5 mg DNA/well. Transfections were performed with FuGene6
using the manufacturer’s protocol. Cells were lysed 48 hrs after
transfection and lysates were assayed for firefly and renilla
luciferase activities using the Dual Luciferase Reporter Assay
System (Promega), according to the manufacturer’s protocol. All
transfections and measurements were performed in triplicate.
Absence of Lrp1b in Mice Results in Early Embryonic
We generated two different Lrp1b null alleles – the first targeting
the N-terminus with duplication of exons 6–8 (Lrp1btm1wtsimice),
and the second targeting the C-terminus with duplication of exon
mice); both mutations result in premature
termination through frameshift (Figure 1). ES cells carrying these
alleles were used to generate chimaeras, which transmitted the
targeted alleles to their progeny. Heterozygous mice (Lrp1btm1wtsi/+
and Lrp1btm2wtsi/+) were healthy at birth and both males and
females were fertile. However, no homozygous mice of either allele
were observed at weaning (Table 1). To analyze this in more
detail, we focused on just one of the alleles. Using the Lrp1btm2wtsi
allele, a total of 146 mice were genotyped at weaning (4 weeks old).
No homozygous Lrp1btm2wtsimice were detected, suggesting that
homozygous Lrp1b mice were not viable (Table 1). We then
isolated embryos at E8.5 and E10.5 for genotyping by Southern
hybridization but did not find any homozygous Lrp1b embryos at
these timepoints indicating that Lrp1b disruption caused early
embryonic lethality (Table 1).
Lrp1b-deficient Blastocysts Are Viable
Pre-implantation embryos do not provide sufficient material for
Southern analysis and PCR genotyping cannot distinguish
homozygous embryos for each of the mutant alleles individually
from heterozygous embryos. Therefore in order to narrow the
timepoint when embryos in which Lrp1b had been disrupted die
we intercrossed Lrp1btm1wtsiand Lrp1btm2wtsimice, flushed blasto-
cysts at E3.5 and cultured these to form blastocyst outgrowths. In
total, 25 blastocyst outgrowths were analyzed by PCR for the
presence of both mutant alleles which would indicate homozygous
null embryos. Three of these were genotyped as homozygotes
(p.0.1). As shown in Figure 2 these blastocysts showed normal
morphology with a time-and size-appropriate expansion of the
inner cell mass and outgrowth of trophoblast structures. This result
suggests that loss of Lrp1b does not result in a cell lethal phenotype.
Extracellular Domains Are Expressed by Truncated Lrp1b
and Lrp4 Alleles
The expression of the extracellular domains (ECDs) in the
previously reported knockout models of Lrp1b  and Lrp4
[11,13] was predicted but never confirmed. To confirm the
expression of Lrp1b and Lrp4 ECDs, we utilized whole brain
lysates and antibodies against the extracellular and intracellular
ligand binding domains. For Lrp1b, only a slight size difference
was detectable between the protein products from the wild-type
and the Lrp1b truncation allele (Figure 3A). However, the
intracellular domain epitope was only detectable in the wild type.
For Lrp4, the size difference confirmed the expression of the
predicted 180 kDa Lrp4-ECD in the absence of an ICD
(Figure 3B). Expression levels of the truncated ECDs were
equivalent to wild type full-length protein. We thus confirmed
that the extracellular domains are expressed normally and are
stable in both mutant strains.
Lrp4 Undergoes Regulated Intramembranous Processing
It has previously been reported that the ECD of Lrp1b is shed
into the extracellular space in an in vitro model and the ICD is
released by c-secretase activity . To investigate whether Lrp4 is
similarly processed and the extracellular domain shed into the
extracellular space, the supernatants of Lrp4-transfected cells were
analyzed by immunoblotting using an antibody against the ECD of
Lrp4 (Figure 4A). Cell lysates were used to verify transfection
efficiency using the Lrp4 ICD antibody. No shedECD was detected
in the supernatant from untransfected cells (lane 1) or cells that had
been transfected with either ADAM10 (lane 2) or the full length
Lrp4 construct (lane 3) alone. When the Adam10 metalloproteinase
was co-transfected together with Lrp4 to facilitate cleavage of the
extracellular domain, Lrp4-ECD was released from the cell and
became readily detectable in the culture supernatant (lane 4).
Transfection of Lrp4 reveals different protein products at
,20 kDa, 75 kDa and 250 kDa (Figure 4B, lanes 2 and 4); while
the 250 kDa band represents full length Lrp4, the two smaller
bands appear to be processing products of the receptor. No bands
were detected in the untransfected conditions (Figure 4B, lanes 1
and 3). In analogy to other members of the LDL receptor gene
family, the processing of Lrp4 includes extracellular domain
cleavage by metalloproteases and a release of the ICD by c-
secretase activity. Inhibition of c-secretase by DAPT correlates
with accumulation of the ,20 kDa band.
LRP Ectodomain Functions
PLoS ONE | www.plosone.org3 April 2010 | Volume 5 | Issue 4 | e9960
The Lrp4 ECD Inhibitis Canonical Wnt Signaling
Lrp4 has been reported to be a negative regulator of Wnt
signaling. To investigate whether the ECD can mediate this
inhibition on its own, we used a TOP-Flash assay system and
measured b-catenin dependent promoter activity in vitro. Wnt1 was
used to activate signaling at the extracellular level. Dickkopf-1
(Dkk1) has been reported to be a negative regulator of Wnt
signaling  and an Lrp4 binding partner . As expected,
Lrp4 and Dkk1 do not repress Wnt reporter activity in the absence
of Wnt1 (Figure 5, lanes 3 and 4). However, in the presence of
Wnt1, both Dkk1 (Figure 5, lane 5) and Lrp4 (Figure 5, lane 6) can
independently decrease Wnt signaling significantly. In combina-
tion, Dkk1 and Lrp4 synergistically increase Wnt1 signal inhibition
(Figure 5, lane 7).
In this study, we have presented evidence for an essential role of
Lrp1b during embryonic development. From two different Lrp1b
Table 1. Genotyping of Lrp1b heterozygous intercrosses.
TOTAL Fisher’s exact test
4 weeks 451010 146p,0.0001
E10.55 220 27p,0.01
E8.54 150 19p,0.05
E3.511 113 25 NS
Genotyping data at 4 wks, E8.5 and E10.5 was performed on genomic DNA from
intercrosses of Lrp1btm2wtsimice, whereas genotyping of the E3.5s was
performed on genomic DNA from intercrosses of Lrp1btm1wtsiwith Lrp1btm2wtsi
mice. Experimental data was statistically analyzed using Fisher’s exact test. NS,
Figure 1. Generation of Lrp1b null alleles. (A) Duplication of N-terminal exons 6-8 to generate the Lrp1btm1wtsiallele and Southern blot
hybridization after EcoRV digestion of embryonic stem cell genomic DNA to verify targeting of the allele. (B) Duplication of C-terminal exon 69 to
generate the Lrp1btm2wtsiallele and Southern blot hybridization after BamHI digestion of embryonic stem cell genomic DNA to verify targeting of the
allele. A, AflIII; B, BamHI; EV, EcoRV; S, SwaI.
LRP Ectodomain Functions
PLoS ONE | www.plosone.org4 April 2010 | Volume 5 | Issue 4 | e9960
null alleles, no viable offspring or embryos were obtained.
Although blastocyst outgrowths appeared normal, we were unable
to identify viable homozygous Lrp1b mutant embryos at or beyond
E8.5, suggesting that loss of Lrp1b causes early embryonic lethality
and underscoring the importance of this gene for embryonic
development. We have previously reported that mice carrying a
truncated form of Lrp1b exclusively expressing a secreted ECD, are
born at normal Mendelian ratios and are phenotypically
essentially normal. In this earlier study, we had used insertion of
a ‘neomycin’ cassette to replace the transmembrane domain at
exon 88 of Lrp1b, resulting in the predicted truncation of the
receptor and the secretion of a fully folded and functionally
apparently intact ECD . Under physiological conditions,
LRP1b is anchored through its transmembrane domain in the cell
membrane where it can undergo regulated intramembrane
proteolysis (RIP) . The ECD of LRP1b is cleaved by several
metalloproteinases, including ADAM17 and other members of the
ADAM family, in the initial step of receptor processing and leads
to shedding into the extracellular space where its function has not
Figure 2. Blastocyst outgrowth assay. Time course of Lrp1b wildtype (Lrp1b+/+) compared to Lrp1b knockout (Lrp1btm1wtsi/tm2wtsi) trophoblast
explant growth, showing expansion of inner cell mass and trophoblast formation. Images were taken on days 1, 2, 4, and 6.
Figure 3. Expression of Lrp1b and Lrp4 ECDs. Whole brain lysates
(50 mg) from (A) Lrp1b and (B) Lrp4 truncation mutants were analyzed
for expression of the ECD. For the Lrp1b truncation (‘‘Lrp1b EC Stop’’),
the ECD is expressed at approximately the same size as the full-length
receptor (‘‘Wt’’) due to the negligible reduction in predicted protein
mass. However, as expected the ICD epitope is only present in wild-type
tissues. By contrast, in the Lrp4 truncation (‘‘Lrp4 EC Stop’’), there is a
significant shift in size of the ECD protein band compared to full-length
receptor. As for Lrp1b, no ICD is detected in the truncated Lrp4 strain.
b-Actin was used as a loading control.
Figure 4. Lrp4 undergoes regulated intramembraneous pro-
cessing. (A) Lrp4 ECD release is induced by ADAM10 in vitro.
50 mL of concentrated supernatant and 50 mg of cell lysate were
analyzed with a polyclonal antibody detecting either the Lrp4
extracellular domain (Lrp4 ECD) domain (supernatant) or the Lrp4
intracellular domain (cellular extracts). The extracellular domain is
present in the supernatant after transfection with Lrp4 and co-
transfection with the metalloproteinase Adam10 (lane 4), but not in
the absence of Adam10 (lane 3). Immunoblotting for b-actin was used
to demonstrate equal loading. (B) Lrp4 ICD is cleaved by c c-
secretase. Lrp4 expression in 293T cells reveals bands at 20, 75, and
250 kDa (lanes 2 and 4). The protein levels of 250 and 75 kDa species
are independent of DAPT (i.e. c-secretase inhibitor) treatment. By
contrast, the membrane bound ICD at 20 kDa accumulates in the
presence of DAPT. No protein products were detected in the
untransfected lanes (1 and 3). b-Actin was detected to demonstrate
LRP Ectodomain Functions
PLoS ONE | www.plosone.org5 April 2010 | Volume 5 | Issue 4 | e9960
yet been determined . Subsequently, c-secretase activity
mediates the release the intracellular domain from the membrane.
LRP1 and other members of the LDL receptor gene family are
known to bind a wide variety of ligands, including growth factors,
membrane receptors, the amyloid precursor protein, bacterial
toxins, and other proteins . Given their structural similarities,
LRP1b is also likely to bind a comparable spectrum of ligands. In
fact, the amyloid precursor protein , Pseudomonas exotoxin A
 and some other ligands have been reported to also bind to the
ECD of LRP1b. Our gene targeting study to disrupt Lrp1b by
duplicating internal exons of the gene suggests that the ECD can
function independently from the membrane anchored receptor to
regulate critical developmental processes required for embryonic
viability. The shedding of the ECD into the extracellular space
might therefore serve as a soluble ligand scavenger. This event
presumably preserves a critical signaling threshold at an early stage
of embryonic development.
For other members of the LDL receptor gene family, it has been
demonstrated that the cleavage of the extracellular domain can
occur in the native receptor . Interestingly, we have found a
comparable rescue of a severe perinatally lethal phenotype by a
truncated form of Lrp4, where only the ECD remains expressed
[11,13]. Here, we confirmed Lrp4 ECD expression in this mutant
mouse strain and present in vitro evidence that Lrp4 undergoes
regulated intramembraneous processing (RIP) by cleavage and
shedding of the ECD by metalloproteases and ICD release after c-
secretase cleavage. Both steps have important physiological
functions in other LDL gene family members including signal
modulation and transcriptional inhibition.
Furthermore, our in vitro results suggest that Lrp4 ECD can
negatively modulate Wnt signaling. Whether this happens through
cooperation with inhibitory ligands or scavenging of activating
ligands extracellularly remains to be determined. It also remains
presently unclear whether shedding occurs in vivo and on which
Figure 5. Lrp4 ECD Inhibits Wnt signaling in vitro. HEK-293 cells
were transfected using the TOP-Flash reporter system in the presence
of the indicated plasmids (0.5 mg/construct as indicated, where required
empty pcDNA3.1 plasmid DNA was added to bring the DNA
concentration up to a total amount of 2.5 mg plasmid DNA/condition).
Dkk1 and Lrp4 independently inhibit Wnt signaling (lane 5 and 6).
Inhibition of Wnt1 induced reporter activation by co-transfection of
Lrp4 ECD and Dkk-1 is synergistic (lane 7). Lrp5 and Lrp6 are co-
receptors of the frizzled complex and required for Wnt1 mediated
activation, however, HEK-293T cells do not express Lrp5 or 6
endogenously and thus need to be co-transfected with the respective
Figure 6. Summary of known mutations and their respective phenotypes. The known mutations in murine models for (a) Lrp1b and (b) Lrp4
are shown. The presence of the extracellular domain (ECD) rescues the lethality caused by the complete functional null mutation.
LRP Ectodomain Functions
PLoS ONE | www.plosone.org6 April 2010 | Volume 5 | Issue 4 | e9960
physiological processes this may impact. However, anchorage- Download full-text
independent modulation of extracellular conditions seems to play
a crucial role in preserving a threshold for proper cellular signal
input. No specific signaling mechanisms, which are modulated by
Lrp1b are currently known. This hypothesis thus requires further
confirmation once such pathways have been identified.
Deletion of Lrp4 causes perinatal death due to an inability to
form neuromuscular junctions and subsequent respiratory failure
. This phenotype is mitigated in the truncated Lrp4 receptor
expressing only the ECD, allowing the animal to breathe and
move, despite general muscular weakness and hypotrophy.
Another prominent phenotype, involving abnormal distal limb
development, appears to be identical in the null and hypomorph
There are several reports of LRP1b being deleted or epigenet-
ically silenced in a variety of human tumors [3,4,5,7]. The exact
mechanistic role of LRP1b in tumor suppression and development
has remained elusive. The previously reported functional insights
into tumor suppression at the molecular level overlap with its close
relative LRP1. They include the regulation of uPA, uPAR and
PDGF receptor tyrosine kinase [8,25]. However, the lack of
mutations in LRP1  indicate important functions that have
diverged from those of LRP1b. These differences could be
attributed to the distinct selective pressure on the LRP1b gene in
the process of tumor development. It is thus possible that the same
unknown mechanisms that are regulated by the Lrp1b ECD are
involved in tumorigenesis as well as development.
While the release of the intracellular domain and its effect on
inflammatory signaling and proliferation has been described for
both LRP1b  and LRP1 , no such independent function has
been described for the isolated ECDs of either receptor. Our data,
obtained from two distinct mouse models suggest that the ECD of
Lrp1b can function to some extent to maintain signaling
homeostasis even in the absence of membrane integration. In
analogy to LRP1, this might occur through binding of soluble
ligands in the extracellular space .
In summary, we have reported an essential role for Lrp1b in
embryonic development and propose a novel role for Lrp1b and
Lrp4 as signal modulators through ligand scavenging (Figure 6).
Further elucidation of the molecular functions of the LRP1b and
LRP4 ECDs has the potential to provide novel and functionally
significant insights into the role of LRP1b in embryogenesis and
Conceived and designed the experiments: MFD LvdW HMP AB JH DJA.
Performed the experiments: MFD LvdW HMP. Analyzed the data: MFD
LvdW HMP AB JH DJA. Contributed reagents/materials/analysis tools:
HMP AB JH DJA. Wrote the paper: MFD JH.
1. Herz J, Chen Y, Masiulis I, Zhou L (2009) Expanding functions of lipoprotein
receptors. J Lipid Res 50 Suppl: S287–292.
2. Liu CX, Musco S, Lisitsina NM, Forgacs E, Minna JD, et al. (2000) LRP-DIT, a
putative endocytic receptor gene, is frequently inactivated in non-small cell lung
cancer cell lines. Cancer Res 60: 1961–1967.
3. Langbein S, Szakacs O, Wilhelm M, Sukosd F, Weber S, et al. (2002) Alteration
of the LRP1B gene region is associated with high grade of urothelial cancer. Lab
Invest 82: 639–643.
4. Cengiz B, Gunduz M, Nagatsuka H, Beder L, Gunduz E, et al. (2007) Fine
deletion mapping of chromosome 2q21-37 shows three preferentially deleted
regions in oral cancer. Oral Oncol 43: 241–247.
5. Nakagawa T, Pimkhaokham A, Suzuki E, Omura K, Inazawa J, et al. (2006)
Genetic or epigenetic silencing of low density lipoprotein receptor-related
protein 1B expression in oral squamous cell carcinoma. Cancer Sci 97:
6. Sonoda I, Imoto I, Inoue J, Shibata T, Shimada Y, et al. (2004) Frequent
silencing of low density lipoprotein receptor-related protein 1B (LRP1B)
expression by genetic and epigenetic mechanisms in esophageal squamous cell
carcinoma. Cancer Res 64: 3741–3747.
7. Rahmatpanah FB, Carstens S, Guo J, Sjahputera O, Taylor KH, et al. (2006)
Differential DNA methylation patterns of small B-cell lymphoma subclasses with
different clinical behavior. Leukemia 20: 1855–1862.
8. Boucher P, Liu P, Gotthardt M, Hiesberger T, Anderson RG, et al. (2002)
Platelet-derived growth factor mediates tyrosine phosphorylation of the
cytoplasmic domain of the low Density lipoprotein receptor-related protein in
caveolae. J Biol Chem 277: 15507–15513.
9. Loukinova E, Ranganathan S, Kuznetsov S, Gorlatova N, Migliorini MM, et al.
(2002) Platelet-derived growth factor (PDGF)-induced tyrosine phosphorylation
of the low density lipoprotein receptor-related protein (LRP). Evidence for
integrated co-receptor function betwenn LRP and the PDGF. J Biol Chem 277:
10. Marschang P, Brich J, Weeber EJ, Sweatt JD, Shelton JM, et al. (2004) Normal
development and fertility of knockout mice lacking the tumor suppressor gene
LRP1b suggest functional compensation by LRP1. Mol Cell Biol 24: 3782–3793.
11. Johnson EB, Hammer RE, Herz J (2005) Abnormal development of the apical
ectodermal ridge and polysyndactyly in Megf7-deficient mice. Hum Mol Genet
12. Weatherbee SD, Anderson KV, Niswander LA (2006) LDL-receptor-related
protein 4 is crucial for formation of the neuromuscular junction. Development
13. Johnson EB, Steffen DJ, Lynch KW, Herz J (2006) Defective splicing of Megf7/
Lrp4, a regulator of distal limb development, in autosomal recessive mulefoot
disease. Genomics 88: 600–609.
14. Croy JE, Shin WD, Knauer MF, Knauer DJ, Komives EA (2003) All three LDL
receptor homology regions of the LDL receptor-related protein bind multiple
ligands. Biochemistry 42: 13049–13057.
15. Ohazama A, Johnson EB, Ota MS, Choi HY, Porntaveetus T, et al. (2008) Lrp4
modulates extracellular integration of cell signaling pathways in development.
PLoS One 3: e4092.
16. Adams DJ, Biggs PJ, Cox T, Davies R, van der Weyden L, et al. (2004)
Mutagenic insertion and chromosome engineering resource (MICER). Nat
Genet 36: 867–871.
17. Ramirez-Solis R, Liu P, Bradley A (1995) Chromosome engineering in mice.
Nature 378: 720–724.
18. McMahon AP, Bradley A (1990) The Wnt-1 (int-1) proto-oncogene is required
for development of a large region of the mouse brain. Cell 62: 1073–1085.
19. Glinka A, Wu W, Delius H, Monaghan AP, Blumenstock C, et al. (1998)
Dickkopf-1 is a member of a new family of secreted proteins and functions in
head induction. Nature 391: 357–362.
20. Choi HY, Dieckmann M, Herz J, Niemeier A (2009) Lrp4, a novel receptor for
Dickkopf 1 and sclerostin, is expressed by osteoblasts and regulates bone growth
and turnover in vivo. PLoS One 4: e7930.
21. Liu CX, Ranganathan S, Robinson S, Strickland DK (2007) gamma-Secretase-
mediated release of the low density lipoprotein receptor-related protein 1B
intracellular domain suppresses anchorage-independent growth of neuroglioma
cells. J Biol Chem 282: 7504–7511.
22. Marzolo MP, Bu G (2009) Lipoprotein receptors and cholesterol in APP
trafficking and proteolytic processing, implications for Alzheimer’s disease.
Semin Cell Dev Biol 20: 191–200.
23. Pastrana DV, Hanson AJ, Knisely J, Bu G, Fitzgerald DJ (2005) LRP 1 B
functions as a receptor for Pseudomonas exotoxin. Biochim Biophys Acta 1741:
24. Willnow TE, Moehring JM, Inocencio NM, Moehring TJ, Herz J (1996) The
low-density-lipoprotein receptor-related protein (LRP) is processed by furin in
vivo and in vitro. Biochem J 313(Pt 1): 71–76.
25. Tanaga K, Bujo H, Zhu Y, Kanaki T, Hirayama S, et al. (2004) LRP1B
attenuates the migration of smooth muscle cells by reducing membrane
localization of urokinase and PDGF receptors. Arterioscler Thromb Vasc Biol
26. Zurhove K, Nakajima C, Herz J, Bock HH, May P (2008) Gamma-secretase
limits the inflammatory response through the processing of LRP1. Sci Signal 1:
27. Herz J, Strickland DK (2001) LRP: a multifunctional scavenger and signaling
receptor. J Clin Invest 108: 779–784.
LRP Ectodomain Functions
PLoS ONE | www.plosone.org7 April 2010 | Volume 5 | Issue 4 | e9960