The lipoprotein receptors (including the LDL receptor,
VLDLR, ApoER2/LRP8, LRP1 and LRP2/megalin) comprise
a family of single-pass type I membrane proteins that mediate
uptake of various protein cargoes into cells via the endocytic
pathway (Krieger and Herz, 1994). Each receptor binds many
different cargo proteins (more than 35 for megalin) and
continuously recycles to and from the cell surface. The
receptors cluster in clathrin-coated pits, are internalized and
sorted to early endosomes. Following dissociation of ligands
in late endosomes, the receptors are returned to the cell surface.
The whole cycle occurs rapidly, just 5 minutes to internalize
and another 15 minutes or so to recycle, depending on the cell
type and the receptor.
Endocytosis of the prototype lipoprotein receptor, the LDL
receptor, requires an FxNPxY signal, with crucial F, N, P and
Y residues, in the cytoplasmic tail (Chen et al., 1990). Most
lipoprotein receptors contain a similar sequence, although the
importance of this signal for endocytosis of some receptors has
been questioned (Li et al., 2000). The identity of the protein or
proteins that interact with the FxNPxY signal and mediate
endocytosis has been unclear until recently.
The first reported interaction, with clathrin, is still of
unknown functional significance (Kibbey et al., 1998).
However, two protein-interaction–phosphotyrosine-binding
(PID-PTB) domain-containing proteins, ARH and Dab2, also
bind (Bork and Margolis, 1995; He et al., 2002; Mishra et al.,
2002; Morris and Cooper, 2001; Oleinikov et al., 2000), and
ARH is required genetically for efficient LDL receptor uptake
in liver and lymphocytes (Garcia et al., 2001; Norman et al.,
1999). Dab2 is important for megalin transport in the kidney
proximal tubule but its role in other tissues is unclear (Morris
et al., 2002b; Nagai et al., 2005).
The dab2 gene is alternatively spliced to produce two protein
products (Xu et al., 1995), one of which, p96, binds to clathrin
and the clathrin adaptor AP2, and localizes to clathrin-coated
pits, whereas the other, p67, does not (Mishra et al., 2002;
Morris and Cooper, 2001). Significantly, overexpression of a
dimer of the PTB domain inhibits internalization of LDL,
indicating that the Dab2 PTB domain can displace the protein
or proteins that normally mediate LDLR endocytosis (Mishra
et al., 2002). However, absence of Dab2 does not affect LDLR
endocytosis in a variety of cultured cells (M.E.M. and J.A.C.,
Dab2 is essential in the visceral endoderm (VE) for
embryonic development (Morris et al., 2002b; Yang et al.,
2002). The VE is a polarized epithelial tissue, with a well-
defined brush border composed of dense apical microvilli, that
surrounds the developing
implantation. Between embryonic days (E) 5.5 and 7.5,
nutrients supplied by the VE support the rapid proliferation of
the epiblast (Bielinska et al., 1999; Snow, 1977). The VE also
plays an active role in patterning the early embryo (Beddington
and Robertson, 1999; Coucouvanis and Martin, 1999; Rossant
and Tam, 2004). Conditional knockout of the dab2 gene from
most embryonic cells but not the VE allows normal
mammalian embryo after
Rapid endocytosis of lipoprotein receptors involves NPxY
signals contained in their cytoplasmic tails. Several
proteins, including ARH and Dab2, can bind these
sequences, but their importance for endocytosis may vary
in different cell types. The lipoprotein receptor megalin is
expressed in the visceral endoderm (VE), a polarized
epithelium that supplies maternal nutrients to the early
mammalian embryo. Dab2 is also expressed in the VE, and
is required for embryo growth and gastrulation. Here, we
show that ARH is absent from the VE, and Dab2 is
required for uptake of megalin, its co-receptor cubilin, and
a cubilin ligand, transferrin, from the brush border of the
VE into intracellular vesicles. By making isoform-specific
knock-in mice, we show that the p96 splice form of Dab2,
which binds endocytic proteins, can fully rescue
endocytosis. The more abundant p67 isoform, which lacks
some endocytic protein binding sites, only partly rescues
endocytosis. Endocytosis of cubilin is also impaired in VE
and in mid-gestation visceral yolk sac when p96 is absent.
These studies suggest that Dab2 p96 mediates endocytosis
of megalin in the VE. In addition, rescue of embryonic
viability correlates with endocytosis, suggesting that
endocytosis mediated by Dab2 is important for normal
Key words: Dab2, Megalin, Endocytosis, Visceral endoderm,
Lipoprotein receptor traffic, Endocytic adaptor protein
Endocytosis of megalin by visceral endoderm cells
requires the Dab2 adaptor protein
Meghan E. Maurer and Jonathan A. Cooper*
Division of Basic Sciences and Molecular and Cellular Biology Program, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Seattle,
WA, 98109, USA
*Author for correspondence (e-mail: firstname.lastname@example.org)
Accepted 16 August 2005
Journal of Cell Science 118, 5345-5355 Published by The Company of Biologists 2005
Journal of Cell Science
JCS ePress online publication date 1 November 2005
development (Morris et al., 2002b). However, the essential
function of dab2 in the VE has previously been unclear.
Here we show that the p96 isoform of Dab2 is essential for
normal endocytosis and development. Endocytosis of
transferrin (Tf) is decreased and the lipoprotein receptor
megalin is mislocalized in the VE of dab2 mutants. The
scavenger receptor cubilin lacks a cytoplasmic domain and is
dependent on megalin for endocytosis (Christensen and Birn,
2002; Kozyraki et al., 2001). Accordingly, cubilin is also mis-
localized in the VE of dab2 mutants. Even though Dab2 p67
is more highly expressed than p96 in the VE, expression of p67
alone led to decreased endocytosis, delayed development and
reduced viability compared to control embryos, whereas
expression of p96 was sufficient for normal endocytosis and
development. These results indicate that Dab2 p96 mediates
endocytosis of megalin and cubilin in the VE.
Materials and Methods
dab2 knockout mice
Previously described dab2–/–mice were used for these studies (Morris
et al., 2002b). These mice were maintained on a mixed
129Sv/C57BL/6 genetic background. The phenotype of these mice is
similar to that of embryos in which dab2 is not expressed because of
the absence of GATA6, a zinc-finger transcription factor that directs
expression of dab2 in the VE (Morrisey et al., 2000; Morrisey et al.,
1998). Other alleles of dab2 and gata6, both of which were disrupted
by ?-galactosidase, cause earlier embryonic lethality with a
disorganized VE, suggesting that expression of ?-galactosidase
increases the severity of the phenotype when dab2 is not expressed
(Koutsourakis et al., 1999; Yang et al., 2002).
E6.5 and E7.5 embryos were dissected in PBS and allowed to recover
for 1 hour in 75% fetal bovine serum (FBS) with 25% Dulbecco’s
modified Eagle’s medium (DMEM) at 37°C in 5% CO2. Horseradish
peroxidase (HRP) and Texas-red-labeled transferrin (TR-Tf) were
added at 2 mg/ml and 25 ?g/ml, respectively, for 5 minutes at 37°C.
Following uptake, embryos incubated with TR-Tf were incubated in
ice-cold acid stripping solution (150 mM NaCl, 10 mM NaOAc pH
5.0) for 5 minutes to remove surface-bound Tf. Embryos were fixed
in 4% Paraformaldehyde (PFA) for 1 hour at 4°C, followed by three
5-minute washes in PBS. HRP uptake was detected by incubating
embryos in diaminobenzidine tetrahydrochloride (DAB) solution plus
nickel until a color change was observed (~10 minutes), followed by
a water rinse to stop the reaction. TR-Tf uptake was detected using
fluorescence microscopy. To score uptake, a central region of each
embryo was used to generate a histogram from which the average
pixel intensity was determined. These values were averaged for each
genotype, and the standard error was calculated. Significance was
determined by using the Mann-Whitney test. Embryos were
genotyped by PCR.
E6.5 embryos were fixed in Karnovsky’s half-strength fixative for 36
hours and post-fixed in osmium s-collidine for 8 hours. Samples were
dehydrated for 1 hour each in 35, 70 and 95% ethanol, followed by
two washes with 100% ethanol and propylene oxide. Samples were
then infiltrated with 50:50 propylene oxide:Epon 812 and placed in a
vacuum oven overnight. Fresh Epon 812 was added and samples were
returned to the vacuum oven overnight. Embryos were embedded in
fresh Epon 812 and allowed to harden in the oven for 48 hours.
Sections (400-600 nm) were placed on 150 mesh grids and stained for
2 hours with 6% saturated uranyl acetate, then with Millonig’s lead
stain for 4 minutes. Sections were viewed using the JEOL 100SX
transmission electron microscope.
Embryos were fixed in 4% PFA, paraffin embedded and sectioned.
Immunohistochemistry was performed using the Vectastain Elite
ABC Kit (Vector Laboratories, Inc.) as described (Morris et al.,
2002b). Briefly, 6-?m paraffin sections were de-waxed in Histoclear,
rehydrated in serial dilutions of alcohol and steamed for 20 minutes
in 30 mM citrate buffer pH 4.8, for antigen retrieval. Sections were
rinsed in PBS and incubated in 3% hydrogen peroxide for 5 minutes
to block endogenous peroxidases. Sections were rinsed and blocked
for 30 minutes in 5% normal serum with 2% bovine serum albumin
(BSA) in PBS. Slides were then incubated overnight at 4°C with a
1:200, 1:200 or 1:400 dilution of mouse anti-Dab2 (p96) (BD
Transduction Labs), and goat anti-cubilin (Santa Cruz) or sheep anti-
megalin (kind gift from R. Nielsen, University of Aarhus, Aarhus,
Denmark) diluted in 5% BSA in PBS. Following three 5-minute
washes in PBS plus 0.05% Tween-20 (PBST), sections were incubated
for 30 minutes with horse anti-mouse, rabbit anti-goat, or rabbit anti-
sheep biotinylated secondary antibodies diluted 1:200 in 2% BSA in
PBS. Slides were washed again in PBST and incubated for 30 minutes
with Vectastain Elite ABC Reagent. Following the last wash of PBST,
sections were incubated in DAB solution plus nickel until a color
change was observed (~5 minutes) and slides were rinsed in water.
Sections were counterstained with hematoxylin and mounted using
Immunofluorescence was performed on whole-mount, paraffin- and
cryostat-sectioned E6.5 embryos, and cryostat-sectioned kidneys.
Embryos were isolated and fixed in 4% PFA-PBS for 2 hours at 4°C,
and kidneys were fixed by perfusion with 4% PFA-PBS. Prior to
freezing in optimal cutting temperature compound (OCT, Tissue-Tek),
cryostat-sectioned embryos and kidneys were taken through a series
of 30% sucrose-PBS:OCT incubations (100%:0%, 50%:50%,
25%:75%, and 0%:100%). 7 ?m cryostat sections were rehydrated in
PBS. Whole-mount and cryostat-sectioned tissues were permeabilized
in 0.1% Triton X-100 in PBS for 20 minutes at 25°C. Tissues were
blocked in 5% normal serum with 2% BSA in PBS for 1 hour before
incubating with mouse anti-Dab2 (1:200), rabbit anti-ARH (1:20, kind
gift from M. Farquhar, University of California, San Diego, CA), goat
anti-cubilin (1:200), sheep anti-megalin (1:1000) and/or rabbit anti-
EEA1 (1:200) antibody (Affinity BioReagents) overnight at 4°C.
Paraffin-embedded embryos were prepared as described above, and
immunofluorescence for Dab2
immunohistochemistry up to the point of secondary antibody addition.
Following three 5-minute washes in PBST, whole-mount, cryostat-
and paraffin-sectioned tissues were incubated for 1 hour with the
appropriate AlexaFluor-labeled secondary antibody (Molecular
Probes) diluted 1:1000. Following three 5-minute washes in PBST,
4?,6-diamidino-2-phenylindole (DAPI, 1:1000, Sigma) was added for
10 minutes. Sections were rinsed with water and mounted using the
ProLong Antifade Kit (Molecular Probes). For visualization, 0.2 ?m
serial sections were taken using a Delta Vision microscope (Applied
Precision), and the images were deconvolved and analyzed using the
softWoRx program (Applied Precision).
was performed like
Tissues were lysed on ice in lysis buffer (1% Triton X-100, 10 mM
HEPES pH 7.4, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 0.2 M
Na3VO4, 1% aprotonin, 1 mM PMSF, 10 mg/ml leupeptin) followed
by centrifugation at 20,000 g for 10 minutes at 4°C. Samples were
Journal of Cell Science 118 (22)
Journal of Cell Science
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Journal of Cell Science