1 Max-Delbrueck-Center for Molecular Medicine and 2Medical Faculty of
the Free University of Berlin, Germany
2 Department of Medical Biochemistry, University of Aarhus, Aarhus,
Denmark
3 Center for Human Genetics, K. U. Leuven and Flanders Interuniversity Institute
for Biotechnology, Leuven, Belgium
* Author for correspondence (e-mail: willnow{at}mdc-berlin.de)
Accepted 29 October 2002
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Summary |
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Key words: Scaffold protein, Endocytosis, Forebrain development, LDL receptor gene family, Vitamin D metabolism
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Introduction |
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Remarkably, endocytic as well as signaling functions of LDL receptor gene
family members are controlled by adaptor proteins that recognize binding
epitopes in the cytoplasmic tail of the receptors. Because these adaptors
simultaneously interact with other components of the cellular machinery, they
assemble multimeric protein complexes that determine receptor activities. For
example, binding of the adaptor complex AP-2 couples receptors to the clathrin
coat of endocytic vesicle and facilitates endocytosis
(Kirchhausen et al., 1997).
Interaction with the adaptor disabled (Dab)1 may link receptors with
intracellular tyrosine kinases of the Src and Abl family and modulates
downstream signal transduction pathways
(D'Arcangelo et al., 1999
;
Hiesberger et al., 1999
).
The molecular mechanisms that control interaction of the receptors with
adaptor proteins and the consequences for endocytosis and signal transduction
are far from being fully understood. Specific epitopes in the receptor tails
serve as high-affinity binding site for cytoplasmic scaffold or adaptor
proteins. Among others, NPxY motifs, (S/T)xY elements, as well as SH3 binding
sites have been identified in the receptor tails
(Gotthardt et al., 2001;
Rader et al., 2001
).
Interestingly, each receptor species harbors a unique combination of binding
motifs that may even be changed by alternative splicing, further supporting
the concept that adaptor interactions play a crucial role in the regulation of
individual receptor functions (Stockinger
et al., 2002
). As far as interacting partners are concerned, a
number of adaptor proteins have been uncovered that bind to the LDL receptor
gene family. They represent three main types of proteins: proteins with
phosphotyrosine-binding domain (PTB)
(Hiesberger et al., 1999
;
Gotthardt et al., 2001
;
Oleinikov et al., 2001
), with
PDZ domain (Gotthardt et al.,
2001
; Patrie et al.,
2001
) or with an ankyrin repeat
(Rader et al., 2001
). The
significance of most of these adaptors for receptor function remains
unknown.
To better understand the molecular interactions of adaptors with LDL
receptor gene family members, we have performed new yeast two-hybrid screens
to identify novel interaction partners for megalin, a member of the gene
family that plays an important role in brain development and in renal vitamin
D metabolism; functions that probably involve signal transduction
(Willnow et al., 1996;
Nykjaer et al., 1999
;
McCarthy et al., 2002
). We
identified a novel class of adaptor molecule with tetratrico peptide repeat
domains, designated megalin-binding protein (MegBP) that functionally
interacts with megalin and links this receptor to transcription regulation
pathways.
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Materials and Methods |
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Yeast two-hybrid screen
The cDNA sequence of the human megalin tail or truncations thereof were
produced by RT-PCR from total RNA of human embryonic kidney cell line 293 and
confirmed by sequencing. The following primers were used:
5'-GATCCTCATCATATGCACTATAGAAGGACCGGCTC-3' and
5'-GGTGGTGGGATCCCTATTACTATACTTCAGAGTCTTCTTTAACAAGATTTGCGGTGTCTTT-3'
(full-length tail), 5'-GATCCTCATCATATGCACTATAGAAGGACCGGCTC-3' and
5'-GGTGGTGGGATCCCTATTACACTTTGACAGCACTGCTCTG-3' (fragment AB),
5'-GATCCTCATCATATGAAAGTGGTTCAGCCAATCC-3' and
5'-GGTGGTGGGATCCCTATTACTATCTTCAGAGTCTTCTTTAACAAGATTTGCGGTGTCTTT-3'
(fragment CD), 5'-GATCCTCATCATATGCACTATAGAAGGACCGGCTC-3' and
5'-GGATCCCTATTACACTCCAATATCCATGTTAAGATC-3' (fragment A) and
5'-CATATGGGAGTGTCTGGTTTTGGACCT-3' and
5'-GGTGGTGGGATCCCTATTACACTTTGACAGCACTGTCTCTG-3' (fragment B). The
sequences were cloned into vector pAS2-1 (Clontech,
www.clontech.com)
via EcoRI and BamHI restriction sites and used as bait to
screen a GAL4 Matchmaker library from human brain tissue (HY4028AH, Clontech)
according to the manufacturers' recommendations. Positive clones were isolated
from the yeast strain Y190, sequenced, and their interaction with the megalin
tail confirmed by retransformation of the purified plasmids in the absence or
presence of the megalin bait vector. Similarly, truncations of the megalin
tail sequence were generated by PCR cloning approach, introduced into vector
pAS2-1, and their interaction with MegBP tested by transformation of the
respective constructs into yeast strain Y190.
Expression of recombinant proteins
GST and GST-fusions of full-length murine MegBP or the protein interacting
domain of Dab1 (Gotthardt et al.,
2001) were obtained by cloning of the respective gene sequences
into expression vector pGEX-4T-1 (Amersham,
www.amershambiosciences.com)
and expression in DH5
bacteria. Recombinant proteins were purified
according to standard procedures using glutathione-agarose affinity
chromatography. An N-terminal fusion of the megalin tail with a hexahistidine
epitope (His-megalin tail) was produced by cloning of the receptor sequence
into vector pET14b (Novagen,
www.novagen.com)
and by purification of the fusion protein from BL21 bacteria by routine Ni-NTA
affinity chromatography. A fusion protein of GST, Discosoma red (DsRed)
fluorescent protein and rat RAP (GST-DsRed-RAP) was generated by introducing
the DsRed sequence (from vector pDsRed, Clontech) in-frame into a pGEX
expression construct containing the GST-rat RAP sequence (provided by J.
Herz). The fusion protein was purified from DH5
bacteria by
glutathione-agarose affinity chromatography. The purity of all protein
preparations was confirmed by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE).
In vitro protein interaction
For ligand blot analysis, equal amounts of purified GST or full-length
GST-fusion proteins were subjected to reducing 10% SDS-PAGE and transferred
onto nitrocellulose membranes. Membranes were blocked for 2 hours at room
temperature in buffer A containing 100 mM Tris/HCl, pH 8.0, 0.9% (w/v) NaCl,
and 2% (v/v) Tween-10 (binding buffer). Thereafter, the membranes were
incubated for 16 hours at 4°C in buffer B (50 mM Tris/HCl, pH 7.5, 150 mM
NaCl, 2 mM CaCl2, 2 mM MgCl2, 0.5% bovine serum albumin)
containing 125I-labeled His-megalin tail protein
(1x106 cpm/ml), washed four times for 15 minutes in buffer B
at room temperature and exposed to X-ray film. Iodination of the His-megalin
tail protein followed the protocol of Fraker and Speck
(Fraker and Speck, 1978). For
coprecipitation experiments, 110 µg of anti-GST IgG (G-7781; Sigma,
www.sigma.com)
was incubated with either 35 µl GST-MegBP (0.869 mg/ml) or 35 µl binding
buffer for 2 hours at 4°C. Then, 455 µl renal membrane extracts (0.824
mg/ml) were added to each sample and incubated for an additional 2 hours at
4°C. IgG-bound proteins were recovered using the Seize Classic
Immunoprecipitation Kit (#45213; Pierce,
www.piercenet.com).
BIAcore analysis of the interaction of adaptor proteins with immobilized
rabbit megalin or the megalin tail protein has been described in detail before
(Nykjaer et al., 1999
).
Cell culture studies
Sequences encoding the full-length mouse MegBP or the partial human clone
isolated from the GAL4 Matchmaker library were introduced into vector pEGFP-C1
(Clontech) using XhoI and EcoRI restriction sites and
expressed as a C-terminal fusion with the enhanced green fluorescent protein
(EGFP). For protein expression, cell lines 293 (human embryonic kidney cells),
BN16 (rat choriocarcinoma cells), LLCPK-1 (Lewis lung carcinoma porcine kidney
cells), L2 (rat yolk sac cells) and MEF (murine embryonic fibroblasts) were
grown in standard DMEM containing 10% fetal calf serum (FCS). Cells were
transfected with 0.5-1 µg/ml of plasmids encoding EGFP or EGFP-MegBP by
biolistic particle delivery (BN16; Biolistic PDS-100 delivery system,
www.biorad.com)
or liposomal transfection technology (Fugene 6; Roche,
www.roche.com).
Expression of the proteins was confirmed by fluorescence microscopy (Olympus
BX51 microscope) or confocal fluorescence microscopy (Leica TCS SP2). For
immunodetection of megalin, cells were fixed in 4% (w/v) paraformaldehyde,
blocked for 1 hour at 37°C with 10% FCS/PBS and incubated for 1 hour at
37°C with a goat anti-rabbit megalin antibody (1:1000 in 10%FCS/PBS).
Bound IgG was detected by subsequent incubation for 1 hour at 37°C with a
Cy5-conjugated rabbit anti-goat antibody (10 µg/ml in 10% FCS/PBS;
#81-1616, ZYMED;
www.zymed.com).
The endocytic uptake of GST-DsRed-RAP was tested by incubation of the cells in
phenol red-free DMEM containing 30 µg/ml of purified fusion protein
followed by fluorescence microscopy.
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Results |
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In the following experiments, we focused our attention on MegBP because
TPR-containing proteins have previously not been considered as adaptors of the
LDL receptor gene family. We confirmed the interaction of MegBP with the
receptor employing ligand blot and surface plasmon resonance (BIAcore)
analysis. To do so, a fusion protein of glutathione S-transferase (GST) and
full-length murine MegBP (GST-MegBP) was constructed and expressed in
bacteria. The intact protein could be recovered from bacterial extracts by
glutathione-agarose affinity chromatography
(Fig. 2, lane 1). Additional
lower molecular weight proteins in the GST-MegBP preparation represented
degradation products of the fusion protein that included the GST moiety as
demonstrated by western blot analysis using an anti-GST antibody
(Fig. 2, lane 4). As a positive
control for the binding assays, we expressed and purified a GST fusion of the
protein interacting domain of Dab1, an established adaptor of megalin
(Gotthardt et al., 2001)
(Fig. 2, lanes 3 and 6). By
ligand blot analysis, we demonstrated strong binding of the iodinated megalin
tail to full-length GST-MegBP and to GST-Dab1 but not to GST or GST-MegBP
degradation products (Fig. 2,
lanes 7 to 9). By coprecipitation experiments, we specifically precipitated
endogenous megalin from renal mouse extracts using the GST-MegBP fusion
protein and glutathione agarose beads (Fig.
3). Finally, by BIAcore analysis, we tested binding of a dilution
series of GST-MegBP and GST-Dab1 preparations to the full-length receptor
protein and to the recombinant tail domain immobilized on the sensor chip
surface (Fig. 4). GST-MegBP
reversibly interacted with the full-length receptor and the receptor tail
domain with a Kd of 1.1 and 0.8 µM, respectively
(Fig. 4A,B). Similar affinities
were obtained when binding of GST-Dab1 to the receptor (0.25 µM) or to the
tail fragment was analyzed (0.15 µM)
(Fig. 4C,D). GST alone did not
interact with the receptor polypeptides
(Fig. 4B,D). As GST-MegBP bound
with the same affinity to native megalin and to the recombinant tail, these
data indicated a specific binding site for MegBP in the cytoplasmic but not in
the extracellular receptor domain.
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Next, we mapped the binding site of MegBP on the receptor tail to gain information about recognition motifs for TPRs. We generated truncations of the human megalin tail sequence and tested their interaction with MegBP in the yeast two-hybrid system. As summarized in Fig. 5, MegBP specifically bound to the N-terminal half of the megalin tail (fragment AB) that encompasses one NPxY and a PxxP motif. No binding to the C-terminal region (fragment CD), which includes the second NPxY motif and the (S/T)xY element of the receptor, was seen. In further fine mapping, the binding site of MegBP was narrowed down to stretch of 45 amino acids adjacent to the membrane anchor of the receptor (fragment A). This polypeptide sequence included the PxxP motif but lacked the NPxY element.
|
To test whether there is a functional interaction between megalin and MegBP in vivo, we tried to transiently express MegBP as a fusion with the enhanced green fluorescent protein (EGFP) in mammalian cell lines. Surprisingly, no or very faint EGFP-MegBP expression could be detected 24 to 48 hours after transfection of cell lines BN16 and L2 (Fig. 6). These cells expressed large amounts of megalin as demonstrated by western blot analysis (Fig. 7). By contrast, the cell lines readily produced significant amounts of EGFP as shown by fluorescence microscopy (Fig. 6). Contrary to BN16 and L2 cells, strong expression of EGFP-MegBP was achieved in a number of cell lines that did not produce endogenous megalin, including 293 cells, LLCPCK1 cells or murine embryonic fibroblasts (MEF) (Figs 6 and 7). In these cells, MegBP was seen as a punctuate and vesicular staining pattern. The signal was distinctly different from the cytoplasmic staining seen for EGFP in all the cell lines (Fig. 6).
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|
The data obtained so far indicated that high levels of MegBP may be incompatible with megalin expression in BN16 and L2 cells. Therefore, we tested expression of MegBP in BN16 cells at shorter time points after transfection. Consistent with our hypothesis, low levels of MegBP expression could be seen in BN16 cells 4 hours after transfection (Fig. 8). The protein was detected in a vesicular staining pattern close to the plasma membrane and throughout the cytoplasm (Fig. 8B). A localization close to the plasma membrane partially overlapped with the localization of megalin (Fig. 8A,C), suggesting interaction of the proteins in cells. Consistent with an impaired viability, all MegBP-expressing BN16 cells died within 24 hours of transfection. Cell death was not caused by apoptosis as was demonstrated by the absence of cells positive for double-stranded low molecular weight DNA (TUNEL reaction). Rather, MegBP-expressing cells were characterized by disintegration of cellular architecture and necrosis (data not shown).
|
Previously, it was speculated that binding of adaptors to the tail of
megalin may block binding of the AP-2 complex to the receptor and, thus,
interfere with endocytosis (Rader et al.,
2001). To test whether MegBP binding to megalin has such an effect
we established an in vivo fluorescence assay to analyze endocytic uptake of
ligands via megalin. As a model ligand we used the receptor-associated protein
(RAP), a high-affinity ligand of the receptor
(Christensen et al., 1992
). RAP
was expressed as a fusion protein with Discosoma red (DsRed) fluorescent
protein (GST-DsRed-RAP). GST-DsRed-RAP bound to megalin in BIAcore assays
(data not shown) and was taken up via this receptor pathway in BN16 cells as
shown by immunofluorescence microscopy
(Fig. 9A,B). We tested uptake
of this ligand in BN16 cells that were transiently transfected with the
EGFP-MegBP construct. Four hours after transfection, internalization of
GST-DsRed-RAP was seen in all cells, including those that expressed the
adaptor (Fig. 9C,D). These
findings demonstrated that MegBP expression interfered with the viability of
megalin-positive cells; however, it did not inhibit the endocytic activity of
the receptor.
|
The presence of TPRs in MegBP suggested a role for them in protein-protein
interaction and perhaps in the assembly of protein complexes at the
cytoplasmic domain of megalin. To identify candidates that may interact with
MegBP and thereby be recruited to the receptor tail, we used the MegBP
sequence as a bait to screen a human brain library. A number of clones was
identified by yeast two-hybrid screening that specifically bound to this
adaptor (Table 2). In keeping
with the proposed role of megalin in signaling processes, potential
interaction partners for MegBP could be grouped into two distinct
physiological pathways: (i) transcriptional regulators such as SKI-interacting
protein (SKIP), transforming growth factor (TGF)-ß-stimulated clone 22
homologous gene (THG-1) and Hlark; or (ii) components of signal transduction
cascades including protein kinase C -binding protein (PRKCABP),
mitogen-activated protein kinase kinase kinase 10 (MAP3K) and brain-specific
angiogenesis inhibitor 2 (BAI2).
|
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Discussion |
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Recently, a surprising role for the LDL receptor gene family in cellular
signal transduction has been uncovered
(Trommsdorff et al., 1999).
This function has been characterized in detail for the very low-density
lipoprotein (VLDL) receptor and the apolipoprotein (apo) E receptor-2, two
family members expressed in migrating neurons in the embryonic brain. These
neurons receive positional information through binding of the extracellular
factor reelin to both receptors. Binding of reelin results in phosphorylation
of Dab1 bound to the receptor tails and in activation of downstream signaling
pathways (Hiesberger et al.,
1999
; D'Arcangelo et al.,
1999
). Genetic defects in the VLDL receptor and in the apo E
receptor-2 in mice result in abnormal layering of neurons in the cortex,
hippocampus and cerebellum (Trommsdorff et
al., 1999
). The same phenotype is seen in mice with Dab1 or reelin
gene defects (D'Arcangelo et al.,
1995
; Sheldon et al.,
1997
).
As well as the VLDL receptor and the apoE receptor-2, megalin also plays a
crucial role in brain development as judged by the forebrain formation defects
seen in knockout mice (Willnow et al.,
1996). In addition, the receptor is essential for the retrieval of
filtered vitamin D metabolites in the proximal tubules of the kidney and for
regulation of the systemic vitamin D and bone metabolism
(Nykjaer et al., 1999
;
Hilpert et al., 2002
). Several
lines of evidence suggest that adaptors may contribute to these receptor
functions. Thus, a patient was identified that suffered from
holoprosencephalic syndrome in combination with renal tubular resorption
defects and vitamin D deficiency (Muller
et al., 2001
). Although this syndrome did not involve the megalin
gene directly, as shown by haplotype analysis, the close phenotypic similarity
with murine megalin deficiency suggested receptor malfunction caused by an
adaptor defect as the underlying disease mechanism
(Muller et al., 2001
). A
similar finding was obtained by Hobbs and colleagues who detected mutations in
the adaptor ARH (autosomal recessive hypercholesterolemia) as the reason for
LDL receptor dysfunction in a kindred with familial hypercholesterolemia
(Garcia et al., 2002
).
Additional support for a role of megalin in signaling processes through
adaptor proteins came from studies that uncovered megalin as a receptor for
sonic hedgehog, a central factor in forebrain development
(McCarthy et al., 2002
), and
from the identification of a number of proteins that interacted with the
receptor tail. These adaptors included the scaffold proteins JIP-1, JIP-2 and
Dab1 in the brain (Gotthardt et al.,
2001
), as well as Dab2
(Oleinikov et al., 2001
),
ANKRA (Rader et al., 2001
) and
MAGI-1 (Patrie et al., 2001
)
in the kidney.
Although little is known about the physiological significance of the
identified adaptors for megalin function, these studies have already provided
some insights into the molecular structures required for receptor and adaptor
interaction. So far, three major types of adaptors have been shown to bind to
the receptor tail. PTB-containing proteins (e.g. Dab1) bind to NxPY motifs in
the receptor tail, whereas PDZ domain proteins interact with the motif (S/T)xY
(e.g., MAGI-1). A third group of adaptors, represented by the
ankyrin-repeat-containing protein ANKRA, may recognize a proline-rich PxxP
element (Rader et al., 2001).
MegBP constitutes a novel class of megalin adaptor with TPR structure. It
binds to a 45 amino-acid motif in the N-terminal region of the receptor tail
characterized by a proline-rich PxxP motif. The very same element is
recognized by ANKRA and may also serve as a docking site for factors with SH3
domain (Rader et al., 2001
).
Because TPRs are the only obvious structural element in MegBP, these repeats
probably represent the binding domain of the adaptor. Whether one or both
repeats contribute to receptor recognition remains to be shown. The inability
of the megalin tail to bind to C-terminal GST-MegBP truncation products
(Fig. 2) suggests that both
repeats may be required to do so.
TPRs are found in a number of nuclear, cytoplasmic and mitochondrial
proteins. It is assumed that they serve as interaction sites with non-TPR
sequences and facilitate the assembly of multimeric protein complexes
(Lamb et al., 1995;
Das et al., 1998
). Examples of
such protein complexes include the mitochondrial import complex, peroxisomal
import receptor complex and transcription regulation complexes
(Lamb et al., 1995
;
Das et al., 1998
). In support
of a role of TPR proteins in transcription regulation, proteins that bound to
MegBP in the yeast two-hybrid system included those involved in RNA binding
(Hlark) (Jackson et al., 1997
)
and in transcriptional activation (SKIP)
(Zhang et al., 2002
) or
repression (THG-1) (Kester et al.,
1999
). This observation raises the intriguing hypothesis that
megalin may directly regulate gene transcription through controlled binding or
release of transcription factors. Overexpression of MegBP in megalin producing
cells may thus result in uncontrolled sequestration of transcriptional
regulators (or other MegBP interaction partners) on the receptor tail and in
impaired cellular viability.
What is the physiological relevance of the megalin and MegBP interaction?
Two of the identified MegBP-binding partners provide some insights into that
question. TGF-ß-stimulated clone 22 (TSC-22) and its homologue the
TGF-ß-stimulated clone 22 homologous gene 1 (THG-1) are two related
leucine zipper proteins (Kester et al.,
1999). When fused to a GAL4 DNA-binding domain, they inhibit
transcription from a heterologous promoter, suggesting that these proteins act
as endogenous transcription repressors when sequestered at the DNA
(Kester et al., 1999
). A link
to megalin function is provided by the fact that TSC-22 is highly upregulated
in megalin-deficient kidneys as uncovered by expression profiling
(Hilpert et al., 2002
). The
second interaction partner with obvious relevance for megalin function is
SKIP. SKIP, also known as NcoA-62, is a co-activator of the vitamin D receptor
(VDR). It binds to the hormone receptor and stimulates its activity in a
ligand-independent manner (Baudino et al.,
1998
; Zhang et al.,
2002
). A role for megalin in cellular vitamin D metabolism is well
established. It binds vitamin D metabolites complexed with the carrier
vitamin-D-binding protein (DBP) and mediates their uptake into cells
(Nykjaer et al., 1999
;
Nykjaer et al., 2001
). Thus,
endocytosis of vitamin D metabolites through megalin may be directly linked to
activation of the nuclear hormone receptor. In such a model, SKIP may be
sequestered via MegBP to the receptor tail and released upon endocytic uptake
of vitamin D metabolites. Unbound SKIP then interacts with the VDR and primes
it for incoming ligands. Although speculative, this model incorporates data
obtained in independent experimental systems (including receptor-deficient
mice) and provides a working hypothesis.
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Acknowledgments |
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