From the Department of Pharmacology and of Cell and Molecular Physiology, Bowles Center for Alcohol Studies, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599
Received for publication, May 15, 2000, and in revised form, December 4, 2000
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ABSTRACT |
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Hepatocyte growth factor-regulated tyrosine
kinase substrate (Hrs) is a mammalian homologue of yeast vacuolar
protein sorting (Vps) protein Vps27p; however, the role of Hrs in
lysosomal trafficking is unclear. Here, we report that Hrs interacts
with sorting nexin 1 (SNX1), a recently identified mammalian homologue
of yeast Vps5p that recognizes the lysosomal targeting code of
epidermal growth factor receptor (EGFR) and participates in lysosomal
trafficking of the receptor. Biochemical analyses demonstrate that Hrs
and SNX1 are ubiquitous proteins that exist in both cytosolic and membrane-associated pools, and that the association of Hrs and SNX
occurs on cellular membranes but not in the cytosol. Furthermore, endogenous SNX1 and Hrs form a ~550-kDa complex that excludes EGFR.
Immunofluorescence and subcellular fractionation studies show that Hrs
and SNX1 colocalize on early endosomes. By using deletion analysis, we
have mapped the binding domains of Hrs and SNX1 that mediate their
association. Overexpression of Hrs or its SNX1-binding domain inhibits
ligand-induced degradation of EGFR, but does not affect either
constitutive or ligand-induced receptor-mediated endocytosis. These
results suggest that Hrs may regulate lysosomal trafficking through its
interaction with SNX1.
Vesicular trafficking, the process by which a transport vesicle
buds from a donor membrane and fuses with its target, is fundamental to
the function of eukaryotic cells. For example, it is becoming increasingly clear that vesicular trafficking of ligand-activated receptor tyrosine kinases such as epidermal growth factor receptor (EGFR)1 plays a critical role
in controlling diversity, intensity, and duration of tyrosine kinase
signaling (1, 2). Binding of EGF triggers the dimerization of EGFR and
the activation of the tyrosine kinase at the cytoplasmic domain of the
receptor, which then activates downstream signal transduction pathways
(3). After ligand binding, the ligand-receptor complexes are recruited to clathrin-coated pits and internalized. Following endocytosis, the
ligand-receptor complexes are transported to early endosomes, where a
sorting decision must be made between recycling back to the cell
surface or delivery to lysosomes for degradation. The internalized
EGF·EGFR complexes are primarily transported to lysosomes, and
their degradation represents a major mechanism for attenuating EGF
signaling (4). Moreover, accumulating evidence indicates that the
internalized EGF·EGFR continues to bind and phosphorylate downstream
signaling proteins in pre-degradative intracellular compartments,
leading to activation of signaling pathways that are distinct from
those originated at the cell surface (2, 5). To ensure proper temporal
and spatial signaling, the endocytic and lysosomal trafficking of EGF
receptors is tightly regulated. Whereas the major events in endocytosis
are fairly well understood, the molecular mechanisms underlying
lysosomal trafficking of these receptors remain poorly characterized.
Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) was
identified originally as a phosphoprotein whose tyrosine phosphorylation is induced upon stimulation by hepatocyte growth factor
(6). Subsequent studies demonstrate that the tyrosine phosphorylation
of Hrs is also induced by a variety of other growth factors and
cytokines, including epidermal growth factor (EGF), platelet-derived
growth factor (PDGF), interleukin 2, and granulocyte-macrophage colony-stimulating factor (6, 7). Hrs exists in cytosolic and
membrane-associated forms, and appears to function in both signaling
and vesicular trafficking (6-9). Hrs is thought to function in cell
growth signaling by cytokines via its interaction with signal
transducing adaptor molecule (STAM) (7). Recently, it was reported that
Hrs also interacts with Hbp/STAM2, an STAM isoform involved in cytokine
signaling and degradation of PDGF receptors (10, 11). In addition, our
previous work has demonstrated that Hrs regulates the exocytotic fusion
process via its interaction with SNAP-25, an essential component of the
membrane fusion machinery (9). Hrs shares 20% sequence identity and
similar domain structure with Vps27p, a yeast protein that is required
for trafficking of proteins from a prevacuolar/endosomal compartment to
Golgi and vacuole, the yeast equivalent of the lysosome (8, 9, 12).
Targeted disruption of Hrs gene in mice leads to abnormally enlarged
early endosomes that are reminiscent of exaggerated "class E"
compartment in yeast vps27 mutant, suggesting that Hrs may have an analogous function in vesicular trafficking through mammalian endosomes (8, 13). However, it has yet to be demonstrated whether Hrs
actually acts in endosome-to-Golgi and endosome-to-lysosome trafficking
in mammalian cells.
To understand the action of Hrs in vesicular trafficking and signaling,
we performed a search for proteins that interact with Hrs using a yeast
two-hybrid screen. We report here the isolation and characterization of
a Hrs-interacting protein that is the rat counterpart of the human
sorting nexin 1 (SNX1) (14). SNX1 was first identified as a protein
that interacts with the lysosomal targeting signal-containing
cytoplasmic region of EGFR (14). Overexpression of SNX1 accelerates
degradation of EGFR, suggesting a role for SNX1 in endosome-to-lysosome
trafficking (14). It remains controversial as to whether SNX1 interacts
only with EGFR (14) or additionally with multiple types of other cell
surface receptors, including the receptors for PDGF, insulin, leptin, and transferrin (15). Interestingly, SNX1 is homologous to Vps5p, a
yeast protein that is required for endosome-to-Golgi trafficking (16-18). Recent evidence indicates that Vps5p is a molecular component of a multimeric membrane-associated protein complex termed the retromer
complex, which serves as a novel membrane coat acting in the formation
of vesicles for endosome-to-Golgi trafficking (18). It thus likely that
SNX1 may function in a similar manner in mammalian cells, acting as a
key component of the lysosomal sorting machinery by incorporating cargo
proteins into a retromer-like membrane coat.
In the present study, we demonstrate that Hrs interacts with SNX1 both
in vitro and in vivo. We define the structural
requirement for this novel interaction and show that the Hrs-binding
site of SNX1 overlaps with its EGFR-binding site. In addition, gel filtration analysis and coimmunoprecipitation studies reveal that SNX1
and Hrs form a ~550-kDa complex that excludes EGFR. We characterize the expression pattern and subcellular localization of SNX1 and show
that it colocalizes with Hrs on early endosomes. Furthermore, we show
that Hrs and SNX1 are involved in the regulation of the ligand-induced
degradation of EGF receptors, but not in the internalization of these
receptors. Our data suggest that Hrs may regulate lysosomal sorting and
trafficking pathways via its interaction with SNX1.
Yeast Two-hybrid Screens and Interaction Assays--
A bait
plasmid, pPC97-Hrs, was constructed by subcloning the entire open
reading frame of rat Hrs (9) into the pPC97 vector (19, 20). For the
two-hybrid screen, the yeast strain CG-1945 (CLONTECH) was transformed sequentially with
pPC97-Hrs and a rat hippocampal/cortical two-hybrid cDNA library
(20). Positive clones were selected on 3-aminotriazole-containing
medium lacking leucine, tryptophan, and histidine, and confirmed by a
filter assay for cDNA Cloning--
For cloning of the full-length rat
SNX1, a rat hippocampal cDNA library in Antibodies--
An anti-SNX1 antibody was raised in chicken
against the COOH-terminal peptide of rat SNX1, CKYWEAFLPEARAIS. The
NH2-terminal cysteine residue was added for the coupling
purposes. The antibody was affinity-purified using the immunogen
peptide coupled to a SulfoLink column (Pierce). Other antibodies that
were used in this study are: anti-Hrs (9); anti-EGFR (1005 and 528, Santa Cruz Biotechnology, Inc.), anti-SNAP-25 (SMI 81, Sternberger
Monoclonals, Inc.); anti-Rab5, anti-Rab11, and anti-EEA1 (Transduction
Laboratories); anti-HA (3F10, Roche Molecular Biochemicals); anti-actin
(C4, Roche Molecular Biochemicals); and secondary antibodies coupled with Texas Red (Jackson ImmunoResearch Laboratories). The anti-LAMP1 (H4A3) and anti-LAMP2 (H4B4) antibodies were obtained from the Developmental Studies Hybridoma Bank maintained by the University of
Iowa (Iowa City, IA).
Western Blot Analyses--
Rat tissues were homogenized in 1%
SDS and subjected to SDS-PAGE. The proteins were transferred onto
nitrocellulose membranes, and probed with the anti-SNX1 and other
antibodies. Antibody binding was detected by using the enhanced
chemiluminescence system (Amersham Pharmacia Biotech).
Production of Recombinant Proteins--
The full-length rat SNX1
was subcloned into the prokaryotic expression vectors pET32c (Novagen)
to obtain the construct pET32-SNX1. This plasmid encodes a fusion
protein, S-tag-SNX1, which consists of (from amino to carboxyl
terminus) the 109-amino acid thioredoxin protein, a hexahistidine tag,
and a 15-amino acid S-tag peptide fused in frame with the SNX1 protein.
For the production of glutathione S-transferase (GST)-Hrs
fusion proteins, rat Hrs (residues 225-776) was subcloned into the
vector pGEX-5X-2 (Amersham Pharmacia Biotech). Fusion proteins were
expressed in bacteria, and purified as described previously (21).
In Vitro Binding Assays--
GST-Hrs fusion protein or GST
control was immobilized on glutathione-agarose beads, and incubated
with S-tag-SNX1 fusion protein for 3 h at 4 °C under gentle
rocking in 50 mM Tris-HCl (pH 8.0), 150 mM
NaCl, and 0.1% Triton X-100. After extensive washes with the same
solution, bound proteins were eluted by boiling in Laemmli sample
buffer, subjected to SDS-PAGE, and immunoblotting using horseradish
peroxidase-conjugated S-protein (Novagen), and visualized by enhanced
chemiluminescence (ECL, Amersham Pharmacia Biotech). For detection of
the interaction of Hrs with endogenous SNX1, GST-Hrs fusion proteins
immobilized on glutathione-agarose beads were incubated with rat brain
homogenates for 2 h at 4 °C as described (21). Bound proteins
were analyzed by SDS-PAGE and immunoblotting.
Expression Constructs and Transfections--
Conventional
molecular biological techniques (23) were used to generate the
following expression constructs: pcDNA3.1-SNX1 and
pcDNA3.1-Hrs, which direct the expression of full-length SNX1 and
Hrs, respectively; pCHA-SNX1, pCHA-Hrs, and
pCHA-Hrs225-776, which direct the expression of
NH2-terminal HA epitope-tagged, full-length SNX1,
full-length Hrs, and a Hrs fragment (residues 225-776), respectively;
and pEGFP-SNX1, pEGFP-Hrs, and pEGFP-Hrs225-776, which
direct the expression of NH2-terminal GFP-tagged,
full-length SNX1, full-length Hrs, and a Hrs fragment (residues
225-776), respectively. Transfections of HEK293 and HeLa cells were
performed using LipofectAMINE (Life Technologies, Inc.) according to
the manufacturer's instructions.
Immunoprecipitation--
Extracts were prepared from HeLa cells
transiently transfected with pAlterMAX-EGFR alone (0.1 µg) or in
combination with pCHA-SNX1 or pCHA-Hrs (1 µg), and
immunoprecipitation were performed as described previously (24), using
anti-HA antibody (3F10), anti-EGFR antibody (528), or corresponding
control IgG. For coimmunoprecipitation of SNX1 and Hrs in cellular
fractions, HA-SNX1-transfected cells were homogenized in buffer A (50 mM Tris-HCl, pH 7.6, 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and
10 µg/ml benzamide) and centrifuged at 16,000 × g
for 15 min. After removal of the supernatant (cytosol) fraction, the
particulate fraction was resuspended with buffer A containing 1%
Nonidet P-40 and incubated at 4 °C for 30 min to solubilize
membrane-bound proteins. The cytosol and membrane fractions were
centrifuged at 100,000 × g for 20 min at 4 °C, and
the supernatants were incubated with rat monoclonal anti-HA antibody
(3F10) or control rat IgG for 1 h at 4 °C. The immunocomplexes
were recovered by incubation with protein G-Sepharose beads (Sigma) for
1 h at 4 °C. After extensive washes with the same solution, the
immunocomplexes were dissociated by boiling in the Laemmli sample
buffer, and analyzed by SDS-PAGE and immunoblotting.
Membrane Association Analyses--
Separation of PC12 cells into
cytosol fraction (100,000 × g supernatant) and
membrane particulate fraction (100,000 × g pellet) were performed as described previously (21). The membrane fractions were subjected to extraction studies as described (21), using 1.5 M NaCl or 4 M urea.
Subcellular Fractionation--
HeLa cells were treated with 100 ng/ml EGF for 10 min at 37 °C and then washed twice with PBS at
4 °C. Cells were gently scraped from culture plates and collected by
centrifugation. They were then homogenized in 1 ml of ICT (78 mM KCl, 4 mM MgCl2, 8.37 mM CaCl2, 10 mM EGTA, 50 mM HEPES/KOH, pH 7.0) plus 250 mM sucrose (25),
and centrifuged at 1,000 × g for 5 min. The
supernatant was placed on a 5-20% linear Optiprep (Nycomed) gradient
formed in ICT, and centrifuged at 4 °C for 20 h at 125,000 × g in a SW40 rotor (Beckman). Following centrifugation,
the gradient was harvested into 300-µl fractions using an Auto
Densi-Flow gradient harvester (Labconco).
Size Exclusion Chromatography--
HeLa cells were treated with
100 ng/ml EGF for 10 min at 37 °C, and then lysed for 30 min at
4 °C in a lysis buffer (50 mM Tris-HCl, pH 7.6, 50 mM NaCl, 0.1% Triton X-100, 1% Nonidet P-40) containing
protease inhibitors (1 mM phenylmethylsulfonyl fluoride and
10 µg/ml each of leupeptin, aprotinin, benzamidine, and pepstatin). After centrifugation at 16,000 × g for 15 min, the
supernatant was concentrated into 7.8 µg/ml protein in the elution
buffer (40 mM HEPES, pH 7.8, 2 mM EDTA, 10%
glycerol, 50 mM KCl, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride). The extract (500 µl)
was loaded on a Superose 6 HR 10/30 column and fractionated by
size-exclusion chromatography, using a ÄKTAdesign advanced chromatography system (Amersham Pharmacia Biotech). The column was
eluted using a flow rate of 0.5 ml/min, and 0.25-ml fractions were
collected. The column was calibrated with protein standards (Sigma),
including blue dextran, thyroglobulin, apoferritin, Immunofluorescence Microscopy--
HeLa cells were fixed with
4% paraformaldehyde and processed for indirect immunofluorescence
microscopy as described previously (21). The distributions of GFP-Hrs
and GFP-SNX1 were compared with the distribution of marker proteins
using a Leica TCS-NT confocal microscope with fluorescein
isothiocyanate and Texas Red filters. To avoid bleed-through, images
were collected by sequential scanning for fluorescein isothiocyanate or
Texas Red while turning off the other laser. The images were then
processed using Adobe Photoshop 5.0 (Adobe Systems, Inc.).
Endocytosis Assays--
For measurement of transferrin
endocytosis, transfected HeLa cells were incubated in serum-free medium
for 1 h, followed by addition of 100 µg/ml Texas Red-conjugated
transferrin (Molecular Probes) and incubation at 37 °C for 30 min in
the absence or presence of 100 ng/ml EGF. The cells were then were
washed with phosphate-buffered saline, fixed with 4% paraformaldehyde,
and processed for indirect immunofluorescence microscopy as described
above. For measurement of EGFR endocytosis, HeLa cells were incubated
in serum-free medium for 1 h and then incubated in the absence or
presence of 100 ng/ml EGF for 10 min at 37 °C. After washing with
phosphate-buffered saline, the cells were fixed and stained with
anti-EGFR antibodies followed by Texas Red-conjugated secondary
antibodies (21).
Assays for Degradation of EGFR--
Cultures of HeLa cells in
10-cm dishes were transiently transfected with 0.10 µg of
pAlterMAX-EGFR (24) in combination with 2 µg of pCHA vector,
pCHA-Hrs, pCHA-Hrs225-776, or pCHA-SNX1. After 24 h,
cells were incubated in serum-free medium for 1 h and then
incubated in the absence or presence of 100 ng/ml EGF for 45 min at
37 °C. Cell lysates were prepared as described (24). Equal amount
(600 µg) of protein from each lysate was immunoprecipitated using
anti-EGFR antibody 528. The immunoprecipitates were subjected to
immunoblotting with anti-EGFR antibody 1005 and enhanced
chemiluminescence (ECL) detection. Signals were digitized using MTI
CCD-72S camera (DAGE-MTI, Inc.), and the intensity of the EGFR band
(180 kDa) was quantitated using MCID M4 software (Imaging Research
Inc.).
Identification of Rat SNX1 as a Hrs-binding protein in Yeast
Two-hybrid Screens--
To identify proteins that interact with Hrs,
we screened a rat hippocampal/cortical cDNA library using a
full-length Hrs as bait in the yeast two-hybrid selection. Positive
clones were rescued, and confirmed by re-transformation experiments. Of
the 22 positive clones sequenced, 4 clones encoded STAM, a
signal-transducing adaptor molecule that is known to interact with Hrs
(7). Three independent overlapping clones encoded portions of a protein
that is the rat homologue of the human sorting nexin 1 (Fig.
1A). The specificity of the
interaction between Hrs and SNX1 was confirmed by yeast two-hybrid
analysis demonstrating that SNX1 only interacts with Hrs, but not with
a coiled-coil domain-containing protein SNAP-25 or the proline-rich
cytoplasmic region of synaptophysin (data not shown).
By screening a rat hippocampal Tissue Distribution and Membrane Association of SNX1--
To
analyze SNX1 expression at protein level and biochemically characterize
its association with Hrs, we raised a chicken polyclonal antibody
against the COOH-terminal 14-amino acid peptide of rat SNX1. To
characterize this antibody, HEK293 cells were transfected with an
expression construct pCHA-SNX1, which directed the expression of a HA
epitope-tagged, full-length SNX1 protein (HA-SNX1) (Fig. 2A). Immunoprecipitation of
HA-SNX1 with an anti-HA antibody followed by immunoblotting with the
anti-SNX1 antibody demonstrated that the antibody is able to recognize
the recombinant SNX1 protein (Fig. 2A). The specificity of
the anti-SNX1 antibody was confirmed by the following experiments. 1)
The preimmune chicken IgY did not react with the recombinant or
endogenous SNX1 proteins (data not shown). 2) Pre-absorption of the
anti-SNX1 antibody with recombinant SNX1 protein or the peptide
immunogen completely eliminated its immunoreactivity to both
recombinant and endogenous SNX1 protein (data not shown).
Although the tissue distribution of SNX1 mRNA expression has
previously been determined (15), the distribution of SNX1 protein expression has not yet been reported. To analyze the tissue
distribution of SNX1 at protein level, affinity-purified anti-SNX1
antibodies were used on Western blots of various rat tissues. A doublet
of protein bands with an apparent molecular masses of 66 and 61 kDa was
detected (Fig. 2B). The size of the upper band agrees well with that of the endogenous SNX1 protein detected in murine NIH 3T3 and
human HeLa cells (14). The lower band is likely to be the alternatively
spliced isoform SNX1A, which has an NH2-terminal internal
65-amino acid deletion compared with SNX1 (15). As shown in Fig.
2B, SNX1 is abundantly expressed in pancreas, spleen, kidney, intestine, and lung, and moderately expressed in brain, liver,
and ovary. Longer exposure of the same blot demonstrated that SNX1 is
also present at low level in heart and skeletal muscle (data not
shown). The additional immunoreactive bands of low molecular weights
observed in pancreas and intestine are likely to be the degradation
products of SNX1/1A since the relative intensity of these bands as
compared with the SNX1/1A bands varied from preparation to preparation.
To examine the intracellular distribution of endogenous SNX1,
postnuclear supernatant of PC12 cells was separated into cytosol and
membrane particulate fraction, and then subjected to Western blot
analysis with the anti-SNX1 antibody (Fig. 2C). SNX1
immunoreactivity was detected in both cytosol and membrane particulate
fraction, although the relative amount of SNX1 in the cytosol fraction
was severalfold more than that in the particulate fraction. To
investigate the nature of SNX1 association with membranes, the membrane
particulate fraction was extracted with 1.5 M NaCl or 4 M urea (Fig. 2C). Unlike the integral membrane
protein EGFR, which was resistant to extraction by high salt and urea,
SNX1 was extracted by these treatments, suggesting that SNX1 is
peripherally associated with membranes.
Hrs and SNX1 Associate in Vitro--
To obtain independent
evidence for the interaction between Hrs and SNX1, GST-Hrs fusion
protein or GST control was immobilized on glutathione-agarose beads,
and incubated with S-tag-SNX1 fusion protein. Proteins that bound to
the GST-Hrs or GST control were detected with the horseradish
peroxidase-conjugated S protein against the S-tag (Fig.
3A). The results demonstrate
that S-tag-SNX1 bound to GST-Hrs, but not to the GST control,
indicating that recombinant Hrs and SNX1 are capable of interacting
directly with each other. To determine whether recombinant Hrs is able
to bind endogenous SNX1, GST-Hrs fusion protein immobilized on
glutathione beads was used to affinity-purify ("pull-down")
endogenous SNX1 from brain homogenates. As shown in Fig. 3B,
the GST-Hrs fusion protein was able to pull down endogenous SNX1. In
contrast, control GST protein was unable to pull down SNX1. These
results confirm the Hrs-SNX1 interaction detected in the yeast
two-hybrid system.
Association of Hrs and SNX1 Occurs on Cellular Membranes but Not in
Cytosol--
Coimmunoprecipitation experiments were performed to
determine whether Hrs interacts with SNX1 in vivo. Since
both Hrs and SNX1 exist in a cytosolic pool and a membrane-associated
pool (Fig. 2C) (9), we were interested to know whether the
association of Hrs and SNX1 occurs in the cytosol or on membranes. To
examine these possibilities, HA-SNX1-transfected HeLa cells were
fractionated into the cytosol and membrane fractions. These fractions
were then subjected to immunoprecipitation with a rat anti-HA antibody or control rat IgG, and the immunoprecipitates were analyzed by Western
blot analysis using antibodies against Hrs and HA tag (Fig.
3C). The results demonstrate that Hrs and SNX1
coimmunoprecipitate only in the membrane fraction, but not in the
cytosol fraction. Moreover, control IgG was unable to precipitate
either SNX1 or Hrs, confirming the specificity of the observed Hrs-SNX1 association.
Identification of the Hrs-binding Domain in SNX1--
The three
SNX1 clones isolated from yeast two-hybrid screens encode residues
123-522, 178-522, and 253-522 of rat SNX1 (Fig. 1A),
indicating that the NH2-terminal 252 residues including
most of the PX domain of SNX1 is dispensable for the association with Hrs. To further determine the specific region in SNX1 that is responsible for binding Hrs, we made a series of SNX1 deletion mutants
that were expressed in yeast as fusion proteins with the GAL4 DNA
binding domain (Fig. 4A). The
interaction of these SNX1 deletion mutants with the full-length Hrs was
analyzed by the ability to grow on histidine-deficient media and a
Identification of the SNX1-binding Domain in Hrs--
To further
understand the structural requirements that underlie the interaction
between Hrs and SNX1, we performed similar deletion analysis to map the
specific region of Hrs involved in binding SNX1 (Fig. 4B).
Deletions of the VHS domain, FYVE finger, and the COOH-terminal
proline-rich domain had little effect on the ability of Hrs to bind
SNX1, indicating that these domains are dispensable to the Hrs-SNX1
interaction. As shown in Fig. 4B, only the fusion proteins
that contain the central region (residues 225-541) encompassing the
two predicted coiled-coil domains H1 and H2 of Hrs were able to
interact with SNX1. Unlike the interaction of Hrs with STAM, Hbp, or
SNAP-25, which is mediated by the H2 domain (7, 8, 11), we found that
the H2 domain (residues 443-541) by itself is not sufficient to bind
SNX1. Moreover, a Hrs fusion protein (residues 225-449) containing the
H1 domain and the proline-rich linker region was unable to bind SNX1.
Taken together, these data suggest that multiple domains and/or a
complex folded structure of the Hrs central region (residues 225-541) are involved in binding SNX1.
Cofractionation of SNX1 with Hrs and with Early Endosomal Markers
on a Density Gradient--
Since both Hrs and SNX1 exist in cytosolic
and membrane-associated pools, we sought to determine whether Hrs and
SNX1 associate with the same population of membranes by using
subcellular fractionation. Postnuclear supernatants were prepared from
HeLa cells and fractionated on a 5-20% linear Optiprep gradient using
a well established protocol (25). Fractions were collected and analyzed
by SDS-PAGE and Western blot analysis (Fig.
5). As expected, a portion of Hrs and
SNX1 were detected as soluble proteins in very low density regions
(fractions 3-9). Furthermore, there is clear cofractionation between
Hrs and SNX1 in the denser regions (fractions 26-28), suggesting that
the membrane-bound pools of these two proteins associate with the same
population of membranes.
We next examined the organellar origin of the membrane compartment to
which Hrs and SNX1 are colocalized. Previous studies using
immunofluorescence staining and immunoelectron microscopy demonstrate
that Hrs is localized to the cytoplasmic surface of early endosomes
(8). Based on these studies, the population of membranes to which Hrs
and SNX1 are colocalized is likely to represent the early endosomes. To
confirm this possibility, we determined the distributions of Rab5,
Rab11, and LAMP2 marker proteins in the same fractions of the Optiprep
gradient using Western blot analysis (Fig. 5). Rab5, a marker for early
endosomes (32), was detected in a membrane-associated pool with a peak at fraction 27. In contrast, Rab11, a marker for recycling endosomes (33), exhibited a membrane-associated peak around fraction 25. Consistent with the results of Sheff et al. (25), we
observed the presence of a large fraction of Rab5 and Rab11 in very
low-density regions (fractions 4-7). Unlike Rab5 and Rab11 in the
denser regions (fractions 24-29), which could be quantitatively
pelleted when centrifuged at 165,000 × g, both Rab
proteins in the light fractions did not pellet after the
ultracentrifugation, indicating that the low density Rab5 and Rab11 are
soluble instead of membrane-bound. LAMP2, a marker for late endosomes
and lysosomes (34), was localized to high density fractions (fractions
32-34). Comparison of the distribution of Hrs and SNX1 with these
markers suggests that Hrs and SNX1 primarily associate with early
endosomes, although we cannot exclude the possibility that a small
percentage of these proteins are also localized to recycling endosomes.
Colocalization of Hrs and SNX1 on Early Endosomes by
Immunofluorescence Microscopy--
To further confirm the
colocalization of Hrs and SNX1 within the cell, full-length,
amino-terminally HA- or GFP-tagged Hrs and SNX1 were expressed in HeLa
cells, and their intracellular distribution was analyzed by indirect
immunofluorescence and confocal microscopy. The tagged Hrs and SNX1,
when expressed at low levels, exhibited vesicular staining patterns
(Fig. 6, A and B)
which are indistinguishable from those observed for nontagged Hrs and SNX1 when visualized using the antibodies against Hrs and SNX1 (data
not shown). These staining data are consistent with the results
reported by Komada et al. (8) for Hrs and by Kurten et
al. (14) for SNX1. The SNX1-positive vesicular compartment overlapped to a significant extent with Hrs, showing that at least a
subpopulation of SNX1 colocalizes with Hrs (Fig. 6, compare A and B). Furthermore, recombinant Hrs, when
expressed at high levels, caused the formation of enlarged vesicular
structures (Fig. 6D), which are believed to be exaggerated
early endosomes (8, 35). Although overexpression of recombinant SNX1
alone does not lead to formation of enlarged vesicular structures, SNX1 colocalized with Hrs to the exaggerated vesicular structures resulted from high level Hrs expression (Fig. 6, compare C and
D). The colocalization of SNX1 with Hrs is in agreement with
the results of subcellular fractionation studies (Fig. 5) and provides
supporting evidence for an in vivo association of Hrs with
SNX1.
To investigate the identities of the vesicular structures labeled by
Hrs and SNX1, we performed double immunofluorescence experiments to
compare the distribution of these proteins with early endosome antigen
1 (EEA1). EEA1, a core component of early endosome docking and fusion
machinery, has been widely used as a marker for early endosomes (36,
37). Consistent with previous studies (8) and our results of
subcellular fractionation (Fig. 5), a substantial overlap was observed
between Hrs distribution and EEA1 immunoreactivity (compare Fig.
7, A and B).
Similarly, a majority of SNX1-positive vesicular structures also
contain EEA1 (compare Fig. 7, C and D). Moreover,
many of the vesicular structures labeled by Hrs and SNX1 were stained
by Rab5, another marker for early endosomes (Fig. 7, I and
J; data not shown). However, no colocalization was observed
between the distribution of Hrs or SNX1 and that of LAMP1 and LAMP2,
markers for late endosomes and lysosomes (data not shown). Together,
these results suggest that Hrs and SNX1 primarily associate with early
endosomes.
To further confirm the early endosomal localization of Hrs and SNX1,
HeLa cells were stimulated for 10 min with EGF or Texas Red-conjugated
EGF, and the internalized EGF·EGFR complexes were visualized by
anti-EGFR antibodies (Fig. 7, E-H) or by Texas Red-EGF labeling (data not shown). It is known from previous studies using immunofluorescence and immunoelectron microscopy that, under similar conditions, the internalized EGF·EGFR complexes are almost
exclusively localized to early endosomes (38, 39). As shown in Fig. 7, most of the Hrs- and SNX1-positive structures contained internalized EGF·EGFR complexes (Fig. 7, compare E and F,
and G and H), providing further evidence for the
early endosomal localization of Hrs and SNX1. To examine possible
association of Hrs and SNX1 with recycling endosomes, HeLa cells were
treated with Texas Red-conjugated transferrin for 30 min. It is well
established that the internalized transferrin-receptor complexes
accumulate in recycling endosomes, which are often concentrated at the
microtubule organizing center (40) (Fig. 7L). The punctate staining pattern of SNX1 (Fig. 7K) or of Hrs (data not
shown) exhibited limited overlap with the distribution of internalized transferrin (Fig. 7L), suggesting that most of Hrs and SNX1
does not associate with recycling endosomes. These data, together with the results of subcellular fractionation (Fig. 5), provide strong evidence for the colocalization of Hrs and SNX1 on early endosomes.
Hrs and SNX1 Coexist in a Large Protein Complex Within the
Cell--
To further characterize the in vivo association
of Hrs and SNX1, we performed gel filtration analysis to assess the
relative sizes of SNX1 and Hrs and their associated complexes in HeLa
cells. As shown in Fig. 8, fractionation
of HeLa cell extracts by size-exclusion chromatography on a Superose 6 high resolution column reveals that SNX1 exists in two distinct protein
complexes that eluted from the column with apparent molecular masses of
~260 and ~550 kDa. In addition, a significant portion of SNX1
eluted in the void volume fractions. No SNX1 immunoreactivity was
detected in the fractions corresponding to the size of monomeric form
of SNX1 (predicted molecular mass of 59 kDa and apparent molecular mass of 66 kDa on SDS-PAGE), suggesting that SNX1 does not exist as monomers
in vivo. The ~260-kDa complex may represent a SNX1
tetramer or a heteromeric complex of SNX1 with other sorting nexin
isoforms. Consistent with this possibility, HA-tagged SNX2 has been
shown to interact with itself and with SNX1, SNX1A, and SNX4 (15). Similarly, it appears that Hrs does not exist in the monomeric form
(predicted molecular mass of 86 kDa and apparent molecular mass of 110 kDa on SDS-PAGE). The lowest apparent molecular weight of Hrs detected
by the size-exclusion chromatography is ~185 kDa, which may
correspond to a Hrs dimer or a heteromer of Hrs with another protein
such as STAM (7) or STAM2/Hbp (10, 11). In contrast, monomeric form of
EGFR was detected in the fractions corresponding to its expected
size.
The ~550-kDa SNX1-containing complex seems to also include Hrs, as
suggested by the coelution of Hrs with SNX1 in the same fractions
(fractions 44-50). EGFR did not coelute with SNX1 and Hrs in these
fractions, suggesting that EGFR is not part of the ~550-kDa complex.
The size of ~550-kDa complex is significantly larger than a simple
heterodimer of Hrs·SNX1, which has a combined molecular mass of
~175 kDa, suggesting that the ~550-kDa complex contains either
multiple subunits of SNX1 and Hrs, or additional proteins such as
mammalian homologue of Vps17p (18).
As shown in Fig. 8, SNX1, Hrs, and EGFR coeluted in the void volume of
the Superose 6 column. This is reminiscent of the coelution of yeast
retromer components (Vps5p, Vps29p, and Vps35p) in the void volume,
which is thought to represent a very large (>1000 kDa) complex (18).
Thus, one possible explanation of the data is that SNX1, Hrs, and EGFR
may be components of an analogous high molecular weight complex in
mammalian cells. Alternatively, the coelution of SNX1, Hrs, and EGFR in
the void volume could be the result of nonspecific aggregation. The
latter possibility seems to be more likely since no heterotrimeric
Hrs·SNX1·EGFR complex could be detected in the
coimmunoprecipitation experiments as shown in Fig.
9.
To further investigate the nature of Hrs- and SNX1-containing
complexes, we performed coimmunoprecipitation experiments using antibodies against EGFR, HA-Hrs, and HA-SNX1 (Fig. 9).
Immunoprecipitation of HA-Hrs with an anti-HA antibody was able to
bring down endogenous SNX1 (Fig. 9, lane 3), further
confirming the presence of a Hrs·SNX1 complex in vivo.
However, EGFR did not coprecipitate with HA-Hrs and SNX1 (Fig. 9,
lane 3), indicating that Hrs, SNX1, and EGFR do not coexist
in a single complex. These results are consistent with the gel
filtration data (Fig. 8) showing the presence of a ~550-kDa complex
that contains Hrs and SNX1 but excludes EGFR. Immunoprecipitation of
EGFR with an anti-EGFR antibody resulted in the coprecipitation of SNX1
but not Hrs (Fig. 9, lane 5), providing further evidence for
the presence of the SNX1·EGFR complex but not the Hrs·SNX1·EGFR
complex in vivo. As expected, immunoprecipitation of HA-SNX1
with an anti-HA antibody was able to bring down both Hrs and EGFR (Fig.
9, lane 1) due to the ability of the antibody to precipitate
both SNX1·Hrs and SNX1·EGFR complexes. Together, these
coimmunoprecipitation data demonstrate that there are two mutually
exclusive complexes containing SNX1, one with Hrs and one with EGFR.
This is in agreement with the results of deletion analysis (Fig.
4A) indicating that the Hrs-binding site of SNX1 overlaps
with its EGFR-binding site (14). It is interesting to note that,
although the SNX1·EGFR complex was first reported by Kurten et
al. (14), and subsequently confirmed by Haft et al.
(15) and this study, no such complex could be detected in the gel
filtration analysis (Fig. 8). Thus, the relative abundance of the
SNX1·EGFR complex seems to be very low compared with the abundance of
the SNX1·Hrs complex in HeLa cells.
Hrs and SNX1 Are Involved in Degradation but Not in Internalization
of EGFR--
Since SNX1 has been shown to accelerate down-regulation
of EGFR (14), the association and colocalization of Hrs with SNX1 raise
the possibility that Hrs may also have a role in the down-regulation of
EGFR. To test this possibility, we used a well established assay (14,
24, 41) to evaluate the effect of overexpression of Hrs on the
down-regulation of EGFR (Fig. 10). In
agreement with previous reports (14, 41), we observed that, in
vector-transfected control cells, stimulation with EGF for 45 min led
to a large decrease in the number of mature EGFR. The EGF-induced
down-regulation of EGFR was significantly enhanced in cells
overexpressing SNX1, which is consistent with the results of Kurten
et al. (14). In contrast, overexpression of Hrs resulted in
a significant reduction in the EGF-induced down-regulation of EGFR.
Similar extent of reduction in the down-regulation of EGFR was also
observed in cells expressing a Hrs fragment (amino acids 225-776)
containing the SNX1-interacting domain. These results suggest that Hrs
is involved in the ligand-induced down-regulation of EGFR, perhaps via
its interaction with SNX1.
To determine whether the effect of overexpressing SNX1 and Hrs on the
down-regulation of EGFR is due to a change in the internalization of
EGFR, HeLa cells overexpressing SNX1 or Hrs were tested for their
capacity to internalize EGF·EGFR complexes in the presence of EGF or
Texas Red-conjugated EGF. We found that, compared with untransfected
cells, cells overexpressing SNX1, Hrs, or Hrs225-776 internalized similar amounts of EGFR and Texas Red-conjugated EGF (Fig.
7 (E-H) and data not shown), indicating that
overexpression of these proteins has little effect on ligand-induced
endocytosis/internalization of EGFR. Furthermore, no difference in the
amount of internalized transferrin was observed between cells
overexpressing SNX1, Hrs, or Hrs225-776 and untransfected
cells (Fig. 7L, and data not shown), suggesting that Hrs and
SNX1 are not involved in constitutive receptor-mediated endocytosis.
Taken together, these data suggest that SNX1 and Hrs alter the
down-regulation of EGFR by affecting lysosome trafficking of the
receptor for degradation.
Trafficking of EGFR from endosome to lysosome plays a key role in
attenuating EGF signaling. However, little is known at the molecular
level about the mechanisms that regulate the lysosomal trafficking
pathway. Previous studies have defined the lysosome-targeting signals
within the cytoplasmic domain of EGFR that are responsible for the
EGF-induced lysosomal degradation (42, 43). The first sorting nexin,
SNX1, which recognizes the EGFR lysosome-targeting signals, has
recently been identified and shown to function in lysosomal trafficking
of EGFR (14). In this paper, we describe a novel interaction between
SNX1 and Hrs, a protein that is implicated in both vesicular
trafficking and cell growth signaling. The interaction of SNX1 with Hrs
was demonstrated in the yeast two-hybrid system and confirmed by
in vitro binding studies and coimmunoprecipitation experiments. Deletion analysis reveals that the Hrs-SNX1 interaction involves multiple coiled-coil domains and complex folded structures of
the Hrs central region (residues 225-541) and of the SNX1
COOH-terminal region (residues 300-522). Several lines of evidence
support a physiological significance of the observed interaction
between SNX1 and Hrs. 1) Hrs and SNX1 are ubiquitously expressed
proteins that exist in both cytosolic and membrane-associated pools. 2) Coimmunoprecipitation experiments demonstrate that the association of
Hrs and SNX occurs on cellular membranes but not in the cytosol. 3) Gel
filtration analysis reveals the presence of an endogenous ~550-kDa
protein complex containing SNX1 and Hrs. 4) Subcellular fractionation
studies show that SNX1 cofractionates with Hrs and early endosomal
markers on an Optiprep density gradient. 5) Double immunofluorescence
analysis demonstrates that Hrs and SNX1 colocalize on early endosomes.
6) Overexpression of Hrs or its SNX1-binding domain inhibits
ligand-induced degradation of EGFR, but has no effect on EGFR
internalization. Together, these data suggest that the interaction
between SNX1 and Hrs may be involved in the regulation of
endosome-to-lysosome trafficking of EGFR.
Our results indicate that SNX1 and Hrs share similar properties with
their yeast homologues, Vps5p and Vps27p. In yeast, both Vps5p and
Vps27p are localized to the prevacuolar/endosomal compartment, although
it is not known whether they colocalize with each other (17, 18, 44).
In mammalian cells, we found that Hrs and SNX1 colocalize on the early
endosome, a sorting compartment where membrane proteins destined for
degradation are sorted away from proteins that are recycled back to
cell surface. The involvement of SNX1 and Hrs in lysosomal trafficking
of EGFR is consistent with the role of Vps5p and Vps27p in yeast
vesicular trafficking. Recently, it was reported that Vps5p assembles
with Vps17p, Vps26p, Vps29p, and Vps35p to form a novel coat complex
called the retromer complex (18). The presence of mammalian homologues
of other retromer components Vps26p, Vps29p and Vps35p suggest that
SNX1 may function in a manner that is analogous to Vps5p by forming a
retromer-like coat complex in mammalian cells (18). Since SNX1 directly
interacts with lysosome-targeting signals on cargo proteins such as
EGFR (14), it is likely that SNX1 performs its sorting function by
selectively recruiting specific cargo proteins into the retromer-like
coat complex.
The mutually exclusive interaction of Hrs and of EGFR with SNX1
indicates that the association of Hrs with SNX1 is likely to interfere
with the ability of SNX1 to bind and recruit EGFR into a functional
coat complex for delivery to lysosomes. Supporting this view,
overexpression of Hrs or its SNX1-binding domain in HeLa cells leads to
an inhibition of lysosomal trafficking of EGFR for degradation. Based
upon these data, a model for the role of Hrs in lysosomal trafficking
can be envisaged. Hrs, by interacting with SNX1, might serve as a
regulator for the assembly of functional sorting machinery. The
association of Hrs with SNX1 keeps SNX1 in an inactive state,
unavailable to interact with cargo proteins and/or with other
components of the retromer-like coat complex. Disruption of this
association by protein phosphorylation (6, 7) or interaction with
signaling proteins such as STAM and STAM2/Hbp (7, 10, 11) would then
increase the availability of SNX1 and promote cargo recruitment and
assembly of functional coat complexes, and hence facilitate lysosomal
sorting and trafficking. Future studies will test this model and
determine the molecular mechanisms by which Hrs and SNX1 regulate
vesicular trafficking.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity (21). Prey plasmids from positive clones were rescued and re-transformed into fresh yeast cells
with the Hrs bait or various control baits to confirm the specificity
of the interaction. For analysis of Hrs-SNX1 interaction, deletion
constructs of Hrs and SNX1 were made by polymerase chain reaction and
were subcloned into the pPC97 vector. The interactions between these
Hrs and SNX1 fragments were tested in the yeast two-hybrid assay by
using HIS3 and
-galactosidase as the reporter genes.
Quantitative
-galactosidase assay was performed on the yeast
extracts by using the substrate chlorophenol red
-D-galactopyranoside as described previously (21,
22).
ZAPII (Stratagene) was
screened using a partial SNX1 cDNA probe from the yeast two-hybrid
prey clone, according to standard procedures (23). The cDNA inserts
from positive SNX1 clones were sequenced multiple times on both
strands, using an Applied Biosystems 373A DNA sequencer.
-amylase, alcohol dehydrogenase, albumin, and carbonic anhydrase.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (43K):
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Fig. 1.
Structure of rat sorting nexin 1. A, domain structure of rat SNX1. The following domains are
indicated: PX domain (residues 161-272); H1, H2, and H3, predicted
coiled-coil domain 1 (residues 309-342), coiled-coil domain 2 (residues 356-388), and coiled-coil domain 3 (residues 440-476). The
locations of the Hrs-interacting SNX1 clones isolated from the yeast
two-hybrid screens are indicated below the domain structure.
B, sequence of rat SNX1. The nucleotide sequence of rat SNX1
(not shown) has been deposited in GenBankTM with the accession number
AF218916. The deduced amino acid sequence of rat SNX1 is shown in
single-letter code and numbered on the
left. In-frame stop codon is denoted with an
asterisk. Indicated are the phox homology domain
(dashed underline), the predicated coiled-coil domains
(underline), and potential SH3 domain interaction motifs
(boxes).
ZAP cDNA library, we isolated two
full-length and four partial cDNA clones of rat SNX1. The full-length rat SNX1 cDNA (accession no. AF218916) contains an open
reading frame encoding a 522-amino acid protein (Fig. 1B)
that is 95% identical to human SNX1 (14, 15). Sequence analysis
demonstrated that rat SNX1 is hydrophilic with a theoretical isoelectric point (pI) of 5.15 and a high percentage (30%) of charged
amino acids distributed over the entire length. Like human sorting
nexins, rat SNX1 contains a Phox homology (PX) domain (15, 26). The PX
domain, whose function is as yet unknown, is an evolutionarily
conserved sequence that is present in a number of proteins with diverse
function, including proteins involved in vesicular trafficking (15-17,
26-29). By using the algorithm of Lupas et al. (30), we
identified three regions with high probability (p = 0.94, 0.98, and 1.00, respectively) of forming a coiled-coil structure.
In addition, rat SNX1 contains three putative SH3 domain-binding
(PXXP) motifs (31). Thus, SNX1 could potentially interact
with multiple proteins or be involved in the formation of multi-protein
complexes via coiled-coil interactions, associations of its
proline-rich motifs with the SH3 domain-containing proteins, and/or
other types of protein-protein interactions through its PX domain.
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Fig. 2.
Characterization of expression and membrane
association of SNX1. A, specificity of the anti-SNX1
antibody. HEK293 cells were transfected with pCHA-SNX1 or pCHA vector
control. Cell lysates prepared from the transfected cells were
immunoprecipitated (IP) with an anti-HA antibody. The
precipitates were then analyzed by immunoblotting (IB) with
anti-HA antibody and anti-SNX1 antibody. B, Western blot
analysis of SNX1 expression in rat tissues. Equal amounts of
homogenates (30 µg of protein/lane) from indicated rat tissues were
analyzed by immunoblotting using the anti-SNIP antibody.
Sk., Skeletal. C, nature of SNX1 association with
cellular membranes. Postnuclear supernatant (T) from PC12
cells was separated into cytosol (C) and membrane
(M) fractions. The membranes were extracted with 1.5 M NaCl or 4 M urea, and then separated into
soluble (S) and pellet (P) fractions. Aliquots
representing an equal percentage of each fraction were analyzed by
SDS-PAGE and immunoblotting for SNX1 and EGFR.
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Fig. 3.
Biochemical characterization of the
association of Hrs with SNX1. A, recombinant Hrs and
SNX1 associate in vitro. GST or GST-Hrs fusion proteins were
immobilized on glutathione-agarose beads and then incubated with
S-tag-SNX1 (Input). After extensive washes, bound proteins
were analyzed by SDS-PAGE and probing with the horseradish
peroxidase-conjugated S-protein that specifically binds the S-tag.
B, binding of endogenous SNX1 to immobilized GST-Hrs fusion
proteins. Rat brain homogenate (Input) was incubated with
immobilized GST control or GST-Hrs fusion proteins. Bound proteins were
analyzed by immunoblotting with the affinity-purified anti-SNX1
antibody. C, in vivo association of Hrs and SNX1.
HEK293 cells transfected with HA-tagged SNX1 and FLAG-tagged Hrs were
separated into cytosol and membrane fractions, and then subjected to
immunoprecipitation using a rat antibody against HA tag or control rat
IgG. The immunoprecipitates were then immunoblotted with antibodies
against Hrs and HA tag.
-galactosidase filter assay (data not shown) as well as a
quantitative
-galactosidase assay (Fig. 4A). The results
demonstrated that the SNX1 COOH-terminal region (residues 300-522)
interacts strongly with Hrs, whereas neither the PX domain (residues
158-282) nor COOH-terminal fragments (residues 300-399 or residues
429-522) is able to bind Hrs. These data suggest that multiple domains
and/or a complex folded structure of the SNX1 COOH-terminal region
(residues 300-522) are required for binding Hrs. The Hrs-binding
domain of SNX1 overlaps with its EGFR-binding site, which has been
mapped to the COOH-terminal 58 amino acids (residues 465-522) of SNX1
(14).
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Fig. 4.
Identification of interacting domains of Hrs
and SNX1. A, mapping of the Hrs-binding domain of SNX1.
Expression plasmids encoding full-length SNX1 and the indicated SNX1
deletion mutants fused to the GAL4 DNA binding domain were
cotransformed into yeast CG-1945 cells with pPC86-Hrs, a plasmid
encoding full-length Hrs fused to the GAL4 activation domain. The
interactions of SNX1 deletion mutants with Hrs were tested using yeast
two-hybrid assays. The, .-galactosidase activity of each sample was
determined using the substrate chlorophenol red
-D-galactopyranoside, normalized to its protein content,
and expressed as a percentage of the activity of full-length SNX1.
B, mapping of the SNX1-binding domain of Hrs. Expression
plasmids encoding full-length Hrs and the indicated Hrs deletion
mutants fused to the GAL4 activation domain were cotransformed into
yeast CG-1945 cells with pPC97-SNX1. Yeast two-hybrid assays were
performed as in A. The
-galactosidase activity of each
sample was expressed as a percentage of the activity of full-length
Hrs. Data are shown as mean ± S.D. of the results from triplicate
determinations.
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Fig. 5.
Cofractionation of SNX1 with Hrs on an
Optiprep gradient. Postnuclear supernatants were prepared from
HeLa cells and fractionated on a 5-20% linear Optiprep gradient as
described under "Experimental Procedures." The gradient was divided
into 38 fractions, with fraction 1 corresponding to the top of the
gradient. Equal volumes of each fraction were analyzed by SDS-PAGE,
followed by immunoblotting for Hrs, SNX1, Rab5, Rab11, and LAMP2.
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Fig. 6.
Comparison of the intracellular distribution
of SNX1 with Hrs by confocal fluorescence microscopy. HeLa cells
were transiently cotransfected with pEGFP-Hrs and pCHA-SNX1
(A and B) or pEGFP-SNX1 and pCHA-Hrs
(C and D). The distributions of GFP-Hrs
(A) and GFP-SNX1 (C) were directly visualized by
the green fluorescence emitted by the GFP. The same cells were stained
with primary antibodies against HA tag (B and D),
followed by detection with secondary antibodies conjugated to Texas
Red. The arrows indicate vesicular structure clearly visible
in both panels (colocalized), whereas the arrowhead marks
the vesicular structure visible in one panel but not the other (not
colocalized). Scale bar = 10 µm.
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Fig. 7.
Comparison of the distribution of Hrs and
SNX1 with markers for early endosomes and recycling endosomes.
HeLa cells were transiently transfected with either pEGFP-Hrs
(A, B, E, and F) or
pEGFP-SNX1 (C, D, and G-L).
Transfected cells were identified by the green fluorescence emitted by
the GFP (A, C, E, G,
I, and K). Some of the cells were treated at
37 °C with either EGF for 10 min (E-H) or Texas
Red-conjugated transferrin for 30 min (K and L).
Internalized transferrin was visualized by the red fluorescence emitted
by the Texas Red (L). Cells were stained with primary
antibodies against EEA1 (B and D), EGFR
(F and H), or Rab5 (J), followed by
detection with secondary antibodies conjugated to Texas Red. The
arrows indicate vesicular structure clearly visible in both
panels (colocalized), whereas the arrowhead marks the
vesicular structure visible in one but not the other panel (not
colocalized). Scale bar = 10 µm.
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Fig. 8.
Gel filtration analysis of protein complexes
containing Hrs and SNX1. HeLa cell extracts were prepared as
described under "Experimental Procedures" and fractionated by
size-exclusion chromatography using a Superose 6 high resolution
analytical gel filtration column. Equal volumes of each fraction were
analyzed by SDS-PAGE, followed by immunoblotting for SNX1, Hrs, and
EGFR. Standards used for column calibration are blue dextran (2000 kDa), thyroglobulin (670 kDa), apoferritin (443 kDa), -amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), and carbonic
anhydrase (29 kDa).
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Fig. 9.
Coimmunoprecipitation analyses reveal the
presence of the Hrs·SNX1 and SNX1·EGFR complexes but not the
Hrs·SNX1·EGFR complexes. Extracts from HeLa cells transiently
transfected with pAlterMAX-EGFR alone (lanes 5 and 6) or in combination with pCHA-SNX1 (lanes
1 and 2) or pCHA-Hrs (lanes
3 and 4) were subjected to immunoprecipitation
(IP) with anti-HA antibody 3F10 (lanes
1 and 3), anti-EGFR antibody 528 (lane
5) or corresponding control IgG (lanes
2, 4, and 6). The immunoprecipitates
were analyzed by immunoblotting (IB) with anti-EGFR,
anti-Hrs, or anti-SNX1 antibodies.
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Fig. 10.
Effect of overexpression of SNX1, Hrs, and
Hrs225-776 on degradation of EGFR. HeLa cells were
transiently transfected with pAlterMAX-EGFR in combination with pCHA
vector control (Cont.), pCHA-Hrs (Hrs),
pCHA-Hrs225-776 (Hrs N), or
pCHA-SNX1 (SNX1). At 24 h after transfection, cells
were subjected to serum starvation, followed by incubation in the
absence or presence of 100 ng/ml EGF for 45 min at 37 °C. Cells were
then lysed, and equal amounts (600 µg) of protein from each lysate
were immunoprecipitated (IP) using anti-EGFR antibody 528. The immunoprecipitates were analyzed by immunoblotting (IB)
with anti-EGFR antibody 1005 (A), and the corresponding
lysates (50 µg of protein/lane) were sequentially immunoblotted with
anti-HA antibody (B) and anti-actin antibody (C).
The remaining EGF receptor level after stimulation with EGF for 45 min
was measured by quantification of the intensity of the 180-kDa EGFR
band, and expressed as a percentage of the EGFR level of unstimulated
cells that were identically transfected (D). Data are
mean ± S.E. (error bar) of the results from four
independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Paul Worley (The Johns Hopkins University, Baltimore, MD) and Hamid Band (Harvard Medical School, Cambridge, MA) for providing the rat hippocampal/cortical cDNA library and the pAlterMAX-EGFR construct, respectively. We thank Drs. Yi Zhang and Hengbing Wang for advice and help in the analysis of protein complexes using size-exclusion chromatography.
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FOOTNOTES |
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* This work was supported by a grant from the University of North Carolina Research Council (to L.-S. C.) and by Grant NS37939 from the National Institutes of Health (to L. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF218916.
To whom correspondence should be addressed: Dept. of Pharmacology,
Rm. 1025A Thurston-Bowles, University of North Carolina, Chapel Hill,
NC 27599-7178. Tel.: 919-966-0503; Fax: 919-966-5679; E-mail:
LianLi@med.unc.edu.
Published, JBC Papers in Press, December 7, 2000, DOI 10.1074/jbc.M004129200
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ABBREVIATIONS |
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The abbreviations used are: EGFR, epidermal growth factor receptor; Hrs, hepatocyte growth factor-regulated tyrosine kinase substrate; SNX1, sorting nexin 1; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; STAM, signal transducing adaptor molecule; Hbp, Hrs-binding protein; EEA1, early endosome antigen 1; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; GFP, green fluorescent protein; Vps, vacuolar protein sorting; PX, Phox homology.
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1. | Di Fiore, P. P., and Gill, G. N. (1999) Curr. Opin. Cell Biol. 11, 483-488[CrossRef][Medline] [Order article via Infotrieve] |
2. | Ceresa, B. P., and Schmid, S. L. (2000) Curr. Opin. Cell Biol. 12, 204-210[CrossRef][Medline] [Order article via Infotrieve] |
3. | Schlessinger, J., and Ullrich, A. (1992) Neuron 9, 383-391[Medline] [Order article via Infotrieve] |
4. | Mellman, I. (1996) Curr. Opin. Cell Biol. 8, 497-498[CrossRef][Medline] [Order article via Infotrieve] |
5. | Bergeron, J. J., Di Guglielmo, G. M., Baass, P. C., Authier, F., and Posner, B. I. (1995) Biosci. Rep. 15, 411-418[Medline] [Order article via Infotrieve] |
6. | Komada, M., and Kitamura, N. (1995) Mol. Cell. Biol. 15, 6213-6221[Abstract] |
7. |
Asao, H.,
Sasaki, Y.,
Arita, T.,
Tanaka, N.,
Endo, K.,
Kasai, H.,
Takeshita, T.,
Endo, Y.,
Fujita, T.,
and Sugamura, K.
(1997)
J. Biol. Chem.
272,
32785-32791 |
8. |
Komada, M.,
Masaki, R.,
Yamamoto, A.,
and Kitamura, N.
(1997)
J. Biol. Chem.
272,
20538-20544 |
9. |
Kwong, J.,
Roundabush, F. L.,
Moore, P. H.,
Montague, M.,
Oldham, W.,
Li, Y.,
Chin, L.,
and Li, L.
(2000)
J. Cell Sci.
113,
2273-2284 |
10. |
Takata, H.,
Kato, M.,
Denda, K.,
and Kitamura, N.
(2000)
Genes Cells
5,
57-69 |
11. | Endo, K., Takeshita, T., Kasai, H., Sasaki, Y., Tanaka, N., Asao, H., Kikuchi, K., Yamada, M., Chenb, M., O'Shea, J. J., and Sugamura, K. (2000) FEBS Lett. 477, 55-61[CrossRef][Medline] [Order article via Infotrieve] |
12. | Piper, R. C., Cooper, A. A., Yang, H., and Stevens, T. H. (1995) J. Cell Biol. 131, 603-617[Abstract] |
13. |
Komada, M.,
and Soriano, P.
(1999)
Genes Dev.
13,
1475-1485 |
14. | Kurten, R. C., Cadena, D. L., and Gill, G. N. (1996) Science 272, 1008-1010[Abstract] |
15. |
Haft, C. R.,
de la Luz Sierra, M.,
Barr, V. A.,
Haft, D. H.,
and Taylor, S. I.
(1998)
Mol. Cell. Biol.
18,
7278-7287 |
16. | Horazdovsky, B. F., Davies, B. A., Seaman, M. N., McLaughlin, S. A., Yoon, S., and Emr, S. D. (1997) Mol. Biol. Cell 8, 1529-1541[Abstract] |
17. |
Nothwehr, S. F.,
and Hindes, A. E.
(1997)
J. Cell Sci.
110,
1063-1072 |
18. |
Seaman, M. N.,
McCaffery, J. M.,
and Emr, S. D.
(1998)
J. Cell Biol.
142,
665-681 |
19. | Chevray, P. M., and Nathans, D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5789-5793[Abstract] |
20. | Li, X. J., Li, S. H., Sharp, A. H., Nucifora, F. C., Jr., Schilling, G., Lanahan, A., Worley, P., Snyder, S. H., and Ross, C. A. (1995) Nature 378, 398-402[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Chin, L. S.,
Nugent, R. D.,
Raynor, M. C.,
Vavalle, J. P.,
and Li, L.
(2000)
J. Biol. Chem.
275,
1191-1200 |
22. | Li, L., Suzuki, T., Mori, N., and Greengard, P. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1460-1464[Abstract] |
23. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
24. |
Lill, N. L.,
Douillard, P.,
Awwad, R. A.,
Ota, S.,
Lupher, M. L., Jr.,
Miyake, S.,
Meissner-Lula, N.,
Hsu, V. W.,
and Band, H.
(2000)
J. Biol. Chem.
275,
367-377 |
25. |
Sheff, D. R.,
Daro, E. A.,
Hull, M.,
and Mellman, I.
(1999)
J. Cell Biol.
145,
123-139 |
26. |
Ponting, C. P.
(1996)
Protein Sci.
5,
2353-2357 |
27. |
Lock, P.,
Abram, C. L.,
Gibson, T.,
and Courtneidge, S. A.
(1998)
EMBO J.
17,
4346-4357 |
28. |
Voos, W.,
and Stevens, T. H.
(1998)
J. Cell Biol.
140,
577-590 |
29. |
Sato, T. K.,
Darsow, T.,
and Emr, S. D.
(1998)
Mol. Cell. Biol.
18,
5308-5319 |
30. | Lupas, A., Van Dyke, M., and Stock, J. (1991) Science 252, 1162-1164[Medline] [Order article via Infotrieve] |
31. | Cohen, G. B., Ren, R., and Baltimore, D. (1995) Cell 80, 237-248[Medline] [Order article via Infotrieve] |
32. | Bucci, C., Parton, R. G., Mather, I. H., Stunnenberg, H., Simons, K., Hoflack, B., and Zerial, M. (1992) Cell 70, 715-728[Medline] [Order article via Infotrieve] |
33. | Ullrich, O., Reinsch, S., Urbe, S., Zerial, M., and Parton, R. G. (1996) J. Cell Biol. 135, 913-924[Abstract] |
34. | Chen, J. W., Murphy, T. L., Willingham, M. C., Pastan, I., and August, J. T. (1985) J. Cell Biol. 101, 85-95[Abstract] |
35. |
Hayakawa, A.,
and Kitamura, N.
(2000)
J. Biol. Chem.
275,
29636-29642 |
36. |
Mu, F. T.,
Callaghan, J. M.,
Steele-Mortimer, O.,
Stenmark, H.,
Parton, R. G.,
Campbell, P. L.,
McCluskey, J.,
Yeo, J. P.,
Tock, E. P.,
and Toh, B. H.
(1995)
J. Biol. Chem.
270,
13503-13511 |
37. | Christoforidis, S., McBride, H. M., Burgoyne, R. D., and Zerial, M. (1999) Nature 397, 621-625[CrossRef][Medline] [Order article via Infotrieve] |
38. |
Carter, R. E.,
and Sorkin, A.
(1998)
J. Biol. Chem.
273,
35000-35007 |
39. |
Oksvold, M. P.,
Skarpen, E.,
Lindeman, B.,
Roos, N.,
and Huitfeldt, H. S.
(2000)
J. Histochem. Cytochem.
48,
21-33 |
40. |
Daro, E.,
van der Sluijs, P.,
Galli, T.,
and Mellman, I.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
9559-9564 |
41. |
Levkowitz, G.,
Waterman, H.,
Zamir, E.,
Kam, Z.,
Oved, S.,
Langdon, W. Y.,
Beguinot, L.,
Geiger, B.,
and Yarden, Y.
(1998)
Genes Dev.
12,
3663-3674 |
42. |
Opresko, L. K.,
Chang, C. P.,
Will, B. H.,
Burke, P. M.,
Gill, G. N.,
and Wiley, H. S.
(1995)
J. Biol. Chem.
270,
4325-4333 |
43. |
Kornilova, E.,
Sorkina, T.,
Beguinot, L.,
and Sorkin, A.
(1996)
J. Biol. Chem.
271,
30340-30346 |
44. | Piiper, A., Stryjek-Kaminska, D., Jahn, R., and Zeuzem, S. (1995) Biochem. J. 309, 621-627[Medline] [Order article via Infotrieve] |