From the Laboratory of Cell Regulation and Carcinogenesis, NCI, the
Diabetes Branch, NIDDK, National Institutes of
Health, Bethesda, Maryland 20892, and the § Department of
Surgery, Massachusetts General Hospital, Department of Genetics,
Harvard Medical School, Boston, Massachusetts 02114
Received for publication, January 23, 2001
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ABSTRACT |
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Sorting nexins (SNX) comprise a family of
proteins with homology to several yeast proteins, including Vps5p and
Mvp1p, that are required for the sorting of proteins to the yeast
vacuole. Human SNX1, -2, and -4 have been proposed to play a role in
receptor trafficking and have been shown to bind to several receptor
tyrosine kinases, including receptors for epidermal growth factor,
platelet-derived growth factor, and insulin as well as the long form of
the leptin receptor, a glycoprotein 130-associated receptor. We now
describe a novel member of this family, SNX6, which interacts with
members of the transforming growth factor- The transforming growth factor- Despite extensive work on the characterization of the ligand binding
affinities of the various receptors and determination of downstream
signaling following ligand binding, relatively little is known about
the trafficking of TGF- The sorting nexins are a family of cytoplasmic and membrane-associated
proteins that are hypothesized to function in the intracellular trafficking of plasma membrane receptors. The first sorting nexin, SNX1, was cloned in a yeast two-hybrid assay as an interactor with the
cytoplasmic domain of the EGF receptor (28). SNX1 was subsequently
shown to be homologous to Vps5p, a yeast protein essential for the
correct targeting of carboxypeptidase Y and other soluble hydrolases
from the trans-Golgi network through an
endosomal/prevacuolar compartment to the yeast vacuole (29, 30). Given
this function for the yeast SNX1 homolog, SNX1 itself was proposed to
target the EGF receptor for lysosomal degradation through an
endocytic pathway. Four additional sorting nexins have subsequently
been cloned (31). SNX2, SNX3, and SNX4 were identified in data base
searches through homology with SNX1. Studies with these four proteins
have shown that SNX1, SNX2, and SNX4 bind to multiple receptor tyrosine
kinases, including receptors for EGF, PDGF, and insulin, and to the
long form of the leptin receptor (31). SNX1 additionally binds to the
transferrin receptor. Furthermore, SNX1, SNX2, and SNX4 oligomerize
with each other (31). SNX3 is distinguished from the other SNXs in that
it does not associate with any of the receptors studied or with any of
the other SNXs (31). The most recently reported sorting nexin, SNX5,
was cloned using the yeast two-hybrid system as an interactor with the
Fanconi anemia complementation group A protein (4).
In this paper we report the cloning of a novel sorting nexin, SNX6.
This molecule was identified in a yeast two-hybrid screen to identify
binding partners of Smad1, but studies in mammalian cells have shown
that the interaction with this Smad protein is very weak and that,
instead, SNX6 shows strong interactions with several members of the
TGF- Cell Culture--
COS-1, COS-7, and HepG2 cells were maintained
in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS and
antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin)
(Life Technologies, Inc).
Cloning of SNX6--
SNX6 was originally identified in a yeast
two-hybrid screen using full-length Smad1 as bait and a human fetal
brain cDNA library. Full-length human SNX6 was subsequently cloned
from a heart cDNA library using standard techniques. The
full-length human clone was sequenced in both directions with standard techniques.
Sequence Alignment--
Sequence alignment was performed using
the ClustalW multiple sequence alignment program.
Tissue Distribution of SNX6--
Human multiple tissue Northern
blots (CLONTECH) were hybridized with a full-length
SNX6 probe. The probe was 32P-labeled using a RadPrime DNA
Synthesis (Life Technologies, Inc.) random-primed DNA labeling kit. The
blots were hybridized overnight at 65 °C and then washed 3 times for
30 min each at 65 °C. Washed blots were autoradiographed for several
days at Construction of Epitope-tagged SNX6 Constructs--
5'-Tagged
full-length and deletion constructs of SNX6 were all prepared using a
PCR-based strategy. PCR primers for the gene sequences included
BamHI and ClaI restriction sites. PCR products were gel-purified, restriction-digested, and then ligated into the
BamHI/ClaI sites in the vectors pEBB-FLAG and
pEBB-HA. Each construct was fully sequenced.
Transient Transfection, Immunoprecipitation, and Western
Blotting--
For the coimmunoprecipitation experiments COS-1 cells
were plated in 100-mm dishes at 2 × 106 cells 24 h prior to transfection. Cells were transfected for 4 h in
serum-free medium using 45 µl of LipofectAMINE (Life Technologies, Inc.), washed once with phosphate-buffered saline, and replenished with
fresh medium. After 24 h the medium was replaced with DMEM plus
0.2% FBS, and 48 h after transfection the cells were lysed by the
addition of 0.5 ml of lysis buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, and
10% glycerol) plus phosphatase and protease inhibitors. The cells were
allowed to incubate in the lysis buffer for 20 min on ice and were then
scraped into microcentrifuge tubes. After high speed
centrifugation for 15 min, an aliquot of the lysate was removed for
Western blotting, and the remainder was immunoprecipitated for 2 h
with 0.4-1 µg of an epitope-specific antibody and 35 µl of protein
G-Sepharose (80% suspension) (Amersham Pharmacia Biotech). Lysates and
immunoprecipitates were then separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and transferred onto
Immobilon-P membranes (Millipore) for blotting. Proteins were detected
using horseradish peroxidase-conjugated primary or secondary antibodies
and visualized by chemiluminescence (Pierce). Antibodies used were
anti-c-Myc mouse monoclonal 9E10 (hybridoma supernatant), anti-c-Myc
rabbit polyclonal A-14 (Santa Cruz Biotechnology), anti-HA mouse
monoclonal 12CA5 (hybridoma supernatant), anti-HA rabbit polyclonal
Y-11 (Santa Cruz Biotechnology), horseradish peroxidase-conjugated
anti-HA mouse monoclonal (Roche Molecular Biochemicals), anti-FLAG
mouse monoclonal M2 (Sigma), anti-T Indirect Immunofluorescence--
COS-1 cells were plated at
3 × 105 cells onto 22-mm glass coverslips 24 h
prior to transfection. Cells were transfected for 4 h in
serum-free medium using 8 µl of LipofectAMINE (Life Technologies, Inc.), washed once with phosphate-buffered saline, and replenished with
fresh medium. After 24 h the serum was replaced with DMEM plus
0.2% FBS. 48 h after transfection the cells were fixed in cold
3.5% paraformaldehyde for 5 min, permeabilized in cold absolute methanol for 2 min, and then incubated 5 min in 50 mM
glycine to quench paraformaldehyde autofluorescence. The transfected
constructs were then detected by incubation for 2 h at room
temperature with M2 mouse monoclonal anti-FLAG antibody and Y-11 rabbit
polyclonal anti-HA antibody. After washing in PBS (3 times for 5 min
each), the coverslips were incubated for 2 h at room temperature
with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG and rhodamine (TRITC)-conjugated goat anti-rabbit IgG secondary antibodies. The coverslips were then mounted in medium containing 4,6-diamino-2-phenylindole (DAPI) (Vector Laboratories). The cells were
examined using a Leica laser scanning confocal microscope.
Luciferase Functional Assays--
HepG2 cells were plated at
3 × 105 cells per well in 6-well plates 24 h
prior to transfection. For the 3TP-Lux assays the cells were
transfected with the 3TP-Lux reporter, pSV- SNX6 Is a Member of the Sorting Nexin Family--
Sorting nexin 6 was first cloned from a yeast two-hybrid screen to detect proteins
interacting with Smad1, a signaling intermediate in the BMP pathway
(32). However, attempts to confirm this interaction in a mammalian
system demonstrated that SNX6 associated only weakly with Smad1.
Subsequent data base searches noted the precise sequence for sorting
nexin 6 and identified this clone as a member of the sorting nexin
family. The amino acid sequence alignment for sorting nexins 1-6 is
presented in Fig. 1A, and a
schematic sequence alignment of these same sorting nexins is depicted
in Fig. 1B. The PX domains, which are aligned, are indicated
by the white boxes, and the predicted coiled coil regions
are represented by the smaller black boxes. Each protein
contains only one PX domain but a variable number of coiled coils.
SNX3, unlike the others, has no coiled coil regions, which may
contribute to its general lack of association with other proteins. SNX5
shows the greatest similarity to SNX6 (66% identity at the amino acid
level), and these two proteins structurally resemble each other with
short amino-terminal regions and a relatively long spacer region
between the PX domain and the first coiled coil.
Tissue Distribution of SNX6--
Northern blot analysis of SNX6
using a human multiple tissue blot and full-length SNX6 as probe
disclosed widespread expression of the gene (Fig. 1C).
Expression was highest in heart, skeletal muscle, and placenta, with
little expression noted for lung or liver. Two transcripts were
detected, one of 3.0 kilobase pairs and one of 2.2 kilobase pairs. The
intensity for the smaller transcript was always greater than that for
the larger transcript.
SNX6 Associates with TGF- SNX6 Associates with Receptor Tyrosine Kinases--
Other sorting
nexins have been shown previously to interact with receptor tyrosine
kinases. To investigate associations of SNX6 with receptor tyrosine
kinases, COS-7 cells were cotransfected with various receptors and
HA-tagged SNX6. Each receptor was then immunoprecipitated, and Western
blotting of the immunoprecipitates was performed for the HA-tagged
SNX6. Sorting nexin 6 associated with each receptor examined, the EGF
receptor, the insulin receptor, and the PDGF receptor, and with the
long form of the leptin receptor (Fig.
3).
Other SNX Proteins Show Unique Patterns of Interaction with TGF- SNX6 Forms Homo-oligomeric and Hetero-oligomeric Complexes with
Other SNXs--
As SNX proteins have been shown to complex with each
other, we assessed the ability of SNX6 to form homo-and
hetero-oligomeric complexes by cotransfecting epitope-tagged SNX
expression vectors into COS-1 cells, followed by immunoprecipitation
and Western blot analysis of cell lysates. Homomeric interactions were
tested by cotransfecting HA- and FLAG-tagged SNX6 into COS-1 cells.
Following immunoprecipitation with anti-FLAG antibody, Western blotting with anti-HA antibody demonstrated a strong homomeric association (Fig.
5A) that was not affected by
treatment with TGF-
To map the regions of the protein that mediate the association between
SNX6 and the other sorting nexins, deletion constructs of SNX6 were
tested for their ability to interact with either SNX1 or SNX4. In
contrast to the selective interaction of the PX domain with T Indirect Immunofluorescence of the Sorting Nexins--
Indirect
immunofluorescence was utilized to assess the intracellular
localization of SNX6 and to seek further evidence for the association
of SNX6 with other sorting nexins. Both SNX6 (Fig. 6, A, E, I, and M)
and sorting nexins 1-4 (Fig. 6, B, F, J, and N)
localized specifically to the cell cytoplasm in a diffuse pattern. This
cytoplasmic localization for SNX6 was not altered by cotransfection of
a constitutively active T Luciferase Functional Assays--
One question not yet answered by
the previous experiments was whether alteration of the levels of SNX6
within a cell might perturb TGF- In this paper we show for the first time associations between
members of the sorting nexin family and receptor serine-threonine kinases. Each SNX showed a different pattern of interaction with these
receptors of the TGF- Studies of the association of endogenous SNX2 with T A characteristic structural feature of the sorting nexins is the Phox
homology (PX) domain, a roughly 100-amino acid motif of uncertain
function found in components of the NADPH oxidase system, the sorting
nexins and their orthologs, and the phosphatidylinositol 3-kinases
(33). This domain is typically found in the middle of the molecule or
nearer the amino terminus. All of the sorting nexins except for SNX3,
which consists almost entirely of a PX domain with only minimal amino-
and carboxyl-terminal extensions, also have at least one coiled coil
domain near their carboxyl terminus. Heteromeric interactions of SNX6
with other sorting nexins appear to be mediated roughly to an equal
degree through the PX domain or the coiled coil regions of SNX6. The
receptors can also associate with either domain of SNX6, although the
interaction seems to be stronger with the PX domain. The involvement of
these domains in interactions between SNX6 and another SNX or
between SNX6 and a receptor suggests a model in which a receptor
interacts with the PX domain of SNX6, whereas another sorting nexin
associates through the coiled coil domain, forming a linear trimeric
complex. More probably, additional SNX molecules interact through both their PX and coiled coil domains, allowing larger complex formation, likely including multiple receptor molecules.
The function of the sorting nexins is at this time still unknown.
Mutation of the yeast homologs results in protein missorting, suggesting that they are involved in intracellular trafficking (29,
30). In mammalian cells overexpression of SNX1 was noted to accelerate
EGF receptor degradation, implying a role for this protein in endosomal
transport. Our reporter assays add further evidence that these
molecules play a role in receptor trafficking pathways. The linear
decrease in signal with increasing SNX6 clearly indicates that the
sorting nexin is interfering with TGF- Recently, the sorting nexins have been identified as part of a
molecular complex termed the retromer complex, which may facilitate retrograde transport from endosomes back to the trans-Golgi
network. A core retromer complex, consisting of Vps35p, Vps29p, and
Vps26p, was first identified in yeast and shown to function in the
retrieval of Vps10p (the carboxypeptidase Y receptor) from endosomes to the Golgi (34). Subsequent work demonstrated that Vps5p, the SNX1
homolog, and Vps17p, another PX domain containing protein, also form
part of this complex. In particular, these experiments suggested that a
core complex of Vps35p, Vps29p, and Vps26p provides the receptor
specificity, after which Vps5p and Vps17p assemble onto the membrane to
promote vesicle formation (35). The human orthologs of Vps35p, Vps29p,
and Vps26p have been cloned and investigated recently (36). These
binding experiments demonstrate multiple interactions among these
proteins, again suggesting that Vps35p forms the core of a molecular
complex. But the precise mechanism by which the cargo of a given
transport vesicle is chosen still remains undetermined. Given the
differing specificities the sorting nexins display for the receptor
serine-threonine kinases, the probability seems high that these
molecules, rather than the core retromer complex, either serve to
select the proteins to be conveyed within particular transport vesicles
or associate with and provide a marker for those proteins that actually
do regulate this specificity.
In conclusion, we have cloned a novel sorting nexin, SNX6, that
interacts strongly with specific members of the receptor
serine-threonine kinase and receptor tyrosine kinase families and that
colocalizes and associates with other sorting nexins. These findings
demonstrate for the first time the interaction of sorting nexins with a
family of receptor serine-threonine kinases, both enlarging the scope of receptors that interact with this class of proteins and showing a
higher degree of specificity than that noted for the receptor tyrosine
kinases. The results of these experiments further suggest that the
sorting nexins function in a multimeric complex to enable the specific
selection and transport of molecules within the intracellular vesicular
transport pathways. Whether the levels of this family of proteins are
regulated to modulate physiological responses or to play a role in
disease pathogenesis remains to be determined.
family of receptor
serine-threonine kinases. These receptors belong to two classes: type
II receptors that bind ligand, and type I receptors that are
subsequently recruited to transduce the signal. Of the type II
receptors, SNX6 was found to interact strongly with ActRIIB and more
moderately with wild type and kinase-defective mutants of T
RII. Of
the type I receptors, SNX6 was found to interact only with inactivated
T
RI. SNXs 1-4 also interacted with the transforming growth
factor-
receptor family, showing different receptor preferences.
Conversely, SNX6 behaved similarly to the other SNX proteins in its
interactions with receptor tyrosine kinases. Strong heteromeric
interactions were also seen among SNX1, -2, -4, and -6, suggesting the
formation in vivo of oligomeric complexes. These findings
are the first evidence for the association of the SNX family of
molecules with receptor serine-threonine kinases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-
)1 family includes a
large number of peptides, including the TGF-
s themselves,
activin/inhibin, the bone morphogenetic proteins (BMPs), the growth and
differentiation factors (GDFs), glial-derived neurotrophic factor, and
Müllerian inhibitory substance (1). Although there are no yeast
TGF-
s, homologs have been identified in primitive metazoans,
including Caenorhabditis elegans and Drosophila
(2-4). With the exception of only glial-derived neurotrophic factor,
these ligands signal through heterotetrameric pairs of serine-threonine
kinase receptors. Ligand first interacts with a type II receptor,
which, following ligand binding, recruits a type I receptor (5). The
type II receptors are constitutively active kinases, catalyzing
phosphorylation both of themselves in an autocatalytic reaction and of
the recruited type I receptor (6). Once bound to ligand and
phosphorylated by the type II receptor, the type I receptor then
transduces the signal to the intracellular signaling intermediates,
including the recently described family of Smad proteins (7-13). In
general, one or two closely related type I and one or two closely
related type II receptors are utilized by each class of ligand. For
example, TGF-
1 and TGF-
3 bind to the type II TGF-
receptor
(T
RII), with subsequent recruitment of the type I TGF-
receptor
(T
RI/ALK5 (activin-like kinase 5)) (6). Similarly, activin binds to
either ActRII or ActRIIB, with activin type IB receptor (ALK4) or
possibly ActRI (ALK2) then joining the complex (14-19). BMPs typically
bind to their type II receptor (BMPRII) and then recruit either BMPRIA (ALK3) or BMPRIB (ALK6) (14, 20), but they can also bind to the activin
type II receptors, in which case they recruit ActRI (ALK2) (21).
receptors within the cell. Experiments in
mink lung epithelial cells have shown that endogenous T
RI and
T
RII move from the endoplasmic reticulum to the cell surface along
independent, non-intersecting pathways, so that no heteromeric complex
formation occurs prior to the expression of these receptors on the cell
surface (22, 23). For each receptor, surface binding of ligand both
decreases receptor half-life (22, 23) and also results in
down-regulation of receptor surface expression (24-27). The pathways
available to individual receptors following internalization have not
yet been characterized.
family of receptor serine-threonine kinases, as well as with
receptor tyrosine kinases shown previously to interact with other SNX
proteins. We also show that SNX6 can hetero-oligomerize and colocalize
intracellularly with SNX1, SNX2, and SNX4 and that these other SNXs
also associate with members of the TGF-
receptor family. These
studies are the first to document the interaction of sorting nexins
with receptor serine-threonine kinases, thus broadening the range of
receptor interactions with the SNX family.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C.
RII rabbit polyclonal C16 (Santa
Cruz Biotechnology), anti-T
RII goat polyclonal (R & D Systems), and
anti-SNX2 rabbit polyclonal. The procedure for the immunoprecipitation
of endogenous complexes from HepG2 cells varied only in that the
immunoprecipitation and the primary antibody staining were performed overnight.
-gal to normalize transfection efficiency, the sorting nexin, and pCDNA3 to normalize the amount of transfected DNA. For the FAST1/ARE assays the cells were
transfected with FAST1, the 3A-Luc reporter, pSV-
-gal, the sorting
nexin, and pCDNA3. Cells were transfected for 8 h in
serum-free medium using 8 µl of LipofectAMINE (Life Technologies,
Inc.), washed once with phosphate-buffered saline, and replenished with fresh medium. After 24 h the serum was replaced with DMEM plus 0.2% FBS, and the cells were treated with either 5 ng/ml TGF-
1 or
50 ng/ml Activin A for 24 h. The cells were then lysed, and the
luciferase and
-galactosidase activities were determined. All assays
were performed in triplicate. The
-galactosidase values were used to
correct the luciferase values for transfection efficiency.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The sorting nexins share common PX and coiled
coil domains. A, multiple sequence alignment of sorting
nexins 1-6 was performed using the ClustalW program, and the aligned
sequences are shown. B, sorting nexins 1-6 are depicted
schematically, with the white boxes representing the PX
domain and the black boxes representing coiled coil regions.
The initial amino acids for the PX domains are aligned vertically, and
the remainder of each protein is drawn to scale. The amino acid
positions within each protein are indicated by the numbers.
C, a multiple tissue Northern blot was probed with
full-length SNX6. The numbers adjacent to the blot represent
the size in kilobases (Kb) of each transcript.
Receptor Family Members--
Since
SNX6 interacted only weakly with Smad family members (data not shown)
and since other SNX family members had been shown to interact with cell
surface receptors, we investigated whether SNX6 might interact directly
with receptors of the TGF-
family. Initially, type I receptors
(ALK1-ALK6) and the type II TGF-
receptor (T
RII) were assessed
for their ability to bind SNX6. The kinase-deficient (KD) mutants of
these receptors were utilized in order to maximize the likelihood of
observing what might otherwise be a transient association.
Immunoprecipitation of FLAG-tagged SNX6 followed by blotting for the
HA-tagged receptors demonstrated strong binding of SNX6 to the KD
T
RII (Fig. 2A, lane 9) and
to KD ALK5 (T
RI) (lane 7), with much weaker associations
evident with KD ALK1 (lane 2) and KD ALK6 (lane
8). A panel of type II receptors was also tested for interaction
with SNX6 (Fig. 2B). Wild type (WT) type II receptors were
chosen for this panel, with KD T
RII also included for comparison
with the previous experiments. SNX6 associated more strongly with
ActRIIB than with KD T
RII and showed only a weak association with WT
T
RII. It did not interact with ActRII or BMPRII. Attempts were then
undertaken to map the interacting regions of the proteins. Deletion
mutants of SNX6 were designed to isolate the effects of particular
motifs of the protein, especially the PX or the coiled coil domains
(Fig. 2C). SNX6 deletion mutants were tested with KD T
RI
in coimmunoprecipitation experiments, and in these studies the PX
domain alone (1) bound the receptor as strongly as the full-length
protein, whereas the coiled coil domain showed only a weak association
with the receptor (Fig. 2D).
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Fig. 2.
Sorting nexin 6 associates with type I and
type II TGF- receptors. A,
COS-1 cells were transfected with FLAG-tagged SNX6 and HA-tagged
kinase-deficient (KD) type I receptors or KD type II TGF-
receptor. Cell lysates were immunoprecipitated (IP) with an
anti-FLAG antibody and blotted with an anti-HA antibody to demonstrate
association of KD ALK1, ALK5, ALK6, and TGF-
type II receptors with
SNX6 (top row). The middle and bottom
rows are control Western blots to show expression of HA receptors
and FLAG-SNX6, respectively. B, COS-1 cells were transfected
with FL-SNX6 and HA-tagged receptors (lanes 1-4) or HA-SNX6
and FL-tagged receptors (lanes 5-8). Lanes 1-4
were immunoprecipitated with an anti-FLAG antibody and lanes
5-8 with an anti-HA antibody to immunoprecipitate SNX6.
Lanes 1-4 were then blotted with an anti-HA antibody and
lanes 5-8 with an anti-FLAG antibody to demonstrate
association of the sorting nexin with receptors (top). SNX6
interacts most strongly with ActRIIB, moderately with KD TGF-
RII,
and weakly with WT TGF-
RII. No association was seen with ActRII or
BMPRII. C, the deletion constructs of SNX6 are schematically
depicted with reference to the full-length molecule. 73-406,
153-406, and 253-406 delete all or part of the PX
domain (white box). 1-152 and 1-252
delete the coiled coil domains (solid boxes) and part of the
linker region. 1-72 deletes the coiled coil regions and
nearly the entirety of the PX domain. D, COS-1 cells were
transfected with HA-T
RI KD and the FL-tagged deletion constructs of
SNX6. Cell lysates were immunoprecipitated with an anti-FLAG antibody
and blotted for T
RI KD using an anti-HA antibody. T
RI KD
associated more strongly with PX domain (1-152 and
1-252) than with the coiled coil regions
(253-406). The middle and bottom rows
represent control Western blots for the immunoprecipitations.
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Fig. 3.
Sorting nexin 6 interacts with receptor
tyrosine kinases. COS-7 cells were transfected with HA-tagged SNX6
and untagged receptor constructs. Cell lysates were immunoprecipitated
(IP) with the appropriate anti-receptor antibody and then
blotted for SNX6 with anti-HA antibody to demonstrate association of
SNX6 with each receptor (top row). The middle and
bottom rows are control Western blots to show expression of
HA-SNX6 and receptors, respectively. Odd-numbered lanes
represent controls in which HA-SNX6 was not expressed.
EGFR, EGF receptor; IR, insulin receptor;
PDGFR, PDGF receptor; LR, leptin receptor.
Receptor Family Members--
Given the strong associations of SNX6
with various receptors within the TGF-
family, we examined the
associations between these receptors and the other sorting nexins. The
interactions of SNX1-SNX4 were determined for the panel of KD type I
receptors, with the results tabulated in Table
I. SNX3 showed no interaction with any of
the KD receptors, and SNX1 showed a weak interaction with only KD ALK4.
SNX2 and SNX4 demonstrated more robust associations, with SNX2
interacting with KD ALK4, KD ALK6, and KD T
RII and with SNX4
interacting with KD T
RII and more weakly with KD ALK6. Since SNX6
bound strongly to KD ALK5, we then tested the interaction of this
receptor with the other SNXs. KD ALK5 bound to varying degrees to all
SNXs except SNX3, with SNX6 showing the strongest interaction, and
SNX1, SNX2, and SNX4 showing more modest levels of association (Fig.
4A). A similar experiment was
then performed to assess the binding of the various SNXs with ActRIIB,
which had been shown to interact most strongly with SNX6 (Fig.
4B). Interestingly, SNX1, -2, and -6 also interacted
strongly with this type II receptor. As expected, SNX3 did not interact
but surprisingly neither did SNX4. Finally, in order to demonstrate the
physiologic relevance of these interactions, we sought an association
of an endogenous SNX with an endogenous receptor serine-threonine kinase. HepG2 cells were used since they have both abundant SNX2 and
detectable levels of T
RII. Immunoprecipitation for the receptor using an antibody against the extracellular domain coimmunoprecipitated a portion of the endogenous SNX2 (Fig. 4C). Comparable
experiments using an antibody directed against the cytoplasmic portion
of the receptor failed to show a similar interaction (data not shown), suggesting that the antibody disrupts the association of the sorting nexin with the cytoplasmic region of the receptor.
Association of SNX with kinase-deficient type I and type II receptors
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Fig. 4.
Sorting nexins 1-4 and 6 show different
specificities of interaction with a kinase-deficient type I
TGF- receptor and with the type II receptor
ActRIIB. A, COS-1 cells were transfected with HA-T
RI
and either myc-SNX1-4 or FL-SNX6. The sorting nexin was
immunoprecipitated (IP) using either an anti-Myc or
anti-FLAG antibody, and receptor association was detected with an
anti-HA antibody (top). SNX1, -2, -4, and -6 associated to
varying degrees with this type I receptor. B, COS-1 cells
were transfected with FLAG-ActRIIB and either myc-SNX1-4 or HA-SNX6.
The sorting nexin was immunoprecipitated using either an anti-Myc or
anti-HA antibody, and receptor association was detected with an
anti-FLAG antibody (top). SNX1, -2, and -6 associated
strongly with this type II receptor. The middle and
bottom rows represent control Western blots for the
immunoprecipitations. C, HepG2 cells were plated at 6 × 106 cells and allowed to grow overnight. The lysates
were precipitated with either control IgG (goat), a rabbit
polyclonal anti-T
RII generated against the cytoplasmic portion of
the receptor (cT
RII), or a goat polyclonal
anti-T
RII generated against the extracellular portion of the
receptor (eT
RII). The immunoprecipitates were
detected either with cT
RII or a rabbit polyclonal anti-SNX2.
Ab, antibody.
1 (data not shown). Similar studies were carried
out to determine whether heteromeric interactions could be detected
between SNX6 and the other sorting nexins. Interaction studies using
HA-SNX6 and Myc-tagged SNX1-SNX4 showed strong associations of SNX6
with SNX1, SNX2, and SNX4 but no association with SNX3 (Fig.
5B).
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Fig. 5.
SNX6 associates with itself and with other
sorting nexins. A, COS-1 cells were transfected with
FL- and HA-tagged SNX6. Cell lysates were immunoprecipitated
(IP) with an anti-HA antibody and blotted for SNX6 with an
anti-FLAG antibody to demonstrate the homomeric association of SNX6
with itself (lane 3). B, COS-1 cells were
transfected with HA-SNX6 and Myc-tagged SNX1-4. Cell lysates were
immunoprecipitated with an anti-HA antibody and blotted for SNX1-4
with an anti-Myc antibody to show the heteromeric associations of SNX6
with SNX1, SNX2, and SNX4 (lanes 2, 3, and 5).
C, COS-1 cells were transfected with myc-SNX1 and the
FLAG-tagged deletion constructs of SNX6. Cell lysates were
immunoprecipitated with an anti-FLAG antibody and blotted for SNX1
using an anti-Myc antibody. SNX1 associated strongly with each of the
deletions except 1-72, the smallest (lane 6). The
middle and bottom rows represent control Western
blots for the immunoprecipitations.
RII,
these experiments showed that all regions of the molecule retained
considerable ability to interact (Fig. 5C and data not
shown). Only SNX6-(1-72), the extreme amino terminus lacking
both the PX and coiled coil domains, did not associate. The relative
uniformity of expression of the various deletion constructs suggests
that the degree of association with each region was similar and that
both the PX domain and the coiled coil regions may contribute to the
hetero-oligomerization.
RI to stimulate TGF-
signaling pathways or a constitutively active BMPRIA to stimulate BMP signaling (data not
shown). Cotransfection of SNX6 and sorting nexins 1, 2, and 4 showed
nearly complete overlap of the two staining patterns, as indicated by
the yellow color in the images (Fig. 6, D,
H, and P). Surprisingly, cotransfection of SNX3 with
SNX6 also showed considerable, although not complete, colocalization
(Fig. 6L). For this pair of proteins, the overlap is
strongest at the periphery of the cell, in contrast to the uniform
overlap seen with the other SNXs.
View larger version (52K):
[in a new window]
Fig. 6.
SNX6 colocalizes with other sorting
nexins. COS-1 cells were transfected with HA-SNX6 and myc-SNX1
(A-D), myc-SNX2 (E-H), myc-SNX3
(I-L), or myc-SNX4 (M-P) constructs, fixed in
3.5% paraformaldehyde, permeabilized with methanol, stained, and
analyzed with laser scanning confocal microscopy. The Myc-tagged
sorting nexins were detected using a monoclonal anti-Myc antibody and a
FITC-conjugated goat anti-mouse IgG secondary antibody (A, E,
I, and M). HA-SNX6 was detected using a rabbit
polyclonal anti-HA antibody and a TRITC-conjugated goat anti-rabbit IgG
secondary antibody (B, F, J, and N). DAPI
staining (C, G, K, and O) highlights the location
of nuclei. The FITC, TRITC, and DAPI images were overlapped (D,
H, L, and P) to demonstrate at least partial
colocalization for SNX6 with sorting nexins 1-4.
signal transduction pathways. To
explore this issue, functional assays using TGF-
-responsive reporter
constructs linked to luciferase expression were performed in HepG2
cells. These assays measure the ability of a ligand to induce gene
expression, and they thereby assess the functional capacity of the
entire signaling pathway. Any disturbance along the pathway, including a possible shift in cell surface expression of the TGF-
or activin receptors, might be expected to change the flux through the pathway and
quantifiably modify the read-out. The FAST1/ARE assay uses the 3A-Luc
reporter, a construct generated by the tandem repeat of an activin
response element identified in the promoter for the Mix2
gene. The assay, which is sensitive to both TGF-
and activin,
measures a direct, Smad2-specific response. Assays using this reporter
with stimulation by TGF-
demonstrated a 5-fold reduction in
luciferase activity with increasing amounts of SNX6 (Fig.
7A), and similar assays with
stimulation by activin showed an 8-fold decrease (Fig. 7B).
For both ligands the reduction in luciferase values occurred in a
stepwise, dose-dependent fashion. 3TP-Lux, a segment of the
PAI-1 promoter downstream of three
12-O-tetradecanoylphorbol-13-acetate-responsive elements, is also sensitive to both TGF-
and activin. In assays using this reporter, SNX6 reduced the stimulation by TGF-
about 50%
(Fig. 7C). Interestingly, the basal, unstimulated luciferase readings for both reporters diminished at an even greater rate with
increasing SNX6, with the FAST1/ARE system showing a 12-fold reduction
and the 3TP-Lux system a 6-fold decrease. As a control, similar assays
were performed using increasing amounts of SNX3, the one sorting nexin
that bound none of the tested receptor serine-threonine kinases. In
contrast to SNX6, SNX3 had no effect on reporter activity (data not
shown), suggesting that the reduction in the basal values is not
nonspecific but likely due to interference with autocrine stimulation
of the reporters by TGF-
.
View larger version (14K):
[in a new window]
Fig. 7.
SNX6 inhibits TGF-
and activin signaling. HepG2 cells were transfected with a
3A-Luc reporter plasmid, FAST1, and increasing amounts of SNX6. The
cells were serum-starved for 24 h, during which time half were
treated with 5 ng/ml TGF-
1 (A) or 50 ng/ml activin A
(B). C, HepG2 cells were transfected with the
3TP-Lux reporter plasmid and increasing amounts of SNX6. The cells were
serum-starved for 24 h, during which time half were treated with 5 ng/ml TGF-
1. For each assay
-galactosidase values were used to
normalize for transfection efficiency, and the results are presented as
relative luciferase values. Error bars indicate standard
deviations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
family. Concerning the type I receptors, SNX6
alone bound strongly to ALK5. Likewise, only SNX2 interacted appreciably with ALK4. Differences in association patterns for SNX
binding were again evident in their interactions with the type II
receptors. T
RII associated with SNX2, SNX4, and SNX6 but not with
SNX1 or SNX3, whereas ActRIIB interacted strongly with SNX1, SNX2, and
SNX6 but not with SNX3 or SNX4. In contrast to the distinct
associations of SNXs with receptor serine-threonine kinases, the
pattern of SNX6 binding to the receptor tyrosine kinases was similar to
that of the other sorting nexins. All SNXs (except SNX3) tested to date
have interacted at least to a degree with the tested receptor tyrosine
kinases (28, 31), and SNX6 reproduced this pattern, binding the EGF
receptor, the PDGF receptor, the insulin receptor, and the long form of
the leptin receptor.
RII confirmed
that the above findings have physiologic relevance. We feel that the
somewhat low stoichiometry of the interaction results from the
confluence of several features of sorting nexin biology. First, the
sorting nexins are capable of binding to a range of different
receptors, but probably only one or two simultaneously. In a cell only
a minority of the sorting nexin molecules is likely interacting with a
given receptor. Similarly, most cells express multiple different
sorting nexins, so that at any given time one type of receptor may be
interacting with multiple sorting nexins. Finally, sorting nexins may
have other roles in the cell so that only a small proportion is bound
to receptors in the normal physiologic state. Although the function of
the sorting nexins is not yet clear, the intracellular localization of
these molecules to vesicles and their strong interactions with
receptors suggest that, as a part of their roles within the cell, the
different sorting nexins are involved in the targeting of receptors to
intracellular trafficking pathways.
signaling. Although these
assays provide no direct measure for any single component of this
signaling pathway, when examined in the context of the previous
coimmunoprecipitation data these results suggest that the increasing
amounts of SNX6 within a cell alter plasma membrane receptors. The
sorting nexin may be binding to and sequestering receptor, fostering
increased receptor degradation, or directly inhibiting receptor
function. Despite these findings, definitive evidence for a specific
function remains elusive.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Masa Kawabata for the ALK1-4
and -6, ActRII, ActRIIB, and BMPRII constructs; Jeff Wrana and
Liliana Attisano for TGF- receptor constructs; and Jens
Wuerthner for helpful discussions. Transfection protocols were
generated using Cellputer software.
![]() |
FOOTNOTES |
---|
* 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.
¶ To whom correspondence should be addressed. Tel.: 301-496-5391; Fax: 301-496-8395; E-mail: robertsa@dce41.nci.nih.gov.
Present address: Virginia Mason Research Center, 1201 9th Ave., Seattle, WA 98101, and the Dept. of Immunology, University of
Washington, Seattle, WA 98195.
** Present address: Dept. of Pharmacology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814.
Published, JBC Papers in Press, March 8, 2001, DOI 10.1074/jbc.M100606200
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ABBREVIATIONS |
---|
The abbreviations used are:
TGF-, transforming growth factor-
;
SNX, sorting nexin;
EGF, epidermal
growth factor;
PDGF, platelet-derived growth factor;
ActRIIB, activin
type IIB receptor;
BMP, bone morphogenetic protein;
GDF, growth and
differentiation factor;
ALK, activin-like kinase;
BMPRII, bone
morphogenetic protein type II receptor;
PX domain, phox homology
domain;
FITC, fluorescein isothiocyanate;
TRITC, tetramethylrhodamine B
isothiocyanate;
DAPI, 4,6-diamino-2-phenylindole;
KD, kinase-deficient;
WT, wild type;
DMEM, Dulbecco's modified Eagle's medium;
FBS, fetal
bovine serum;
T
RI, type I TGF-
receptor;
T
RII, type II TGF-
receptor;
HA, hemagglutinin;
PCR, polymerase chain reaction.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Massague, J. (1998) Annu. Rev. Biochem. 67, 753-791[CrossRef][Medline] [Order article via Infotrieve] |
2. | Padgett, R. W., St. Johnston, R. D., and Gelbart, W. M. (1987) Nature 325, 81-84[CrossRef][Medline] [Order article via Infotrieve] |
3. | Padgett, R. W., Savage, C., and Das, P. (1997) Cytokine Growth Factor Rev. 8, 1-9[CrossRef][Medline] [Order article via Infotrieve] |
4. | Otsuki, T., Kajigaya, S., Ozawa, K., and Liu, J. M. (1999) Biochem. Biophys. Res. Commun. 265, 630-635[CrossRef][Medline] [Order article via Infotrieve] |
5. | Wrana, J. L., Attisano, L., Carcamo, J., Zentella, A., Doody, J., Laiho, M., Wang, X. F., and Massague, J. (1992) Cell 71, 1003-1014[Medline] [Order article via Infotrieve] |
6. | Wrana, J. L., Attisano, L., Wieser, R., Ventura, F., and Massague, J. (1994) Nature 370, 341-347[CrossRef][Medline] [Order article via Infotrieve] |
7. | Wrana, J. L., and Attisano, L. (2000) Cytokine Growth Factor Rev. 11, 5-13[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Massague, J.,
and Chen, Y. G.
(2000)
Genes Dev.
14,
627-644 |
9. | Heldin, C. H., Miyazono, K., and ten Dijke, P. (1997) Nature 390, 465-471[CrossRef][Medline] [Order article via Infotrieve] |
10. | Attisano, L., and Wrana, J. L. (1998) Curr. Opin. Cell Biol. 10, 188-194[CrossRef][Medline] [Order article via Infotrieve] |
11. | Derynck, R., Zhang, Y., and Feng, X. H. (1998) Cell 95, 737-740[Medline] [Order article via Infotrieve] |
12. | Padgett, R. W., Das, P., and Krishna, S. (1998) BioEssays 20, 382-390[CrossRef][Medline] [Order article via Infotrieve] |
13. | Christian, J. L., and Nakayama, T. (1999) BioEssays 21, 382-390[CrossRef][Medline] [Order article via Infotrieve] |
14. | ten Dijke, P., Yamashita, H., Ichijo, H., Franzen, P., Laiho, M., Miyazono, K., and Heldin, C. H. (1994) Science 264, 101-104[Medline] [Order article via Infotrieve] |
15. | Attisano, L., Carcamo, J., Ventura, F., Weis, F. M., Massague, J., and Wrana, J. L. (1993) Cell 75, 671-680[Medline] [Order article via Infotrieve] |
16. | Ebner, R., Chen, R. H., Shum, L., Lawler, S., Zioncheck, T. F., Lee, A., Lopez, A. R., and Derynck, R. (1993) Science 260, 1344-1348[Medline] [Order article via Infotrieve] |
17. | Tsuchida, K., Mathews, L. S., and Vale, W. W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11242-11246[Abstract] |
18. | Carcamo, J., Weis, F. M., Ventura, F., Wieser, R., Wrana, J. L., Attisano, L., and Massague, J. (1994) Mol. Cell. Biol. 14, 3810-3821[Abstract] |
19. | Attisano, L., Wrana, J. L., Montalvo, E., and Massague, J. (1996) Mol. Cell. Biol. 16, 1066-1073[Abstract] |
20. | Rosenzweig, B. L., Imamura, T., Okadome, T., Cox, G. N., Yamashita, H., ten Dijke, P., Heldin, C. H., and Miyazono, K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7632-7636[Abstract] |
21. |
Macias-Silva, M.,
Hoodless, P. A.,
Tang, S. J.,
Buchwald, M.,
and Wrana, J. L.
(1998)
J. Biol. Chem.
273,
25628-25636 |
22. |
Wells, R. G.,
Yankelev, H.,
Lin, H. Y.,
and Lodish, H. F.
(1997)
J. Biol. Chem.
272,
11444-11451 |
23. |
Koli, K. M.,
and Arteaga, C. L.
(1997)
J. Biol. Chem.
272,
6423-6427 |
24. |
Frolik, C. A.,
Wakefield, L. M.,
Smith, D. M.,
and Sporn, M. B.
(1984)
J. Biol. Chem.
259,
10995-11000 |
25. | Massague, J. (1985) J. Cell Biol. 100, 1508-1514[Abstract] |
26. | Wakefield, L. M., Smith, D. M., Masui, T., Harris, C. C., and Sporn, M. B. (1987) J. Cell Biol. 105, 965-975[Abstract] |
27. | Zhao, J., and Buick, R. N. (1995) Cancer Res. 55, 6181-6188[Abstract] |
28. | Kurten, R. C., Cadena, D. L., and Gill, G. N. (1996) Science 272, 1008-1010[Abstract] |
29. | Ekena, K., and Stevens, T. H. (1995) Mol. Cell. Biol. 15, 1671-1678[Abstract] |
30. | 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] |
31. |
Haft, C. R.,
de la Luz, S. M.,
Barr, V. A.,
Haft, D. H.,
and Taylor, S. I.
(1998)
Mol. Cell. Biol.
18,
7278-7287 |
32. |
Kim, R. H.,
Wang, D.,
Tsang, M.,
Martin, J.,
Huff, C.,
de Caestecker, M. P.,
Parks, W. T.,
Meng, X.,
Lechleider, R. J.,
Wang, T.,
and Roberts, A. B.
(2000)
Genes Dev.
14,
1605-1616 |
33. |
Ponting, C. P.
(1996)
Protein Sci.
5,
2353-2357 |
34. |
Seaman, M. N.,
Marcusson, E. G.,
Cereghino, J. L.,
and Emr, S. D.
(1997)
J. Cell Biol.
137,
79-92 |
35. |
Seaman, M. N.,
McCaffery, J. M.,
and Emr, S. D.
(1998)
J. Cell Biol.
142,
665-681 |
36. |
Haft, C. R.,
Sierra, M. L.,
Bafford, R.,
Lesniak, M. A.,
Barr, V. A.,
and Taylor, S. I.
(2000)
Mol. Biol. Cell
11,
4105-4116 |