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Molecular and Cellular Biology, December 1998, p. 7278-7287, Vol. 18, No. 12
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification of a Family of Sorting Nexin
Molecules and Characterization of Their Association with
Receptors
Carol Renfrew
Haft,1,*
Maria
de
la Luz Sierra,1
Valarie A.
Barr,1
Daniel H.
Haft,2 and
Simeon I.
Taylor1
Diabetes Branch, National Institutes of
Diabetes and Digestive and Kidney Diseases, National Institutes of
Health, Bethesda, Maryland 20892,1 and
National Biomedical Research Foundation, Washington,
D.C.2
Received 31 December 1997/Returned for modification 6 March
1998/Accepted 8 September 1998
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ABSTRACT |
Sorting nexin 1 (SNX1) is a protein that binds to the epidermal
growth factor (EGF) receptor and is proposed to play a role in
directing EGF receptors to lysosomes for degradation (R. C. Kurten, D. L. Cadena, and G. N. Gill, Science 272:1008-1010,
1996). We have obtained full-length cDNAs and deduced the amino acid sequences of three novel homologous proteins, which were denoted human
sorting nexins (SNX2, SNX3, and SNX4). In addition, we identified a
presumed splice variant isoform of SNX1 (SNX1A). These molecules contain a conserved domain of ~100 amino acids, which was termed the
phox homology (PX) domain. Human SNX1 (522 amino acids), SNX1A (457 amino acids), SNX2 (519 amino acids), SNX3 (162 amino acids), and SNX4
(450 amino acids) are part of a larger family of hydrophilic molecules
including proteins identified in Caenorhabditis elegans and
Saccharomyces cerevisiae. Despite their hydrophilic nature, the sorting nexins are found partially associated with cellular membranes. They are widely expressed, although the tissue distribution of each sorting nexin mRNA varies. When expressed in COS7 cells, epitope-tagged sorting nexins SNX1, SNX1A, SNX2, and SNX4
coimmunoprecipitated with receptor tyrosine kinases for EGF,
platelet-derived growth factor, and insulin. These sorting nexins also
associated with the long isoform of the leptin receptor but not with
the short and medium isoforms. Interestingly, endogenous COS7
transferrin receptors associated exclusively with SNX1 and SNX1A, while
SNX3 was not found to associate with any of the receptors studied. Our
demonstration of a large conserved family of sorting nexins that
interact with a variety of receptor types suggests that these proteins
may be involved in several stages of intracellular trafficking in
mammalian cells.
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INTRODUCTION |
Cell surface receptors mediate a
wide variety of biological processes, including transmembrane
signalling in response to extracellular ligands (7, 19) and
receptor-mediated endocytosis of macromolecules (12, 16,
27). In addition to participating in events initiated at the
plasma membrane, cell surface receptors traverse both the secretory
pathway during their biosynthesis and the endocytic pathway following
internalization from the cell surface (8, 10). Many
different proteins are required to form the complex molecular machinery
used to direct cellular trafficking of receptors and to mediate
signalling downstream of these molecules (6, 36). A variety
of interaction cloning techniques have been used by investigators to
identify some of the proteins involved in these processes (3,
25). Recently, Kurten et al. (21) used the yeast
two-hybrid system to clone a cDNA encoding a protein, sorting nexin 1 (SNX1), that bound to the cytoplasmic domain of the epidermal growth
factor (EGF) receptor. Based primarily on its homology to a protein
(Mvp1p) known to be involved in targeting hydrolases to the vacuole in
yeast, it was hypothesized that SNX1 may be involved in EGF receptor
degradation in lysosomes (15). Moreover, it was reported
that SNX1 promoted degradation of the EGF receptor but did not exert
the same effect on other related tyrosine kinases (21).
We now report the cloning of three novel proteins that are homologous
to human SNX1, which were designated SNX2, SNX3, and SNX4. All four
sorting nexins contain an ~100-amino-acid region, which was termed
the phox homology (PX) domain, that is also contained in at least three
Saccharomyces cerevisiae proteins (32). Two of
these proteins, Mvp1p and Vps5p, are required for the proper sorting of
carboxypeptidase Y to the vacuole (15, 18, 30). A third
yeast protein, Grd19p, has recently been shown to be required to
maintain two late-Golgi enzymes (dipeptidyl amino peptidase A and Kex2)
in their proper locations by retrieving mislocalized molecules from the
prevacuolar compartment (41). Interestingly, Vps5p exhibits
the greatest sequence similarity with SNX1 and SNX2 described here,
while Grd19p is most closely related to SNX3.
In addition, we report that SNX1, SNX2, and SNX4 associate with a
variety of receptors, including receptors for insulin, EGF, platelet-derived growth factor (PDGF), and leptin. In contrast, we did
not detect binding of SNX3 to these different receptor types. Taken
together, these findings suggest that mammalian cells possess multiple
sorting nexins that, by analogy to their yeast homologs, are likely to
play an important role in protein trafficking among various organelles.
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MATERIALS AND METHODS |
Cells.
COS7 cells were obtained from the American Type
Culture Collection (ATCC, Rockville, Md.). NIH 3T3 cells overexpressing
human EGF receptors were kindly provided by J. Schlessinger
(17). The cells were maintained in Dulbecco's modified
medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum (Life
Technologies, Inc., Gaithersburg, Md.), 100 U of penicillin and 100 µg of streptomycin per ml.
Antibodies.
Anti-insulin receptor 
-subunit antibody
(C19), anti-EGF receptor antibody (1005), anti-PDGF receptor,
-isoform antibody (958), and anti-c-myc antibodies (A-14 and 9E10)
were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.).
Anti-hemagglutinin (HA) antibody (12CA5) was from Boehringer Mannheim
Corp. (Indianapolis, Ind.). Anti-transferrin receptor antibody (6F11)
was from Advanced Immuno Chemical Corp. (Long Beach, Calif.).
Anti-leptin receptor antibody directed against the common extracellular
domain of the receptor isoforms (N-term OB-R) was from Research
Diagnostics, Inc. (Flanders, N.J.). Donkey anti-rabbit immunoglobulin G
(IgG)-peroxidase and sheep anti-mouse IgG-peroxidase were obtained from
Amersham Life Science, Inc. (Arlington Heights, Ill.), and mouse
anti-goat IgG-peroxidase was from Jackson ImmunoResearch Laboratories
(West Grove, Pa.).
ESTs related to SNX1.
Using the published amino acid
sequence for human SNX1 (GenBank accession no. U53225), we searched the
expressed sequence tag (EST) nucleotide sequence database of the
National Center for Biotechnology Information for molecules related to
SNX1 at the amino acid level. Four classes of human ESTs were
identified by using the BLAST algorithm that had a high degree of amino
acid homology with SNX1 (1). EST 236890, which was obtained
from an infant brain cDNA library (GenBank accession no. T17214), showed 100% nucleotide identity with SNX1 over 95 amino acids. EST
309337, which was obtained from a human fetal lung cDNA library (GenBank accession no. W40457), showed 71% amino acid identity with
and 81% amino acid similarity to SNX1 over 167 amino acids, while the
translated sequence of EST 324810, which was obtained from a senescent
fibroblast cDNA library (GenBank accession no. W49550), showed 29%
amino acid identity with and 61% amino acid similarity to SNX1 over 54 amino acids. In addition, EST 78509, obtained from a human adult liver
library (GenBank accession no. T60550) showed 30% identity with and
50% similarity to SNX1 over 105 amino acids. The high degree of amino
acid homology between these human ESTs and SNX1 suggested that three
additional human sorting nexin molecules existed in the human genome.
Using these and other overlapping clones, we obtained full-length cDNA molecules and determined the sequences of human SNX2, SNX3, and SNX4
(see Fig. 1 and below).
Cloning of SNX1, SNX1A, SNX2, SNX3, and SNX4 cDNAs.
I.M.A.G.E. Consortium cDNA clones (23) were obtained from
Research Genetics (Huntsville, Ala.) or the ATCC. Plasmid DNA was
isolated with reagents supplied by QIAGEN, Inc. (Chatsworth, Calif.),
and sequenced on one or both strands starting with vector-specific primers, followed by a series of sequence-specific primers as indicated
(see Fig. 1). PCR was performed with the Expand high-fidelity PCR kit
(Boehringer Mannheim Corp.) and 5'-Stretch Plus human pancreas and
skeletal muscle cDNA libraries (Clontech, Palo Alto, Calif.) to obtain
additional sequence information for SNX1, SNX2, and SNX3. A 5'-rapid
amplification of cDNA ends (RACE) strategy (Life Technologies, Inc.,
Gaithersburg, Md.) with total human skeletal muscle as a template was
used to complete the 5' end of the SNX2 cDNA (Clontech). When
necessary, standard molecular biology techniques were used to construct
full-length cDNAs. All sequencing was performed with the ABI PRISM dye
terminator cycle sequencing kit and an ABI automated sequencer (model
373A; Perkin-Elmer Corp., Applied Biosystems Division, Foster City,
Calif.).
Construction of epitope-tagged sorting nexins and receptor
expression vectors.
Epitope tags were introduced at the 5' end of
each sorting nexin molecule and associated mutant proteins by PCR with
the Expand high-fidelity PCR kit (Boehringer Mannheim Corp.). SNX1,
SNX1A, SNX2, SNX3, and SNX4 were myc tagged, and SNX2 was also HA
tagged. Sequences of the primers used are available upon request. The epitope-tagged molecules were then cloned into either pCDNA(3.1±) (Invitrogen Corp., Carlsbad, Calif.) or pCMV5myc-1 expression vectors
(2) by standard techniques. Full-length human insulin receptor (33), PDGF receptor (40), and EGF
receptor (28) were cloned into pCDNA(3.1±) (Invitrogen
Corp.) or pCIS vectors as previously described (33, 34).
Full-length human leptin receptor short and long isoforms (11, 22,
37) were cloned into pCIneo vector (Promega Corp., Madison, Wis.)
by standard techniques.
Tissue distribution of sorting nexins.
Multiple-tissue
Northern blots containing approximately 2 µg of purified
poly(A)+ RNA from human tissues and a multiple tissue dot
blot were obtained from Clontech. The dot blot RNAs were normalized to
the mRNA levels of eight different housekeeping genes, thus allowing
the relative levels of the various sorting nexin mRNAs to be determined
by densitometry of autoradiographs. To obtain probes for the Northern blot analyses, we digested SNX1, -2, and -3 cDNAs with EcoRI
and NotI, while SNX4 cDNA was digested with NcoI
and XhoI. The resulting 1,000- to 2,000-nucleotide fragments
were gel purified, 32P-labeled with a randomly primed DNA
labeling kit (Boehringer Mannheim Corp.), diluted in hybridization
buffer (50% [vol/vol] formamide, 8× Denhardt's solution, 5× SSPE
[1× SSPE is 0.18 M NaCl, 10 mM NaH2 PO4, and
1 mM EDTA; pH 7.7], 0.5% (wt/vol) sodium dodecyl sulfate [SDS],
1-mg/ml salmon sperm DNA), and hybridized for 18 to 24 h at
42°C. The blots were then washed according to the manufacturer's instructions.
Transient transfection of sorting nexins and various
receptors.
Totals of 2 × 105 to 3 × 105 COS7 cells were plated into each well of a six-well
dish at 16 to 20 h before the start of the transfection. The cells
were washed twice with serum-free DMEM; DNA (2 µg of total DNA per
well) was transfected into the cells with Lipofectamine (6 µl per
well) according to the manufacturer (Life Technologies, Inc.). The
cells were incubated for 5 to 6 h with the DNA-Lipofectamine
complexes in serum-free DMEM and then incubated overnight in 10%
(vol/vol) fetal bovine serum. The cells were harvested 28 to 30 h
after the start of the transfection.
Coimmunoprecipitation experiments.
Transfected COS7 cells
were washed in ice-cold phosphate-buffered saline (PBS) and scraped
into 250 µl of lysis buffer (50 mM Tris-HCl [pH 7.5], 0.5%
[vol/vol], Triton X-100, 0.3 M NaCl plus protease inhibitor tablet
[Boehringer Mannheim Corp.]). The cells were then solubilized for 30 min on ice, and insoluble debris was removed by centrifugation at
14,000 × g for 20 min. The proteins were then detected
by immunoblotting with anti-HA, anti-myc, anti-insulin receptor,
anti-PDGF receptor, and anti-EGF receptor antibodies or by
immunoprecipitation of extract (400 µl) with receptor-specific antibodies (1/100 dilution; see Antibody section above), followed by
immunoblotting with anti-HA or anti-myc antibodies (24, 35).
Subcellular localization of sorting nexins.
NIH 3T3 cells
overexpressing human EGF receptors (17) were transiently
transfected with the various epitope-tagged sorting nexin molecules as
described above. At 24 to 26 h posttransfection, the cells
(~5 × 106 to 6 × 106 per well)
were placed on ice and washed twice with ice-cold PBS. Subsequently,
the cells were scraped and lysed in 250 µl of ice-cold homogenization
buffer (10 mM HEPES [pH 7], 0.25 M sucrose, 0.5 mM MgCl2,
and protease inhibitor tablet [Boehringer Mannheim Corp.]) by 15 passages through a 25-gauge syringe. To obtain total membrane and
cytosolic fractions, the lysates were centrifuged at 240,000 × g for 45 min at 4°C in a Beckman TLA 120.2 rotor (Beckman
Instruments, Palo Alto, Calif.). Following the centrifugation step, the
membrane pellet was resuspended in 250 µl of homogenization buffer
with eight strokes with a Teflon pestle and a Potter homogenizer.
Aliquots of the total lysates and cytosolic and membrane fractions were then solubilized in Laemmli buffer and run on 7.5% (vol/vol) SDS gels
and EGF receptors, and the various epitope-tagged sorting nexins were
detected by Western blotting (35).
Nucleotide sequence accession numbers.
The predicted amino
sequences for human SNX1, SNX1A, SNX2, SNX3, and SNX4 cDNAs have been
deposited in the National Center for Biotechnology Information database
under accession no. 3152940, 3152942, 3152938, 3127053, and AF065485, respectively.
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RESULTS |
Identification of human SNX1 homologs.
In an effort to
identify homologs of human SNX1, we searched the dbEST database of the
National Center for Biotechnology Information with the entire amino
acid sequence of human SNX1 or its PX domain (amino acids 161 to 272)
(21, 32). This led to identification of four groups of human
EST sequences which, when translated, showed a high degree of homology
to SNX1 as judged by the BLAST algorithm (1). For each
group, we obtained several EST clones, determined their nucleotide
sequences, and constructed full-length cDNAs (Fig.
1).
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FIG. 1.
Construction and characterization of SNX1, SNX1A, SNX2,
SNX3, and SNX4 cDNAs. Five human sorting nexins (SNX1, SNX1A, SNX2,
SNX3, and SNX4) are depicted. For each full-length cDNA, the nucleotide
numbering is shown. Listed below each molecule are the fragments (ESTs,
PCR, or 5'-RACE products) used to determine the nucleotide sequences of
the cDNAs and to construct full-length cDNAs as detailed in Materials
and Methods. One or both strands of each cDNA fragment were sequenced
as indicated by the directions of the arrowheads. In every case, both
strands of DNA were sequenced from at least one clone.
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Group 1 consisted of ESTs that encode all or part of SNX1 and included
a putative splice variant isoform of SNX1 that we have
designated
SNX1A. The deduced amino acid sequence of our SNX1
cDNA clone showed
complete agreement with the published sequence
of SNX1, except for
three amino acid differences (Fig.
2).
There
was one major difference between the translated sequence of SNX1A
and SNX1, i.e., a deletion of 195 nucleotides that resulted in
an
in-frame deletion of 65 amino acids (Fig.
2, underlined region).
Thus,
SNX1A cDNA is predicted to encode a 457-amino-acid hydrophilic
protein
with a pI of 5.3. PCR was used to confirm the presence
of both SNX1 and
SNX1A mRNAs in multiple tissues (data not shown).
In addition, Northern
blot analyses showed that human SNX1/1A
mRNAs are ubiquitously
expressed, with the highest levels in the
medulla oblongata, prostate,
spinal cord, spleen, pancreas, and
pituitary gland (Fig.
3B).
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FIG. 2.
Predicted amino acid sequences of SNX1, SNX1A, SNX2,
SNX3, and SNX4. The predicted amino acid sequences for human SNX1
(hSNX1), SNX1A, SNX2, SNX3, and SNX4 cDNAs are shown. The SNX1 amino
acid sequence was identical to the published SNX1 sequence
(21), except for the three residues indicated by boldface
and double underlining. We found serine at positions 115 and 117. At
position 211, we found proline (codon CCC) in one clone and serine
(TCC) in two other clones, suggesting that there is a polymorphic amino
acid. The entire coding sequence of SNX1A was shown to be identical to
that of SNX1, except for an in-frame deletion of 195 nucleotides. The
65 amino acid residues (91 to 115) deleted from SNX1A are underlined.
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FIG. 3.
Tissue distributions of SNX1, SNX2, SNX3, and SNX4
mRNAs. Filters containing poly(A)+ mRNA from the indicated
human tissues were hybridized with radiolabeled human SNX2 or SNX3
probes (A). A multiple-tissue dot blot was hybridized with radiolabeled
human SNX1 or SNX4 probes (B) as described in Materials and Methods.
Autoradiographs of the dot blots were scanned with a laser
densitometer, and the individual spots were quantified with ImageQuant
software from Molecular Dynamics. The numbers on the x axis
denote arbitrary units (AU).
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The second group of ESTs encodes pieces of a SNX1 homolog that we have
designated human SNX2. This novel cDNA is predicted
to encode a protein
of 519 amino acids with a pI of 4.8 (Fig.
2). SNX1 and SNX2 are 63%
identical and 73% similar over much
of their lengths (Fig.
4A). As shown by Northern blot analyses,
SNX2 mRNA is ubiquitously expressed, with transcripts of approximately
3.1 and 2.4 kb detected in all human tissues examined. However,
the
ratio of the two SNX2 mRNAs and their absolute amounts varied
among the
tissues studied. The highest levels of SNX2 mRNA were
found in spleen,
heart, skeletal muscle, and peripheral leukocytes,
while very little
SNX2 mRNA was detected in the kidney, liver,
or brain (Fig.
3A).
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FIG. 4.
Sequence identity between human sorting nexins and
related molecules from other species. (A) Schematic illustrations of
the domain architecture of human (h) SNX1, SNX1A, SNX2, SNX3, and SNX4
and related proteins from C. elegans, S. cerevisiae, and S. pombe. The protein labeled CelSNX1
is an ORF found in the nematode sequencing project database (GenBank
accession no. Z69384). The protein labeled SceSNX4 is an S. cerevisiae ORF (accession no. S56808), and the protein labeled
SpoSNX4 is an S. pombe ORF (accession no. 2389002). SceSNX3
(accession no. AF016101) is the newly described S. cerevisiae protein known as Grd19p (41). Vps5p and
Mvp1p are two additional S. cerevisiae molecules involved in
sorting to the yeast vacuole (15, 18, 30). The cross-hatched
boxes represent conserved N-terminal domains of variable lengths. The
black boxes represent a highly conserved domain of ~100 amino acids
which was termed the PX domain (32). The white boxes
represent a conserved domain C terminal to the PX domain. The thin
lines are regions with limited homology. The percentages of amino acid
identity between each human sorting nexin and related proteins from
other species as determined by pairwise alignment with the ALIGN
program (13) are shown below the various domains. The
percentages of amino acid identity between S. cerevisiae and
S. pombe SNX4 are indicated by asterisks. The
carboxyl-terminal regions of the SNX1-like group and the SNX4 group may
be distantly related, but this is not illustrated. (B) Multiple
sequence alignment of the PX domains from each human sorting nexin and
related molecules from other species. The sequences were aligned with
CLUSTAL W (38), followed by manual refinement. Amino acid
residues conserved in at least 6 of 10 proteins of the sorting
nexin-like aligned sequences (top group) or other PX-containing
proteins (bottom group) are highlighted in black. The amino acids that
were predicted to serve as binding sites for SH3 and/or WW
domain-containing proteins are indicated by asterisks (9,
31). Cons, consensus.
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The third and fourth groups of ESTs were identified via their high
degree of sequence homology with a portion of SNX1 called
the PX domain
(amino acids 161 to 272 [Fig.
4B]). The novel cDNA
that we have
designated human SNX4 is predicted to encode a protein
of 450 amino
acids with a pI of 5.6 (Fig.
2). SNX4 mRNA is also
widely expressed,
with its highest levels in the pituitary gland,
testes, and liver (Fig.
3B). In contrast, the protein that we
have designated SNX3 is a
molecule of only 162 amino acids and
a pI of 9.2 (Fig.
2). A single
transcript of ~1.9 kb was detected
in all human tissues hybridized
with an SNX3 probe. The tissue
distribution of SNX3 closely parallels
that seen for SNX2, with
the highest levels of mRNA detected in the
peripheral leukocytes,
spleen, heart, and skeletal muscle, and greatly
reduced levels
in the kidney (Fig.
3A).
Sorting nexin homologs in yeasts and Caenorhabditis
elegans.
To examine the evolutionary conservation of the sorting
nexins, we searched the complete genome of the budding yeast S. cerevisiae and the known open reading frames (ORFs) from C. elegans and Schizosaccharomyces pombe for sequences
similar to those for SNX1, SNX2, SNX3, and SNX4. The best match to
human SNX3 was the recently described S. cerevisiae protein,
Grd19p (ORF YOR357c) (41). Likewise, a search of Grd19p
against libraries of human ESTs showed no closer homolog than SNX3.
Thus, the two appear to be orthologs, and we designate YOR375c as
SceSNX3. Figure 4A shows the percentage of amino acid identity between
SNX3 and SceSNX3 as determined with the ALIGN program
(13). Similar results suggest that human SNX4 and yeast
YJL036w (GenBank accession no. Z49311) may be orthologs; we designate
YJL036w as SceSNX4. Likewise, the S. pombe hypothetical protein that we designate SpoSNX4 (GenBank accession no. 239002) probably represents an SNX4 ortholog, although SpoSNX4 has a shorter C-terminal domain than either the human or S. cerevisiae
SNX4 molecule (Fig. 4A). Finally, the two closely related human
proteins, SNX1 and SNX2, showed a high degree of sequence homology
along most of their lengths with the recently described S. cerevisiae protein, Vps5p (18, 30), and the
hypothetical C. elegans protein that we have designated
CelSNX1 (GenBank accession no. 1439669). The best matches from
searching yeast Vps5p against all human ESTs were the SNX1 and SNX2
groups, again suggesting orthology.
A common feature of the human sorting nexins is the presence of an
~100-amino-acid region that was termed the PX domain. This
domain was
first identified as a conserved sequence in the 40-
and 47-kDa subunits
of NADPH oxidase and has subsequently been
identified in a variety of
proteins with varied functions (
14,
26,
32). Figure
4B shows
a multiple sequence alignment of
the PX domains from the sorting nexins
and their predicted orthologs,
as well as other PX domain-containing
proteins. The alignment
was created with Clustal W (
38) and
was edited by hand. A tree
constructed from the PX domain alignment by
using the unweighted
pair group method with arithmetic mean
(
29) grouped all of the
proteins that we have designated
human sorting nexins or their
orthologs into a single branch of the
tree along with one additional
protein, Mvp1p (data not shown).
Inspection of the PX domain alignment
shows general features of the PX
domain but also some possible
signatures for the sorting nexin-like
subset. The strongest motif
in the alignment, [VIL] X R R [FY] S
[DE] F, is matched well in
most of the members of the alignment, and
not just in the human
sorting nexins and their orthologs (Fig.
4B). In
contrast, the
PX domains of all candidate sorting nexins (including
Mvp1p),
but few other proteins, matched, with one mismatch or fewer, to
the motif [VIM] [VI] P P [LP] P X K. Toward the C terminus of
the
PX domain, the dipeptide motif HP is found in the human sorting
nexins
and Mvp1p, while DP appears in Vps5p, its binding partner
Vps17p
(
18,
20), and a homolog of Vps5p that was designated
YKR078w. No other proteins in the alignment matched the HP or
DP
sequence at that
position.
Sorting nexins exist in both cytosolic and membrane-bound
pools.
To study the functions of the sorting nexin proteins, each
full-length cDNA containing an epitope tag at its 5' end was cloned into a mammalian expression vector and transiently transfected into
either COS7 or NIH 3T3 cells. Western blotting of cell extracts with
anti-tag antibodies showed that each sorting nexin was expressed. However, the apparent molecular mass of each sorting nexin protein differed from its calculated mass by ~8,000 to 12,000 kilodaltons. We
then inquired whether the sorting nexins were located in the cytosol or
whether they were associated with cellular membranes. For these
studies, NIH 3T3 cells were transiently transfected with the various
sorting nexin cDNAs. After homogenization of the cells in
sucrose-containing buffer, we carried out ultracentrifugation to
generate cytosolic and membrane fractions. As judged by Western blotting of the cell fractions, all of the sorting nexins partitioned into both membrane and cytosolic fractions (Fig.
5). SNX1, SNX1A, SNX2, and SNX4 were
associated predominantly with membranes, whereas SNX3 was found mainly
in the cytosol. Similar results were found with endogenous mouse SNX1
and SNX2 when the appropriate SNX-specific polyclonal antibodies were
used (data not shown). In addition, the membrane-associated forms of
the various sorting nexins could be partially extracted with 1 M NaCl,
suggesting that they behave as peripheral membrane proteins (data not
shown).
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FIG. 5.
Analysis of the association of sorting nexins with
membranes. NIH 3T3 cells overexpressing EGF receptors were transiently
transfected with epitope-tagged sorting nexin cDNAs. After 26 to
30 h, total-cell extracts (T), and total membrane (M) and (C)
cytosolic fractions were prepared as described in Materials and
Methods. The distributions of the EGF receptors and the epitope-tagged
sorting nexins were determined by Western blotting with an anti-EGF
receptor antibody and anti-epitope antibodies, respectively.
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Oligomerization of the sorting nexin molecules.
Given that the
yeast protein Vps5p, which is a member of the SNX1/2 subgroup of
sorting nexins, forms a heteromeric complex with another PX
domain-containing protein, Vps17p (20), we examined whether
SNX2 was able to associate with any of the other human sorting nexins.
COS7 cells were transfected with HA-tagged SNX2 alone or in combination
with myc-tagged versions of the other sorting nexins. SNX2 forms
heteromeric complexes with SNX1, SNX1A, and SNX4 but not with SNX3
(Fig. 6, lanes 1, 2, and 5). Likewise, SNX2 was able to oligomerize with itself (lane 3). SNX2 did not associate with SNX3, although SNX3 was readily expressed (lanes 4 and
6).
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FIG. 6.
Oligomerization of the sorting nexin molecules. COS7
cells were transiently transfected with expression vectors encoding
HA-tagged SNX2 in combination with myc-tagged versions of SNX1, SNX1A,
SNX2, SNX3, and SNX4. Total-cell extracts were immunoprecipitated
(ippt) with an anti-myc antibody and electrophoresed, and the resulting
blots were probed with either anti-myc (lane 6) or anti-HA (lanes 1 to
5) antibodies to detect the various immunoprecipitated epitope-tagged
sorting nexin molecules.
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Association with receptor tyrosine kinases.
To test for the
ability of the various sorting nexins to associate with growth factor
receptors, COS7 cells were transiently transfected with expression
plasmids encoding epitope-tagged sorting nexin cDNAs (Fig.
7, lanes 1 to 10). In addition, some
cells were also cotransfected with an expression plasmid for the
insulin receptor (Fig. 7A). Each epitope-tagged sorting nexin molecule was readily detected in cell extracts made from the transfected cells
(Fig. 7A, middle panel). Likewise, overexpression of the insulin
receptor was also detected in the appropriate extracts (Fig. 7A, lower
panel). After incubation of cell extracts with an insulin receptor
-subunit antibody followed by Western blotting of the precipitates
with antibody to the epitope tag, we found that SNX1, SNX1A, and SNX2
and small amounts of SNX4 coimmunoprecipitated with the insulin
receptor (Fig. 7A, upper panel). Kinase-inactive insulin receptors
coimmunoprecipitated amounts of SNX1, SNX2, and SNX4 similar to that
immunoprecipitated with the wild-type receptor (data not shown). We did
not detect association between SNX3 and overexpressed insulin receptors
(lane 8). Neither did we detect association between the transfected
sorting nexin molecules and the small amount of endogenous COS7 insulin
receptors (lanes 1, 3, 5, 7, and 9). However, we consistently observed
that overexpression of insulin receptor decreased the level of
expression of epitope-tagged SNX3 (Fig. 7A, lanes 7 and 8). When these
experiments were performed with extracts from cells overexpressing EGF
receptors and the various sorting nexins (Fig. 7B), we found that SNX1
and SNX1A were readily detected in EGF receptor immunoprecipitates.
SNX2 and SNX4 were detected, but to a lesser degree, and SNX3 was
undetectable (Fig. 7B, upper panel). Likewise, SNX1, SNX1A, and SNX2
were readily coimmunoprecipitated with PDGF receptors (Fig. 7C, lanes
2, 4, and 6), while SNX4 was coimmunoprecipitated to a lesser extent (lane 10). As with the insulin and EGF receptors, no association was
detectable between the PDGF receptor and SNX3 (Fig. 7C, lane 8).
Furthermore, previous work suggested that the last 66 amino acids of
SNX1 are required for this protein, when overexpressed, to accelerate
the turnover of EGF receptors (21). However, we found that
deletion of 66 C-terminal amino acids of SNX1 did not appreciably
disrupt the association of the truncated SNX1 molecule with the
receptor tyrosine kinases for EGF and insulin (data not shown).
Nevertheless, because these C-terminal 66 amino acids bound the EGF
receptor in the yeast two-hybrid system, our findings suggest that the
receptor tyrosine kinases for EGF and insulin have multiple binding
sites for SNX1. In addition, we found that stimulation of cells with
either insulin or EGF for 5 to 60 min did not change the amounts of the
various sorting nexins associated with the respective receptor tyrosine
kinases (data not shown).
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FIG. 7.
Differential association of SNX1, SNX1A, SNX2, SNX3, and
SNX4 with the insulin receptor (IR), EGF receptor, and PDGF receptor.
COS7 cells were transiently transfected with expression vectors
encoding epitope-tagged SNX1 (lanes 1 and 2), SNX1A (lanes 3 and 4),
SNX2 (lanes 5 and 6), SNX3 (lanes 7 and 8), or SNX4 (lanes 9 and 10).
Some cells were also transfected with expression vector encoding the
human insulin receptor (A), EGF receptor (B), and PDGF receptor (C)
(lanes 2, 4, 6, 8, and 10). Total-cell extracts were immunoprecipitated
(ippt) with the appropriate anti-receptor antibody and electrophoresed,
and resulting blots were probed with either anti-myc or anti-HA
antibodies to detect coimmunoprecipitated epitope-tagged sorting nexin
molecules (upper panel). To check for expression of the various
receptors and each of the sorting nexin molecules, each sample was also
immunoblotted with an anti-tag antibody (middle panel) or an
anti-receptor antibody (lower panel). Shown above each lane is the
approximate molecular weight corresponding to the electrophoretic
mobility of each sorting nexin protein. This experiment was repeated
three times with similar results.
|
|
Association with endogenous transferrin receptors.
Having
demonstrated the association of several sorting nexins with receptor
tyrosine kinases, we inquired whether the various sorting nexins also
associated with receptors that constitutively internalize and recycle.
To test this, COS7 cells were transiently transfected with expression
plasmids encoding epitope-tagged sorting nexin cDNAs. Western blotting
of cell extracts made from the transfected cells confirmed that each
epitope-tagged sorting nexin molecule was readily expressed (data not
shown). After incubation of cell extracts with a transferrin receptor
antibody followed by Western blotting of the precipitates with an
anti-tag antibody, we found that only SNX1 and SNX1A
coimmunoprecipitated with endogenous transferrin receptors (Fig.
8, lanes 2 and 3). We did not detect association between endogenous transferrin receptors and SNX2, SNX3, or
SNX4 (lanes 4 to 6).
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FIG. 8.
Differential association of SNX1, SNX1A, SNX2, SNX3, and
SNX4 with endogenous transferrin receptors. COS7 cells were transiently
transfected with expression vectors encoding epitope-tagged SNX1,
SNX1A, SNX2, SNX3, or SNX4. Total-cell extracts were immunoprecipitated
(ippt) with an anti-transferrin receptor antibody and electrophoresed,
and the resulting blots were probed with either anti-myc or anti-HA
antibodies to detect coimmunoprecipitated epitope-tagged sorting nexin
molecules. This experiment was repeated three times with similar
results.
|
|
Association with leptin receptor isoforms.
Then, we asked if
the various sorting nexins associated with a third type of cellular
receptor, cytokine receptors. COS7 cells were transiently transfected
with expression plasmids encoding epitope-tagged sorting nexin cDNAs.
In addition, some cells were also cotransfected with expression
plasmids encoding the two most abundant isoforms of the human leptin
receptor (22, 37). These isoforms share a large ligand
binding domain and transmembrane domain and 29 cytoplasmic domain amino
acids. Following this conserved region, the short form of the human
leptin receptor (hLR34) contains 5 unique carboxyl-terminal amino
acids, and the long form of the human leptin receptor (hLR303) contains
274 unique carboxyl-terminal amino acids. Western blotting of cell
extracts made from transfected cells confirmed that each epitope-tagged
sorting nexin molecule was readily expressed. Likewise, we found that
each leptin receptor isoform was similarly expressed, as shown by
125I-leptin binding to solubilized cell extracts (data not
shown). After incubation of cell extracts with a leptin receptor
antibody directed against the extracellular domain of the receptor
isoforms, followed by Western blotting of the precipitates with an
antitag antibody, we found that SNX1, SNX1A, SNX2, and SNX4
coimmunoprecipitated with the long form of the leptin receptor (Fig.
9, lanes 2, 3, 4, and 6). We did not
detect association between SNX3 and hLR303 (lane 5). In contrast, the
short form of the receptor showed little if any association with the
sorting nexin molecules (Fig. 9, lanes 8 to 12). Two other isoforms of
the human leptin receptor that possess 15 and 67 unique amino acids
following the carboxyl-terminal splice site (4, 12) also
showed little if any association with the sorting nexins (data not
shown).
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FIG. 9.
Differential association of SNX1, SNX1A, SNX2, SNX3, and
SNX4 with the short and long isoforms of the human leptin receptor.
COS7 cells were transiently transfected with expression vectors
encoding epitope-tagged SNX1, SNX1A, SNX2, SNX3, or SNX4. The cells
were also transfected with an expression vector encoding either the
long form (hLR303) or the short form (hLR34) of the human leptin
receptor. Total-cell extracts were immunoprecipitated (ippt) with an
anti-leptin receptor antibody made against the common extracellular
domain of the receptor isoforms. The samples were then electrophoresed,
and the resulting blots were probed with either anti-myc or anti-HA
antibodies to detect coimmunoprecipitated epitope-tagged sorting nexin
molecules. This experiment was repeated three times with similar
results.
|
|
| |
DISCUSSION |
In this report, we have identified three novel human sorting nexin
molecules (SNX2, SNX3, and SNX4) and SNX1A, a presumed splice variant
isoform of SNX1. These mammalian proteins appear to be closely related
to proteins in yeasts and C. elegans, indicating that they
are part of an evolutionarily important family. The sorting nexins are
all hydrophilic proteins that lack obvious hydrophobic regions capable
of serving as membrane anchors, and yet they are found partially
associated with cellular membranes. They also contain a conserved
domain of ~100 amino acids termed the PX domain that may be involved
in protein-protein interactions. The sorting nexins also associate with
each other and with a variety of other cellular proteins, suggesting
that they exist as part of multisubunit complexes. Based on the
functions of their yeast homologs, it is likely that the mammalian
sorting nexins described here serve similar functions related to
intracellular trafficking of proteins to various organelles.
Sorting nexin subgroups.
Human SNX1, SNX1A, and SNX2 are part
of a subgroup of sorting nexins that include a related protein from
C. elegans and the recently described S. cerevisiae protein, Vps5p (18, 30). These proteins
share a high degree of amino acid similarity over their lengths. These
findings suggest that this subclass of sorting nexin proteins may have
evolved from a common ancestral precursor and thus may perform related
functions in humans, yeasts, and invertebrates. Human SNX3, SNX4, and
their respective orthologs make up two additional subgroups of sorting
nexins. The SNX4 subgroup appears to be more closely related to the
SNX1/2 subgroup than the SNX3 subgroup. The SNX4 subgroup contains
amino acids in the PX domains and in the region just C terminal to the
PX domains that are also conserved in the SNX1/2 subgroup. In contrast,
the SNX3 subgroup consists of shorter proteins and has no sequence similarity to the other two groups outside the PX domain. It is interesting to note that yeast has at least five sorting nexin-like proteins. Additional searches of the EST databases suggest that several
additional human sorting nexin proteins also exist.
Functions of yeast sorting nexin-like proteins.
Vps5p, which
is a member of the SNX1/2 subgroup of sorting nexins, is a highly
charged phosphoprotein that forms a heteromeric complex with another PX
domain-containing protein, Vps17p (20). Vps5p is
peripherally associated with a dense membrane fraction distinct from
Golgi, endosomal, and vacuolar membranes. Cells that lack either Vps5p
or Vps17p possess vacuoles of altered morphology and exhibit defects in
the sorting of the vacuolar hydrolase, carboxypeptidase Y, due to
mislocalization of the carboxypeptidase Y sorting receptor, Vps10p.
They also missort late-Golgi membrane proteins to the vacuole. It is
thought that Vps5p and Vps17p and perhaps other proteins may form a
complex on transport vesicles that are responsible for recycling
receptors such as Vps10p from endosomes back to the Golgi protein,
where they then mediate another round of delivery of newly synthesized
vacuolar hydrolases (18, 30). Interestingly, SNX2 is also
able to form homomeric complexes as well as heteromeric complexes with
SNX1, SNX1A, and SNX4. In addition, the sorting nexins are able to
associate with a number of different cell surface receptors (see
below). The regions of the sorting nexin molecules required for
oligomerization, the specificity and degree of oligomerization, and the
functional role of the oligomerization process will require further investigation.
Mvp1p is another
S. cerevisiae protein that contains a
sorting nexin-like PX domain. Its PX domain is more similar to the
PX
domains found in the sorting nexins described in this study
than to
other PX domain-containing proteins. Mvp1p is known to
interact with
Vps1p, a high-molecular-weight GTPase that associates
with Golgi
membranes. Vps1p is part of a family of proteins that
includes the
mammalian protein dynamin, which is involved in the
early stages of
clathrin-mediated endocytosis (
15). Thus, in
yeast cells,
correct sorting of acid hydrolases to the vacuole
appears to require
several multiprotein complexes. One contains
at least a sorting nexin
(Vps5p), another PX domain-containing
protein (Vps17p), and a sorting
receptor (Vps10p), while the second
contains a sorting nexin-like
protein (Mvp1p) and a dynamin-like
GTPase
(Vps1p).
Grd19p is a third
S. cerevisiae sorting nexin-like protein
that appears to be the yeast ortholog of SNX3. Grd19p has recently
been
shown to interact with the cytoplasmic domain of dipeptidyl
aminopeptidase A, a resident late-Golgi enzyme. Unlike the other
yeast
sorting nexin proteins described to date, Grd19p is not
required for
vacuolar protein sorting or the recycling of the
carboxypeptidase Y
sorting receptor Vps10p. Instead, Grd19p is
required to maintain
resident proteins such as Kex2p and dipeptidyl
aminopeptidase A in the
late-Golgi membrane by retrieving escaped
molecules from the
prevacuolar compartment (
41). The molecular
mechanisms used
by these yeast sorting nexin-like molecules to
direct proteins to
specific locations and to maintain them there
are presently unknown.
Further studies will be required to determine
if Grd19p and/or SNX3 is
also part of a multiprotein complex involved
in trafficking to and from
the late-Golgi
membrane.
Association of sorting nexins with receptors.
SNX1 was cloned
by using a two-hybrid screen with a portion of the cytoplasmic domain
of the EGF receptor as bait (21). In the present study, we
inquired whether SNX1 and/or other sorting nexins could bind to EGF
receptors, as well as to other cell surface receptors. In contrast to
the work reported by Kurten et al. (21), our studies
performed with mammalian cells showed that SNX1 binding is not specific
for the EGF receptor. Rather, SNX1 bound to receptors for PDGF, EGF,
and insulin. SNX2 and SNX4 also associated with these three classes of
receptor tyrosine kinases. Interestingly, the SNX-receptor interactions
were not affected by activation of the receptors by ligand. Both active
and inactive receptors associated similarly with the receptor tyrosine
kinases. The various sorting nexins also showed differential
association with several leptin receptor isoforms. SNX1, SNX1A, SNX2,
and SNX4 associated with the long isoform of the leptin receptor but
not with the short and medium isoforms. Genetic evidence suggests that
the long isoform of the leptin receptor mediates most of the
biologically important signalling induced by leptin binding. However,
recent studies have suggested that the short form is also activated
following ligand addition (4, 5). Given that the short and
medium isoforms lack a JAK box found in the long isoform, it is
possible that the sorting nexins associate with the leptin receptor
long form indirectly, possibly through JAKs. Lastly, the transferrin receptor, which internalizes and recycles constitutively, also shows
interactions with a sorting nexin. However, association of transferrin
receptors is restricted to SNX1 and SNX1A. The specific binding of
transferrin receptors to SNX1, but not SNX2 and SNX4, may be important
for determining the fate of the receptor (i.e., recycled versus
degraded) (39).
There are several potential reasons why we did not detect association
of SNX3 with the receptors studied. First, SNX3 may
bind weakly or
transiently to the receptors studied, and, thus,
the amount of complex
formed may be below the level of detection
of our coimmunoprecipitation
assay. Alternatively, SNX3 may bind
to the receptors but be rapidly
degraded after binding. In support
of this idea, we find that
overexpression of PDGF, EGF, or insulin
receptors consistently
diminished the SNX3 overexpression in cell
extracts, whereas the
amounts of SNX1, SNX2, and SNX4 were unchanged.
Thus, it is possible
that SNX3 might associate with the receptors
studied here but then be
rapidly degraded. Finally, SNX3 may bind
to molecules other than the
plasma membrane receptors studied
here. In support of this idea, Kurten
et al. (
21) showed that
SNX1 contained an EGF receptor
binding site in its C-terminal
66 amino acids. SNX3 possesses only a PX
domain and 45 amino acids
N terminal to the PX domain. It does not
contain a large C-terminal
domain like SNX1, SNX2, and SNX4 that may be
important for receptor
recognition. In addition, SNX3 appears to be the
human ortholog
of Grd19p. Thus, it is likely that SNX3 may associate
with late-Golgi
resident proteins rather than cell surface
receptors.
Function of sorting nexins in mammalian cells.
At present, the
functions of SNX2, SNX3, and SNX4 in mammalian cells are not known.
Kurten et al. (21) reported that overexpression of SNX1
resulted in accelerated degradation of EGF receptors. However, we have
been unable to demonstrate that overexpression of SNX1 or any of the
other sorting nexins increased the turnover of insulin or EGF receptors
(unpublished observations). One possible reason for the discrepancy may
be that the sorting nexins appear to exist as subunits of a
multiprotein complex. Therefore, it may be difficult to see accelerated
degradation of receptors in our system because we did not overexpress
all of the components of the complex. Nevertheless, the conclusions of
Kurten et al. (21) are entirely consistent with the role
that Vps5p, the yeast protein most closely related to SNX1 and SNX2, is
thought to play in vacuolar sorting. Further studies are necessary to
elucidate the functions of mammalian sorting nexins.
Conclusion.
The sorting nexins described here, as well as
related proteins from yeast, appear to be a large family of proteins
involved in membrane trafficking. Further studies will be required to
identify the proteins with which the human sorting nexins associate, to determine how sorting nexins associate with cellular membranes and with
each other, and to elucidate the role that these proteins play in
receptor trafficking. Possible roles include involvement in the
recycling of receptors from endosomes back to the plasma membrane,
targeting of receptors from endosomes to lysosomes for degradation,
sorting of receptors from the trans-Golgi network to the plasma
membrane, and retrieval of late-Golgi proteins from endocytic compartments.
| |
ACKNOWLEDGMENTS |
We are grateful to Axel Ullrich for the generous gift of the
insulin receptor cDNA. In addition, we thank Marc Reitman for helpful
discussions and for critical reading of the manuscript. Finally, we
thank Rachel Kulansky, Neinke Grossman, and Jill Sherman for
contributions to this project.
| |
ADDENDUM IN PROOF |
While this paper was under review, Seaman et al. reported that
Vps5p, the yeast ortholog of SNX1 and SNX2, is part of a multiprotein membrane-associated complex. This may serve as a novel coat involved in
vesicle transport from endosomes back to the late-Golgi membrane (M. N. J. Seaman, J. M. McCaffery, and S. D. Emi, J. Cell Biol. 142:665-681, 1998).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: National
Institutes of Health, Building 10, Room 8S-235A, 10 Center Dr.
MSC-1770, Bethesda, MD 20892-1770. Phone: (301) 402-1629. Fax: (301)
402-0573. E-mail: carol_haft{at}nih.gov.
| |
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Molecular and Cellular Biology, December 1998, p. 7278-7287, Vol. 18, No. 12
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
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