From the Department of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark
Received for publication, October 23, 2002, and in revised form, December 12, 2002
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ABSTRACT |
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We previously isolated and sequenced murine
sorCS1, a type 1 receptor containing a Vps10p-domain and a leucine-rich
domain. We now show that human sorCS1 has three isoforms, sorCS1a-c,
with completely different cytoplasmic tails and differential expression in tissues. The b tail shows high identity with that of murine sorCS1b,
whereas the a and c tails have no reported counterparts. Like the
Vps10p-domain receptor family members sortilin and sorLA, sorCS1 is
synthesized as a proreceptor that is converted in late Golgi
compartments by furin-mediated cleavage. Mature sorCS1 bound its own
propeptide with low affinity but none of the ligands previously shown
to interact with sortilin and sorLA. In transfected cells, about 10%
of sorCS1a was expressed on the cell surface and proved capable of
rapid endocytosis in complex with specific antibody, whereas sorCS1b
presented a high cell surface expression but essentially no
endocytosis, and sorCS1c was intermediate. This is an unusual example of an alternatively spliced single transmembrane receptor with
completely different cytoplasmic domains that mediate different trafficking in cells.
SorCS1 is the first identified member of a subgroup of the
mammalian Vps10p-domain
(Vps10p-D)1 receptor family
that comprises an N-terminal Vps10p-D (named after the yeast vacuolar
protein sorting 10 protein), a leucine-rich domain, a single
transmembrane domain, and a short cytoplasmic domain (cd) (1). Two
isoforms of sorCS1, with different cds arising from differential
splicing, have been identified in the mouse (2). The two other known
members of the subgroup are sorCS2 (3, 4) and a highly homologous
receptor tentatively designated sorCS3 (5). The mammalian Vps10p-D
receptors also comprise the previously characterized sortilin, whose
lumenal part consists of a Vps10p-D only (6), and the mosaic receptor sorLA/LR11, which, in addition to an N-terminal Vps10p-D, contains elements also found in the low density lipoprotein
receptor family as well as a cluster of fibronection type III repeats
(7, 8). The N termini of all the Vps10p-D family receptors contain
sequences that conform to the consensus sequence for cleavage by furin
(RX(R/K)R), and recent results (9, 10) have shown
that furin cleaves the precursor forms of sortilin and sorLA and that
removal of the propeptides conditions these receptors for binding of ligands.
Sortilin and sorLA are mainly found in the trans-Golgi network, and
only a few percent of the receptors are on the cell surface (10, 11).
The lumenal domains of the fully processed receptors bind
certain neuropeptides (e.g. neurotensin) (10, 12),
the endoplasmic reticulum-resident receptor associated protein (RAP) (9, 10), as well as lipoprotein lipase (10, 11), apolipoprotein E (8,
10), and notably their own propeptides, which block binding of all
known ligands to the Vps10p-D (9, 10). Both receptors provide
internalization of cell surface-bound ligand (10, 13), and the
sortilin-cd has been shown to convey transport of cargo from Golgi to
late endosomes and to bind GGAs (Golgi-localized SorCS1 is expressed in the murine central nervous system like sortilin
and sorLA, and transcripts have also been observed in kidney, liver,
and heart (1). The receptor prevails in neurons, and during embryonic
development sorCS1 is expressed in a transient and dynamic pattern in
areas of the nervous system where precursors proliferate as well as in
regions where cells differentiate (17).
The purpose of the present work was to begin elucidating sorCS1
function. We cloned human sorCS1b, whose cd is highly similar to that
of the murine orthologue, and identified two new isoforms with distinct
cds and different distributions in tissues. The common lumenal domain
was cleaved by furin in the late synthetic pathway demonstrating that
sorCS1 is synthesized as a proprotein. Analysis of cells stably
transfected with wild type and chimeric receptors showed that the three
cds convey different distributions of sorCS1 in cells and have
different capabilities for internalization. Neither the mature lumenal
domain nor any of the cds bound any of the ligands previously shown to
interact with sortilin and sorLA, demonstrating that sorCS1 is
functionally different from the previously characterized Vps10p-D
family receptors.
Cloning of Human sorCS1 Isoforms--
Murine sorCS1b cDNA
(GenBankTM accession number AF195056) was radiolabeled and
used for screening a human brain cDNA library in the lambda
ZAP vector (Stratagene, La Jolla, CA). Five positive clones were
purified and rescued into the pBK-CMV vector, and sequencing revealed
three different overlapping clones representing bp 462-3504 of human
sorCS1b cDNA in addition to a 3' untranslated region. The 5' part
of the open reading frame was obtained by reverse transcription PCR.
The first strand cDNA was synthesized from 1 µg human fetal brain
RNA (Clontech, Palo Alto, CA) using the primer
5'-AGGTGAAGGTGTAGTGAGCAATAGGG representing bp 1549-1574. RNA was
denatured in the presence of 40 pmol primer for 10 min at 94 °C and
transferred to prewarmed reaction mixture (50 mM Tris, pH
8.3, 75 mM KCl, 3 mM MgCl2, 0.5 mM deoxynucleoside triphosphates, 200 mM
dithiothreitol). Following addition of reverse transcriptase (SuperScript II, Invitrogen), first strand reaction was
performed for 55 min at 42 °C. The reaction was terminated by 15 min
heating to 70 °C followed by 5 min on ice and incubation for 20 min
at 37 °C with RNase-H (AP Biotech, Little Chalfont, UK). A
two-step PCR reaction was performed using Advantage-GC polymerase
(Clontech). The same reverse primer was used as for
the first strand reaction, and the forward primer
(5'-CTCCCGCGATGGGAAAAGTTGGC) was obtained from a sequence in the human
chromosome 10 derived bacterial artificial chromosome RP11-557K21
(GenBankTM accession number AL356439) homologous to the 5' end
of murine sorCS1. The 1.5-kb product was subcloned into the pGem-T Easy
Vector (Promega, Madison, WI) and sequenced. The full-length cDNA
was obtained by ligation of overlapping fragments using the
PstI (bp 1522), SapI (bp 1753), and BamHI (bp
3128) sites and transferred into pBluescript (Stratagene).
Chromosomal localization and organization were determined using the
program tblastn (18) at the NCBI (National Institutes of Health,
Bethesda, MD). Exonic sequences were determined by aligning cDNA
and genomic sequences, and exon/intron boundaries were in agreement
with consensus splice sites (19). Primers corresponding to the 3' ends
of putative terminal exons of sorCS1a and sorCS1c
(5'-TTAATAGAAACCATCACTGCTATG and 5'-TGGTGGCTACTGGGAATCATTTAC) were used
for first strand synthesis on 1 µg of total RNA from human fetal
brain followed by two-step PCR reactions using the forward primer for
generation of full-length sorCS1b.
Analysis of sorCS1 Transcripts--
A multiple tissue blot (AP
Biotech) was hybridized with a [32P]dCTP randomly labeled
probe of sorCS1 (bp 463-1924) and washed under high stringency
conditions followed by autoradiography. For identification of sorCS1
isoforms, reverse transcription PCR reactions were performed
on 1 µg of total human RNA from fetal liver, fetal brain, adult
cerebellum, and adult total brain (Clontech) using the reverse primer 5'-GCCTGTAGCCTTTGGGGGTTTTCC for sorCS1b as
well as the reverse primers designed for isolation of sorCS1a and -c
cDNA. The forward primer (5'-TACCCACCACTGCTGAACTCTTTG) common to
all isoforms was derived from exon 23. The PCR products were verified
by sequencing.
Expression of sorCS1 Constructs--
For generation of wild type
receptor constructs, the sorCS1 cDNA was cut out of pBluescript
using the KpnI site of the polylinker preceding the 5' end
of the cDNA and the natural BamHI site at bp 3128. This
was combined either with a BamHI/ApaI 3' fragment and cloned into pcDNA4/Myc-HisA (Invitrogen) for expression of the
lumenal part of sorCS1 (L-sorCS1) or with
BamHI/NotI 3' fragments and cloned into
pcDNA3.1/Zeo(+) (Invitrogen) for expression of full-length receptor
isoforms. ApaI and NotI sites were introduced by
PCR to the 3' ends using modified primers corresponding to nucleotides
2991-3308 or to segments following the stop codons. Alternatively,
sorCS1a-c cDNA was transferred via NheI and
NotI restriction sites from pcDNA3.1/Zeo(+) to
pcDNA3.1/Hygro(
CHO-K1 cells were cultured in HyQ-CCM5 (HyClone, Logan, UT) and
transfected using FuGENE 6 (Roche Molecular Biochemicals). Stable
transfectants were selected in medium containing 300 µg/ml Zeocin
(Invitrogen) and identified by Western blotting or immunocytochemistry. Double transfectants were generated by transfecting CHO cells expressing IL2R/sorCS1 chimeras with pcDNA3.1/Hygro( Expression of sorCS1 Propeptide and Part of the Leucine-rich
Domain--
The propeptide sequence was amplified from sorCS1 cDNA
using a 5' primer (5'-TCTGGATCCGGCGGCTCCTGCTGC) introducing a
BamHI site to the propeptide N-terminal sequence and a 3'
primer (5'-ATCCTCGAGTCACCGTCTCCTCCG) introducing a stop codon and a
XhoI site after the furin cleavage site 72RRRR.
Following two-step PCR, the product was cloned via
BamHI/XhoI into pGEX-4T-1 (AP Biotech). For
expression of the leucine-rich domain
(Glu942-Ile1023) the native
EcoRI and EcoRV sites were used to generate a
construct in pGEX-4T-1. Both constructs were expressed in the bacterial strain BL21 (DE3), and the resulting GST-tagged proteins were purified
using glutathion-Sepharose beads.
Yeast Two-hybrid Analysis--
EcoRI and
XhoI restriction sites were introduced into cDNA of the
three sorCS1 cytoplasmic tails via PCR followed by insertion into the
pLexA vector to generate bait strains. A Matchmaker LexA two-hybrid
system (Clontech) was used as described before
(13).
Antibodies and Ligands--
Antisera were raised in rabbits
against GST-sorCS1-(942-1023)(anti-Leu sorCS1), L-sorCS1,
and GST-sorCS1-(1-77) propeptide (DAKO, Glostrup, Denmark). Monoclonal
mouse anti-IL2R Metabolic Labeling, Analysis of Glycosylation, and
Immunoblotting--
CHO-K1 cells stably transfected with
L-sorCS1 were grown to 80% confluency and biolabeled
essentially as described previously (9, 10) using 200 µCi/ml
35S-labeled cysteine and methionine (Pro-mix, AP Biotech).
The medium was harvested, and washed cells were lysed in 1% Triton
X-100, 20 mM Tris-HCl, 10 mM EDTA, pH 8.0, supplemented with proteinase inhibitor (CompleteMini, Roche Molecular
Biochemicals). Labeled L-sorCS1 was precipitated
from lysate (200 µl + 600 µl of non-labeled medium) and medium (600 µl + 200 µl of non-labeled lysate) via its His6 tag
using Talon beads. For treatment with PNGase-F (Roche Molecular
Biochemicals), the beads were washed, heated in 10 µl 1% SDS (3 min,
95 °C), heated again after addition of 90 µl 20 mM
NaH2PO4, 10 mM EDTA, 10 mM Na-azide, 0.5% Triton X-100, pH 7.2, and cooled before
the addition of 0.5 units PNGase-F and incubation for 16 h at
30 °C. Alternatively, washed beads were treated with
endoglycosidase-H (Endo-H, Roche) as described (9). For furin cleavage,
beads with bound L-sorCS1 were incubated in 100 µl of 100 mM Hepes, 1 mM CaCl2, 1 mM 2-mercaptoethanol, 0.5% Triton X-100, pH 7.6, and 4 units of furin (Alexis Biochemicals) followed by successive incubations
for 2 h at 30 °C and 2 h at 37 °C. For immunoblotting,
medium or cell lysates were subjected to reducing SDS-PAGE and blotted
following standard procedures. For detection of sorCS1 in blots of
human kidney and spleen (Chemicon), anti-L-sorCS1 was used,
followed by horseradish peroxidase conjugated swine anti-rabbit Ig
(DAKO), and visualization by ECL (AP Biotech).
Surface Plasmon Resonance Analysis--
Measurements were
performed on a BIAcore 2000 instrument using CM5 sensor chips activated
as described (9). L-sorCS1 and the lumenal domains of
sortilin and sorLA were immobilized to an estimated density of ~60
fmol/mm2, and samples for binding (40 µl, 25 °C) were
injected at 5 µl/min in 10 mM Hepes, 150 mM
NaCl, 1.5 mM CaCl2, 1 mM EGTA,
0.005% Tween 20, pH 7.4. Binding was expressed in units as the
response obtained with immobilized receptor minus the response with an
activated but uncoupled chip. The chips were regenerated as described,
and kinetic parameters were determined using BIAevaluation 3.0 software as described (9).
Quantification of Cell Surface Expression--
Cells were
surface-labeled with the impermeable reagent
sulfo-N-hydroxysuccinimidobiotin (Pierce), washed and lysed as
described previously (10, 11), and biotinylated proteins were
precipitated with streptavidin-coupled Sepharose (Zymed
Laboratories Inc., San Francisco, CA). The fractions of
streptavidin-bound and unbound IL2R/sorCS1 chimera or sorCS1 isoforms
in cell lysates were detected by Western blotting and quantified using
a FUJIFILM LAS-1000 luminescence image analyzer.
Immunocytochemistry and Assay for
Internalization--
Transfected or control cells were washed in 10 mM phosphate, 150 mM NaCl, pH 7.3, fixed in the
same buffer with 4% paraformaldehyde, and finally washed in buffer
containing 0.05% Triton X-100 followed by incubation with primary
(anti-Tac or anti-L-sorCS1) and secondary antibodies
(fluorescein isothiocyanate-conjugated rabbit anti-mouse, DAKO, Alexa
488-conjugated goat anti-rabbit or Alexa 568-conjugated goat
anti-mouse, Molecular Probes, Leiden, The Netherlands). To visualize
internalization, cells were surface-labeled with primary antibody at
4 °C for 2 h followed by incubation at 37 °C for various times. Fluorescence microscopy was performed using a laser scanning confocal unit (LSM510, Zeiss). Alternatively, cells transfected with
chimeras were incubated at 4 °C with 125I-labeled
monoclonal anti-Tac (3 × 104 cpm/ml) for 2 h at
4 °C, washed, and reincubated at 37 °C for 0-60 min. Incubations
were stopped by the addition of ice cold acetic acid, 150 mM NaCl, pH 2.5. After 5 min, the supernatant was
recovered, the cells lysed in 1 M NaOH, and radioactivity determined in the two fractions was defined as surface-associated and
internalized antibody, respectively.
Identification of sorCS1 Isoforms--
The human sorCS1b cDNA
(GenBankTM accession number AF284756) encodes a 33 amino acid signal peptide followed by a 1135 amino acid type 1 receptor (Fig. 1A) with 92%
sequence identity to the murine sorCS1b protein. The N-terminal part
contains the sequence (74RRRR77) corresponding
to the optimal multibasic motif (RXR/KR) for cleavage by
furin and the sequence (93RSPR96) corresponding
to the minimal requirements (RXXR) for cleavage. The human
sorCS1b gene maps at 10q23.3, and analysis of genomic sequences
indicated the presence of 26 exons. As two isoforms (sorCS1a and -b)
differing only in their cds have been identified in the mouse (2), and
only the sorCS1b variant was found by human cDNA library screening,
we searched in human genomic databases for sequences corresponding to
the 3' end of murine sorCS1a. However, no such sequence was found, and
genomic sequences between exon 25 (encoding the transmembrane domain
and four residues of the cytoplasmic tail) and exon 26 (encoding the
remaining sorCS1b tail) were therefore analyzed to identify possible
splice variants with little or no homology to murine sorCS1a. Putative
exon sequences identified as open reading frames followed by
polyadenylation signals were analyzed using primers 3' to potential
stop codons and a 5' primer corresponding to exon 23 encoding part of
the leucine-rich domain (cf. Fig. 1B). This
approach revealed two new splice variants in human fetal brain RNA, and
cDNA clones encoding the complete proteins were identified using a
primer corresponding to the 5' untranslated region and the primers
corresponding to sequences 3' to the stop codons.
Fig. 1B shows the generation of human sorCS1 isoforms. Like
in the mouse, human sorCS1a (GenBankTM accession number
AY099453) is generated by using exon 25 as the terminal exon
(stop codon a), but the amino acid sequence encoded by the part of exon
25 that is specific for the sorCS1a-cd is completely different from that of the murine receptor (Fig. 1C and Ref. 2). When the 5' splice site within exon 25 is active, sorCS1b is generated by
skipping the 3' part of this exon and using exon 26 as the terminal
exon. Comparison of the amino acid encoded by exon 26 of human sorCS1b
showed 89% sequence identity with the corresponding segment of the
mouse sorCS1b. Finally, human sorCS1c (GenBankTM accession number
AY099452) is generated by skipping the middle part of exon 25 by
using both splice sites within the exon. Because a murine counterpart
was expected, we subsequently cloned mouse sorCS1c (GenBankTM
accession number AF284755),2
which revealed a cd with 87% amino acid sequence identity to the human
sorCS1c-cd. Searches in databases indicated that the cds of human
sorCS1a and -b show little similarity to other receptor cds, whereas
the sorCS1c-cd exhibits about 50% identity to the cds of sorCS2 and
-3.
Tissue-specific Expression--
Northern blotting of human tissues
using a probe common to all three isoforms showed high levels of
transcripts in adult kidney and comparatively moderate levels in brain,
heart, and small intestine (Fig.
2A). To identify the sorCS1
protein, we performed immunoblots on two human tissue homogenates and
on extracts of CHO cells mock-transfected or stably transfected with
sorCS1a cDNA. A band of ~130 kDa was detected in the
sorCS1a-transfected CHO cells and in kidney, but not in non-transfected
CHO cells and spleen (Fig. 2B), in agreement with the
Northern blots. We next examined expression of the three splice
variants in human adult brain, adult cerebellum, fetal brain, and fetal
liver by reverse transcription PCR. As shown in Fig. 2C, all
three splice variants were detected in the brain samples (lanes
2-4), whereas only sorCS1c was found in human fetal liver
(lane 1), demonstrating that sorCS1 isoforms are
differentially expressed among tissues.
SorCS1 Propeptide Is Removed by Furin-mediated Cleavage--
To
determine whether sorCS1 processing includes propeptide cleavage, CHO
cells were stably transfected with the His6-tagged lumenal
part (amino acid 1-1067) of sorCS1 (L-sorCS1). The
transfectants were biolabeled, and L-sorCS1 secreted into
the medium or present in cell lysates was recovered on Talon beads.
Fig. 3A shows that labeled
L-sorCS1 in the medium (lane 1) has a higher
apparent molecular size than that obtained from lysates (lane
2), whereas, after deglycosylation with PNGase-F, the cellular
form (lane 4) is larger than the secreted form (lane
3), demonstrating cleavage of L-sorCS1. This occurred
within cells because labeled L-sorCS1 isolated from cell
lysates was unchanged by incubation in conditioned CHO medium (not
shown). Fig. 3 further shows that the cellular form of the receptor was
cleaved upon incubation with furin (panel B, lane
4 versus lane 3), whereas the secreted form was
unaffected (lane 2 versus lane 1), and that only
the uncleaved cellular form was sensitive to Endo-H (panel
C, lane 4 versus lane 3), demonstrating that
cleavage occurs in the furin-containing distal synthetic pathway.
Western blotting was then performed, and antibody against sorCS1-(1-77) propeptide reacted only with the cellular form of the
receptor (panel D, lane 1 versus lane
2), whereas both forms reacted with antibody against the
leucine-rich domain (lanes 3 and 4). The results
establish that sorCS1 is synthesized as a proprotein, which is
converted to a mature form by cleavage in the furin-containing late
synthetic pathway.
Characterization of Purified
L-sorCS1--
His6-tagged
L-sorCS1 was purified from the medium of transfected
CHO cells using a Talon column, and sequencing analysis yielded the
N-terminal sequence 78SGADQ, demonstrating cleavage at the
optimal multi-basic motif (74RRRR77). In
addition to this motif, a minimal basic site for potential cleavage by
furin (93RSPR96) is present in the N terminus
of human sorCS1, and optimal basic motifs are present at the same
positions in the murine receptor (1), suggesting that some additional
cleavage might occur after Arg96. To confirm the
involvement of the optimal site for cleavage by furin and to analyze
whether the minimal basic motif might be operational, CHO cells were
transfected with L-sorCS1 mutated in the optimal motif
(74RRRR77 to 74GRGR77).
Fig. 4A shows that wild type
and mutated L-sorCS1 were secreted to a similar extend
(lanes 1 and 2), demonstrating that cleavage of
the propeptide is not necessary for secretion of the receptor as
previously shown for sortilin and sorLA (9, 10). In contrast, propeptide cleavage is a prerequisite for transport of furin (21) and
for most other proprotein convertases (Ref. 22 and references herein)
out of the endoplasmic reticulum. The antibody against propeptide
reacted with mutated sorCS1 (lane 3), and no propeptide immunoreactivity was detected in medium of cells secreting wild type
L-sorCS1 (lane 4) even though GST-sorCS1
propeptide was readily detected following incubation in conditioned
medium (not shown), suggesting that the propeptide is degraded within
the cells. Because the presence of propeptide immunoreactivity in the
medium of cells transfected with mutated L-sorCS1 did not
exclude some cleavage after Arg96, cells were biolabeled,
and mutated and wild type sorCS1 were recovered on Talon beads followed
by treatment with furin. As shown in Fig. 4B, only the wild
type receptor exhibited a smaller size after furin treatment,
demonstrating little or no cleavage after Arg96. We
conclude that L-sorCS1 secreted by CHO cells is cleaved
only at the optimal site for cleavage by furin, i.e. after
Arg77.
Binding of sorCS1 propeptide to mature L-sorCS1-(78-1067)
was measured because the propeptides of sortilin and sorLA bind to the
Vps10p-Ds of their respective receptors with high affinities. Fig.
5A shows that GST-sorCS1
propeptide, but not GST alone, binds to purified L-sorCS1
(inset) with a Kd estimated at about 0.7 µM (range 0.5-1 µM in 3 experiments). This
low affinity binding was abolished by 20 mM EDTA and
reduced to half at pH 6.0 (not shown). We then tested ligands
previously shown to interact with sortilin and sorLA, including RAP
(Fig. 5A), neurotensin, apolipoprotein E3, and lipoprotein
lipase (not shown), and none of them bound to L-sorCS1
(Kd values > 2 µM).
Binding of the sorCS1 propeptide to mature sortilin was measured
because the propeptides of sortilin and sorLA cross-bind to the
respective receptors (10). Surprisingly, GST-sorCS1 propeptide bound to
sortilin (Fig. 5B) with a Kd estimated at
about 20 nM (range 17-30 nM in 3 experiments),
which is comparable with the affinity for binding of sortilin to its
own propeptide (9). The binding was abolished by 5 µM RAP
and markedly reduced by 20 µM neurotensin (Fig.
5B) as previously shown for binding of the sortilin
propeptide (9). In addition, the sorCS1 propeptide bound to mature
sorLA and was inhibited by the 54-residue sorLA propeptide (not shown).
Thus, sorCS1 is different from the previously characterized Vps10p-D
receptors sortilin and sorLA in the sense that it does not bind ligands
common to the two receptors and that it binds its own propeptide with
low affinity. On the other hand, the sorCS1 and sortilin propeptides
bind with similar affinities and apparently to the same or
overlapping sites on sortilin.
Subcellular Distribution of sorCS1a-c--
To determine whether
the three alternative cds might mediate different subcellular
distributions of the receptor, we analyzed transfectants expressing
chimeras of the lumenal and transmembrane parts of IL2R (Tac/CD25) and
the sorCS1a-c cds. Fig. 6 shows that the
IL2R/sorCS1a-cd chimera (chi-a) is predominantly intracellular, whereas
the chimeras containing the sorCS1b and -c tails (chi-b and chi-c)
exhibit distinct cell surface expression in addition to a comparatively
low expression in paranuclear compartments. We also performed surface
biotinylation of the transfectants followed by lysis, recovery of
biotinylated proteins on streptavidin-Sepharose beads, and scanning
densitometry of Western blots of biotinylated and non-biotinylated
chimeras. The estimated cell surface expressions were 10% for chi-a,
46% for chi-b, and 30% for chi-c (mean of two experiments). Parallel
experiments were performed with cells transfected with wild type
sorCS1a-c using polyclonal anti-L-sorCS1, and the
estimated surface expressions were 11% for sorCS1a, 34% for sorCS1b,
and 24% for sorCS1c (mean of three experiments). Thus, the a tail
mediates a much lower cell surface expression of sorCS1 than the b
tail, whereas the c tail appears to be intermediate.
Internalization of sorCS1a-c--
As the different subcellular
distributions could result from differences in capabilities of the cds
to mediate internalization, the chi-a, -b, and -c transfectants were
incubated with 125I-anti-Tac for 2 h at 4 °C
followed by removal of unbound antibody and continued incubation at
37 °C. Internalized antibody was then determined as the amount of
cell-associated radioactivity not removed by a following incubation at
4 °C, pH 2.5. The transfectants bound 10-20% of the added tracer
after the incubation at 4 °C, whereas no binding was observed in
mock-transfected cells. Fig. 7A shows that about 65% of
the initially surface-bound 125I-anti-Tac had been
internalized by chi-a after 60 min, whereas internalization was much
less pronounced in cells transfected with chi-b and chi-c. No
degradation of cell-bound 125I-anti-Tac was observed after
2 h (not shown), suggesting that complexes of anti-Tac and the
chimeric receptors remained stable within the cells. We therefore
followed internalization at 37 °C of anti-Tac bound to cell surfaces
at 4 °C by confocal microscopy. As shown in Fig. 7B,
surface-bound anti-Tac was rapidly internalized to paranuclear
compartments by the chi-a-transfected cells, whereas the chi-b or chi-c
chimeras gave rise to little or no paranuclear staining.
To ensure that the cd is the major determinant for internalization,
i.e. that wild type and chimeric receptors behave similarly, we generated double transfectants co-expressing wild type receptors and
chimeras in different combinations. Fig.
8 presents examples of internalization of
wild type sorCS1 isoforms (green fluorescence) as compared with
chimeras (red fluorescence). The upper panels show that both
sorCS1b and chi-b remain largely on the cell surface. The more patchy
appearance of sorCS1b might suggest that it coalesces more easily than
the chimera or might be related to the polyclonal antibody used. The
middle panels demonstrate that sorCS1a is efficiently internalized in contrast to chi-b, and the reverse experiment (not
shown) confirmed inefficient internalization of sorCS1b as compared
with chi-a. Finally, the lower panels show that the c tail
is capable of mediating some internalization although much less
efficiently than the a tail. Thus, as compared with the b and c tails,
the cytoplasmic tail of sorCS1a mediates a small fractional surface
expression and an efficient internalization of surface-bound
ligand.
Because sorCS1a exhibited a subcellular distribution and
internalization to paranuclear compartments similar to sortilin and sorLA, whose cds bind GGA1 and -2 (13, 16), we tested the sorCS1 cds
for interaction with the GGAs by yeast two-hybrid analysis. However,
none of the sorCS1 cds displayed any binding, suggesting that sorCS1 is
not implicated in GGA-mediated functions in contrast to the two
previously characterized members of the Vps10p-D family.
This study demonstrates alternative splicing of the Vps10p-D
receptor sorCS1 and characterizes three isoforms that are
differentially expressed in human tissues. The isoforms have identical
lumenal and transmembrane domains but different cytoplasmic tails. The cds of both human and murine sorCS1a contain putative internalization signals (Fig. 1C and Ref. 2) and might therefore be
functionally equivalent, although their amino acid sequences are
totally different. In contrast, the cds of human sorCS1b and sorCS1c
are highly similar to their murine orthologues, and the cd of sorCS1c
exhibits about 50% sequence similarity to the cds of sorCS2
and -3.
We show that sorCS1 is synthesized as a proprotein, which is converted
to the mature receptor in late Golgi compartments by cleavage of the
RRRR The propeptides of sortilin and sorLA protect the receptors against
premature ligand binding in the synthetic pathway (9, 10) and might in
addition function as intramolecular chaperones. This is in partial
analogy to the propeptide of furin, which remains bound to the
endoprotease after cleavage and permits its exit from the endoplasmic
reticulum (26). The function of sorCS1 propeptide may be similar even
though the affinity for binding to its own receptor is comparatively
low. Surprisingly, the sorCS1 propeptide showed much higher affinity to
mature sortilin and sorLA, raising the possibility that it might
interact with these members of the Vps10p-D receptor family in the
synthetic pathway.
We suspected that mature sorCS1 might bind one or more of the ligands
previously shown to interact with the Vps10p-Ds of both sortilin and
sorLA, i.e. RAP, neurotensin, and lipoprotein lipase (9-12). However, this was not the case, indicating that the lumenal domain of sorCS1 has a ligand-binding profile different from those of
previously characterized Vps10p-D receptors. In addition, none of the
sorCS1 cds bound GGAs in contrast to those of sortilin and sorLA (13,
16). This is in accordance with the lack of the previously defined
requirements for GGA binding (13, 16, 27-29), although the sorCS1c-cd
contains a reminiscent sequence near the C terminus. It is therefore
unlikely that sorCS1 is implicated in GGA-mediated functions.
Irrespective of the mechanisms involved, we demonstrate that the three
sorCS1 cds generated by alternative splicing mediate different
localization and trafficking of the receptor, as sorCS1a is about 90%
intracellular and sorCS1b exhibits a large cell surface expression.
Because all three cds are expressed in the brain, different
localizations of receptor isoforms might explain that sorCS1
immunoreactivity is seen as punctate cytoplasmic staining in some
neurons, whereas other neurons exhibit significant cell surface
expression (30). The efficient internalization of sorCS1a is in
accordance with the presence of a YXXØ motif and a
dileucine motif in the a tail, whereas the almost non-internalizing b
tail lacks such motifs (Fig. 1C). Putative internalization
signals are also present in the c tail, and it remains to be clarified why this cd only mediates internalization to a moderate extent. In
addition, the presence of a single SH3 domain-binding motif (PXXP) in the a and c tails, and of multiple partially
overlapping motifs in the b tail, suggest that the cds may interact
with scaffold or adaptor proteins that may contribute to signal
transduction events (31).
Alternative splicing is not uncommon among receptors and is an
important mechanism for modulating functions, including those of
cytoplasmic tails. The use of composite internal/terminal exons can
lead to deletions or different lengths of receptor tails (32), and
optional exons to insertion of additional amino acid sequences as
demonstrated for the apolipoprotein E receptor-2 (33). On the other
hand, the creation of completely different cytoplasmic domains is
unusual. Interestingly, analysis of EST databases predicted an exchange
of a transmembrane and cytoplasmic domain of an FC receptor
In conclusion, we show that sorCS1 is synthesized as a proprotein that
is cleaved to mature forms in the trans-Golgi network and expressed in
three isoforms with different cytoplasmic domains capable of mediating
different trafficking of the receptor.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-adaptin ear
containing ADP-ribosylation factor-binding proteins) (13), which are
adaptor proteins believed to be involved in this type of trafficking
(reviewed in Refs. 14 and 15). Considering that sorLA is mainly
intracellular (10) and also binds GGAs (16), it seemed possible that
Vps10p-D receptors other than sortilin might be targeted by similar
sorting mechanisms.
EXPERIMENTAL PROCEDURES
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ABSTRACT
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DISCUSSION
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) (Invitrogen) to generate doubly transfected
cells. To produce L-sorCS1 mutated in the furin cleavage
site (74RRRR to 74GRGR) we performed a
two-step PCR using Advantage-GC polymerase. Overlapping 5' and 3'
fragments were amplified from the original expression construct using
the primers 5'-ATTAATACGACTCACTATAGGGAG, 5'-GATCCGCTCCGCTCCGTCCCCTCCCGC, 5'-CCGGCGGGAGGGGACGGAGCGGAG, and 5'-TTGTTCCATAATCGGTTGACCTCC. The resulting PCR fragment was
digested with KpnI and SacI and used to replace
the 5' end of L-sorCS1 cDNA in the pcDNA4/Myc-HisA
vector. To produce chimeric constructs covering the lumenal and
transmembrane parts of the interleukin-2 receptor-
(IL2R, Tac/CD25)
and the sorCS1 cytoplasmic tails (chi-a, -b, and -c), cDNA encoding
the three tails was amplified by standard PCR technique using primers
generating a 5' HindIII site and a 3' XhoI site.
The primers for the a, b, and c tail constructs were:
5'-TCGTAAGCTTAAGTTTAAAAGGTGCG and
5'-CCTCTCGAGTTAATAGAAACCATCACTGCTATG, 5'-CGTCAAGCTTAAGTTTAAAAGGAGAGTAGCTTTACCC and
5'-GGGGCTCGAGTTAAATTGCATACTGTGCCCCAGCAGATCC, and
5'-CAAGCTTAAGTTTAAAAGGAA GATC and 5'-TACCTCGAGTCATTTACCTATGAGC. The HindIII/XhoI fragments were ligated into
pcDNA3.1/Zeo(+) together with a NheI/HindIII
fragment representing the lumenal and transmembrane parts of IL2R cut
out of pCMV-IL2R/CD25/Tac (20).
) wild
type receptor constructs followed by additional selection using 500 µg/ml Hygromycin (Invitrogen). Secreted His6-tagged
L-sorCS1 was purified by affinity chromatography on Talon
Metal Affinity Resin (Clontech). Prewashed resin (3 ml) was recirculated for 16 h at 4 °C with about 90 ml of
culture medium followed by washings in 50 mM
Na2HPO4, 300 mM NaCl, 0.1% Tween
20, pH 7.0, and elution in 50 mM NaAc, 300 mM
NaCl, pH 5.0.
(anti-Tac) was from Roche. Recombinant RAP,
GST-sortilin and
sorLA propeptides, as well as the lumenal domains of
sortilin (L-sortilin) and sorLA, were produced as described
(9, 10). Neurotensin was from Sigma, recombinant apolipoprotein E3 from
Calbiochem, and bovine lipoprotein lipase was a gift from Dr. G. Olivecrona, Umeå University, Sweden.
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Fig. 1.
Splice variants of human sorCS1.
A, schematic representation of full-length sorCS1. The
Vps10p-D (gray) is followed by the leucine-rich domain
(black), the transmembrane domain, and one of the
cytoplasmic tails. Basic motifs near the N terminus are indicated as
well as the C-terminal residue common to all splice variants.
B, organization of the human sorCS1 gene leading to
generation of different cytoplasmic tails. The black boxes
represent exons 23 and 24 with typical 3' and 5' splice sites. In the
composite internal/terminal exon 25 (gray), the dotted
lines indicate the potential 5' and 3' splice sites. The
alternatively used terminal exon 26 is shown in white.
SorCS1a is generated by using exon 25 and the indicated stop codon a.
The upper broken line indicates generation of sorCS1b (stop
codon b), and the lower broken line indicates generation of
sorCS1c (stop codon c). The horizontal arrows mark the
positions of primers used for cDNA cloning and reverse
transcription PCR. C, amino acid sequences of the
cytoplasmic tails. The dots indicate where the common
sequence ends. YXXØ motifs are underlined, dileucine motifs
are overlined, and SH3 domain-binding motifs are marked by
dashed lines.
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Fig. 2.
Analysis of sorCS1 transcripts.
A, Northern blotting. Nylon membranes carrying immobilized
poly(A)+ RNA (1 µg/lane) from human adult tissues were
hybridized with a 32P-labeled probe encoding part of the
Vps10p-D. Sample origin: 1, kidney (1 day exposed);
2, liver; 3, small intestine; 4,
brain; 5, heart; 6, skeletal muscle;
7, colon; 8, thymus; 9, spleen (all 5 days exposed). B, immunoblotting. Extracts of cells (50 µg
protein/lane) and human tissues (75 µg protein/lane) were analyzed
using anti L-sorCS1 antibody. Sample origin: 1,
CHO cells stably transfected with sorCS1a; 2,
mock-transfected CHO cells; 3, adult kidney; 4,
adult spleen. C, reverse transcription PCR analysis.
Reactions were carried out using total human RNA from: 1,
fetal liver; 2, fetal brain; 3, adult cerebellum;
4, adult total brain. Splice variant-specific primers were
used as indicated in Fig. 1B. The size of the detected
fragments corresponds to the calculated base pairs: a, 404;
b, 450; c, 487. The additional band of 592 base
pairs generated with the primers used for variant c represents sorCS1a
cDNA as the untranslated region of sorCS1a includes the sequence
encoding sorCS1c.
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Fig. 3.
Cleavage of L-sorCS1.
A-C, CHO cells transfected with His6-tagged
L-sorCS1-(1-1067) were biolabeled and receptor secreted
into medium (M) or extracted from cell lysate (L)
was recovered on Talon beads, treated with enzymes as indicated, and
analyzed by reducing SDS-PAGE followed by autoradiography.
D, Western blot analysis of lysate and medium using
anti-sorCS1 propeptide ( -propep) or antibody against the
leucine-rich domain of sorCS1 (
-leu).
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Fig. 4.
SorCS1 cleavage in CHO-cells depends on the
intact consensus sequence 74RRRR. A, medium
of CHO cells expressing L-sorCS1 (WT) or
L-sorCS1 mutated in the consensus cleavage site
(FM) was analyzed by Western blotting using antibody against
the leucine-rich domain ( -leu) or the propeptide
(
-propep). B, cells were biolabeled, and
L-sorCS1 and mutated L-sorCS1 recovered from
lysates on Talon beads were treated with furin followed by SDS-PAGE
and autoradiography of the 2,5diphenyloxazole-treated gel.
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Fig. 5.
Binding of sorCS1 propeptide to sorCS1 and
sortilin. Binding was measured using surface plasmon analysis with
45-60 fmol immobilized L-sorCS1-(78-1067) or
L-sortilin-(45-725). The chip was superfused with
ligand-containing buffer at 100 s followed by buffer alone at
600 s. A, L-sorCS1; sensorgrams of 2 µM GST-sorCS1 propeptide (calculated
Kd, 1.0 µM), 2 µM GST,
or 5 µM RAP at pH 7.4. The inset shows the
purity of L-sorCS1. B, L-sortilin;
sensorgrams of 1 µM GST-sorCS1 propeptide alone
(calculated Kd, 23 nM) and with 5 µM RAP or 20 µM neurotensin. The signals
obtained with RAP and neurotensin alone have been subtracted.
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Fig. 6.
Subcellular Distribution of IL2R/sorCS1-cd
chimeras. CHO cells stably transfected with chimeric receptors
composed of the extracellular and transmembrane domains of IL2R and the
cytoplasmic tail of sorCS1a, -b, or -c (chi-a,
-b, and -c) were permeabilized and immunostained
with anti-Tac Ig using fluorescein isothiocyanate-conjugated anti-mouse
Ig as secondary antibody.
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Fig. 7.
Internalization of IL2R/sorCS1-cd
chimeras. A, CHO cells stably transfected with chi-a
(circles), chi-b (squares), or chi-c
(triangles) were incubated with 125I-labeled
anti-Tac Ig for 2 h at 4 °C, unbound tracer was removed
by washings, incubations were continued at 37 °C and stopped by the
addition of ice-cold acid buffer at the times indicated.
Internalization was determined as the percent cell-associated
radioactivity not released at pH 2.5. The values are averages of 3 experiments ± 1 S.D. B, confocal microscopy of CHO
cells transfected with IL2R/sorCS1a-c chimeras (chi-a,
-b, and -c). Cells were incubated for 2 h at
4 °C with mouse anti-Tac Ig, washed, and re-incubated in 37 °C
warm medium for 0-60 min, fixed and stained with fluorescein
isothiocyanate-conjugated anti-mouse Ig.
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Fig. 8.
Internalization of sorCS1a-c. Cells
doubly transfected with sorCS1b and chi-b, sorCS1a and chi-b, or
sorCS1a and chi-c were incubated for 2 h with rabbit anti-sorCS1
and mouse anti-Tac, washed, re-incubated for 2 h in
37 °C warm medium, stained after fixation (Alexa 488-conjugated
anti-rabbit and Alexa 568-conjugated anti-mouse), and analyzed by
confocal microscopy.
DISCUSSION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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S77 sequence. Furin is present in this compartment
and is likely to be responsible for the cleavage, although
proconvertases of the subtilisin/Kex-2-like family with overlapping
specificities may also participate. Analysis of receptor constructs in
which the typical consensus site was inactivated showed no signs of alternative cleavage after RSPR96 within CHO cells or after
treatment of the precipitated protein with furin under conditions that
readily cleaved after Arg77. This is surprising because
RSPR is thought to be a recognition motif for members of the proprotein
convertase family of the general sequence
(R/K)Xn(R/K), where n = 0, 2, 4, or 6 (23), and because furin is known to cleave the similar sequence
RLPR
D in the mannose 6-phosphate uncovering enzyme (24). In
addition, the pro-form of BACE (beta site amyloid precursor
protein-cleaving enzyme) is cleaved at a RLPR
E sequence (25).
However, the presence of an Arg at position
6 in uncovering enzyme
may greatly facilitate its cleavage (24), and it could not be
completely excluded that a proprotein convertase other than furin
accounted for the cleavage of BACE (25). Alternatively, the presence of
a hydrophobic residue at position
3 (Leu) or an acidic residue
(Asp or Glu) at position +1 as in uncovering enzyme and BACE might
facilitate cleavage by furin. Future studies should establish whether
other members of the proprotein convertase family may cleave sorCS1
after Arg96.
-chain homologue, which might modulate signal transduction activity
(34). However, the present work is to our knowledge the first
demonstration of a receptor with completely different cytoplasmic tails
that mediate different trafficking in cells.
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ACKNOWLEDGEMENTS |
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We thank Mitra Shamsali for expert technical assistance and Drs. M. S. Nielsen and A. Nykjær for valuable suggestions.
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FOOTNOTES |
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* This work is supported by grants from the Novo-Nordic Foundation and the Danish Medical Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF284756, AY099453, and AY099452.
Supported by a Marie Curie Fellowship of the European Community
Programme Improving the Human Research Potential and the Socio-Economic Knowledge Base.
§ To whom correspondence should be addressed: Dept. of Medical Biochemistry, University of Aarhus, Ole Worms Allé Bldg. 170, DK 8000 Aarhus C, Denmark. Tel.: 45-89-422880; E-mail: ghermey@biokemi.au.dk.
Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M210851200
2 G. Hermey, unpublished result.
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ABBREVIATIONS |
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The abbreviations used are:
D, domain;
cd, cytoplasmic domain;
CHO, Chinese hamster ovary;
chi-a-c, chimeric
receptor a-c;
Endo-H, endoglycosidase-H;
GGA, Golgi-localized,
-adaptin ear containing ADP ribosylation factor-binding proteins;
GST, glutathione S-transferase;
IL2R, interleukin-2
receptor-
(Tac/CD 25);
L-sorCS1, lumenal part of sorCS1;
PNGase-F, glycosidase-F;
RAP, receptor-associated
protein.
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