1021
Journal of Cell Science 113, 1021-1032 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
JCS1046
Endo180, an endocytic recycling glycoprotein related to the macrophage
mannose receptor is expressed on fibroblasts, endothelial cells and
macrophages and functions as a lectin receptor
Humma Sheikh1, Helen Yarwood1, Alan Ashworth2 and Clare M. Isacke1
1Department of Biology, Sir Alexander Fleming Building, Imperial College of Science, Technology and Medicine, Imperial College
Road, London SW7 2AZ, UK
2The Breakthrough Breast Cancer Research Centre at The Institute of Cancer Research, Fulham Road, London SW3 6JB, UK
*Author for correspondence (e-mail: [email protected])
Accepted 20 December 1999; published on WWW 21 February 2000
SUMMARY
Endo180 was previously characterized as a novel, cell type
specific, recycling transmembrane glycoprotein. This
manuscript describes the isolation of a full length human
Endo180 cDNA clone which was shown to encode a
fourth member of a family of proteins comprising the
macrophage mannose receptor, the phospholipase A2
receptor and the DEC-205/MR6 receptor. This receptor
family is unusual in that they contain 8-10 C-type lectin
carbohydrate recognition domains in a single polypeptide
backbone, however, only the macrophage mannose
receptor had been shown to function as a lectin. Sequence
analysis of Endo180 reveals that the second carbohydrate
recognition domain has retained key conserved amino
acids found in other functional C-type lectins.
Furthermore, it is demonstrated that this protein displays
Ca2+-dependent binding to N-acetylglucosamine but not
mannose affinity columns. In order to characterize the
physiological function of Endo180, a series of biochemical
and morphological studies were undertaken. Endo180 is
found to be predominantly expressed in vivo and in vitro
on fibroblasts, endothelial cells and macrophages, and the
distribution and post-translational processing in these cells
is consistent with Endo180 functioning to internalize
glycosylated ligands from the extracellular milieu for
release in an endosomal compartment.
INTRODUCTION
membrane protein being taken up into the cell within 2 minutes
of warming to 37°C. Importantly, using these Fab′ fragments
the fate of the internalized receptor could be monitored. It was
found that at least a significant fraction of the protein taken up
from the cell surface into the endosomes recycled back to the
plasma membrane within 60 minutes. Given its molecular size
and recycling endocytic properties, it is proposed that this
receptor is termed Endo180.
Receptor-mediated internalization of ligands is a vital
physiological process carried out by all eukaryotic cells. It
provides a mechanism by which a vast array of components
such as nutrients, chemokines, hormones, toxins and pathogens
are taken up by cells. Receptors that are internalized via
clathrin coated pits can be divided into two groups (Trowbridge
et al., 1993). The first are receptors, such as the epidermal
growth factor receptor, for which internalization is driven by
ligand binding and results in both the ligand and receptor being
targeted for degradation in the lysosome. The second are
receptors such as the transferrin, low density lipoprotein and
asialoglycoprotein receptors whose internalization is
constitutive and independent of ligand binding. In general,
ligands internalized by these receptors dissociate in the low pH
Endo180 was originally identified as an antigen recognized by
four different monoclonal antibodies (mAbs) which were
raised as part of a panel of reagents designed to identify novel
human fibroblast cell surface receptors (Isacke et al., 1990).
Interest in this 180 kDa transmembrane glycoprotein stemmed
from three key observations. First, N-terminal and tryptic
peptide sequences obtained from the purified protein suggested
that this was a novel receptor. Second, this protein was found
to have a restricted cell type expression. Finally, and most
interestingly, was the subcellular distribution and trafficking of
the receptor. Immunofluorescence and immunoelectron
microscopy studies revealed that in cultured fibroblasts, the
Endo180 was concentrated on the plasma membrane into
clathrin-coated pits. Moreover this cell surface protein only
represented 10-30% of the total Endo180 and that the
remaining 70-90% was localized to intracellular vesicles,
identified by co-localization with the transferrin receptor as
being endosomes. Using mAb Fab′ fragments it was then
demonstrated that cell surface receptor could be internalized.
This process was very rapid with greater than 60% of plasma
Key words: Endo180, Macrophage mannose receptor, Endocytosis,
C-type lectin, Endothelial cell
1022 H. Sheikh and others
environment of the endosome leaving the receptors to recycle
back to the plasma membrane. The biochemical,
morphological and kinetic analysis of Endo180 (Isacke et al.,
1990) strongly suggested that this glycoprotein was a novel,
constitutively recycling receptor. In this manuscript we
have made use of the specific anti-Endo180 mAbs and
polyclonal antisera to facilitate cloning of the full length
cDNA and undertake a detailed mechanistic and functional
characterization of this endocytic receptor.
MATERIALS AND METHODS
Isolation and expression of human Endo180 cDNA clones
A human placental λgt11 library (Millán, 1986) was screened using an
anti-Endo180 polyclonal antiserum, inserts were excised from positive
colonies with EcoRI, ligated into pBluescript and partially sequenced.
A human stromal λZAP cDNA library (J. Boulter, UCLA, USA) was
screened with a human EST (I.M.A.G.E. clone number 1252230;
Lennon et al., 1996) which had sequence identity to one of the positive
clones isolated from the λgt11 library. Positives λZAP clones were
rescued by in vivo excision of the pBluescript phagemid and
characterized by PCR, restriction endonuclease digestion and
sequencing. This resulted in the identification of λ10.2 as a putative full
length Endo180 clone. The insert from λ10.2 was excised from
pBluescript using NotI and XhoI and subcloned into the complementary
sites in the pcDNA3 expression vector (Invitrogen). MDCK cells were
transfected with pcDNA3-Endo180 as previously described (Sheikh
and Isacke, 1996) and cultured for 24 hours before analysis.
Cell lines
AG1523, Flow2000 and MRC-5 fibroblasts, RPM-MC melanoma
cells (Byers et al., 1991) and HepG2 cells were maintained in DME
supplemented with 10% FCS. Human umbilical vein endothelial cells
(HUVEC) were isolated from umbilical cords by digestion with
collagenase type II as described previously (Wellicome et al., 1990)
and used at passage 3. Human dermal microvascular endothelial cells
(DMEC) were isolated from human foreskins and cultured on
fibronectin-coated flasks as previously described (Mason et al., 1996)
and used at passage 4 to 6. The human dermal microvascular
endothelial cell line (HMEC-1; Ades et al., 1992), a gift from Dr E.
Ades (CDC, Atlanta, USA) was cultured on 1% gelatin-coated tissue
culture flasks in MCDB-131 growth medium (Gibco) supplemented
with 10% FCS, 10 ng/ml epidermal growth factor (Becton Dickinson),
100 IU/ml penicillin, 0.1 mg/ml streptomycin and 2 mM L-glutamine.
Human monocyte-derived macrophages were prepared by isolating
peripheral blood mononuclear cells on a Percoll gradient, enriching
for monocytes by selective attachment to plasma-coated dishes and
differentiating in vitro by culture in autologous serum for 6 days
(McCutcheon et al., 1998).
Antibodies
The generation and characterization of mAb E1/183 and the
polyclonal anti-Endo180 antiserum have been described previously
(Isacke et al., 1990). mAb A5/158 was generated by immunizing mice
with human AG1523 fibroblasts and screening for mAbs directed
against plasma membrane proteins (Isacke et al., 1986).
Immunoprecipitation of 125I-labelled cell surface proteins followed by
limited proteolytic digestion with 0.25-10 µg Staphylococcus aureus
V8 protease demonstrated that E1/183 and A5/158 recognized the
same 180 kDa antigen.
Endo180 expression
Western blot analysis
Semi-confluent cultures of cells were lysed in sample buffer,
sonicated, resolved on 10% SDS polyacrylamide gels and transferred
to nitrocellulose membranes as previously described (Neame and
Isacke, 1993). Detection of Endo180 was performed by incubation of
the membrane with 4 µg/ml A5/158 anti-Endo180 mAb followed by
150 ng/ml HRP-conjugated anti-mouse Ig (Jackson Immunoresearch).
Blots were developed using the enhanced chemiluminescence (ECL)
kit (Amersham) and exposed to X-ray film (Fuji XR) at room
temperature.
Flow cytometry
Confluent monolayers of endothelial cells were harvested by exposure
to trypsin/EDTA (ICN) for 1 minute at 37°C. After repeated pipetting
to ensure a single cell suspension, cells were incubated with 50 µg/ml
mAb A5/158 or isotype matched irrelevant mAb for 30 minutes at
4°C, washed in HBSS plus 2.5% FCS, incubated with 20 µg/ml FITClabelled rabbit anti-mouse Ig (Dako) for 30 minutes at 4°C, washed
twice and fixed with 1% paraformaldehyde. Samples were analysed
on a Becton Dickinson FACScan flow cytometer by counting 104 cells
per sample. Flow cytometry of macrophages was performed as
described previously (McCutcheon et al., 1998).
Tissue immunostaining
Cryosections of human term placenta were permeabilized with 0.5%
Triton X-100 and stained with mAb E1/183 and counterstained with
biotinylated Ulex europaeus agglutinin-I (UEA) lectin followed by
Cy3-goat anti-mouse Ig and avidin-FITC. Cryosections of human skin
were treated as described above and stained with mAb E1/183
followed by Cy3-goat anti-mouse Ig. Sections were then blocked with
mouse serum and then counterstained with biotinylated anti-CD14
mAb (Serotec) followed by avidin-FITC. Confocal images were
collected on the Leica TCS NT system. In all cases, parallel sections
were stained for each marker separately to establish that the pattern
of expression observed was not due to cross reactivity of second layer
reagents.
Cell immunofluorescence
HMEC-1 cells (cultured on gelatin-coated glass coverslips) and
Flow2000 cells were fixed with 3% paraformaldehyde and processed
as previously described (Neame and Isacke, 1993) using 100 µg/ml
mAb E1/183 followed by 10 µg/ml rhodamine-conjugated anti-mouse
Ig and confocal images were collected on the Leica TCS NT system.
Biochemical analysis
Phosphorylation and phosphoamino acid analysis
35 mm dishes of AG1523 human fibroblasts were washed in
phosphate-free DME and incubated for 16 hours in 2 ml of phosphatefree DME containing 2% FCS, 20 mM HEPES, pH 7.5, and 2 mCi
[32P]orthophosphate (Amersham). Parallel dishes were then left
untreated or treated with 50 ng/ml TPA (12-O-tetradecanoyl-phorbol13-acetate) for 10 minutes prior to lysis and preclearing as described
previously (Neame and Isacke, 1992). Human Endo180 was
immunoprecipitated using mAb E1/183, followed by goat anti-mouse
Ig prebound to Protein A-agarose (Bio-Rad), resolved by SDS-PAGE
and gels were exposed overnight at −70°C. Endo180 bands were
excised from the dried gel and subjected to two-dimensional
phosphoamino acid analysis as previously described (Cooper et al.,
1983).
Pulse chase experiments
35 mm dishes of Flow2000 human fibroblasts were cultured for 1 hour
in methionine-free DME plus 4% dialysed FCS and then 300 µCi of
[35S]methionine (Amersham) per ml was added for a further 15
minutes. The dishes were washed and incubated with DME containing
5% FCS and 3 mM methionine for 0 to 24 hours and Endo180
immunoprecipitated with mAb E1/183 as described above. For endo
H (endo-β-N-acteylglycosaminidase H; Boehringer Mannheim)
treatment, immunoprecipitates were washed three times with PBS,
Endo180, a novel endocytic lectin receptor 1023
boiled for 2 minutes in 25 µl of 50 mM Tris-HCl, pH 5.5, 0.02% SDS,
and then incubated at 37°C for 3 hours with 2.5 mU of enzyme and
1 mM PMSF (phenylmethylsulphonyl fluoride). For endo F (endo-βN-acetylglucosaminidase F) treatment, the immunoprecipitates were
washed 3 times with PBS, boiled for 2 minutes in 25 µl of 100 mM
sodium phosphate buffer, pH 6.1, 50 mM EDTA, 1% 2mercaptoethanol and 0.1% SDS. Nonidet P-40 (NP-40) was added to
1% and samples incubated with 0.5 units of endo F in the presence
of 1 mM PMSF for 3 hours at 37°C. For neuraminidase treatment,
immunoprecipitates bound to Protein A-agarose were washed 3 times
with PBS, resuspended in 25 µl of 50 mM sodium acetate, pH 5.5, 1
mM CaCl2, 1 mM PMSF, 5 units/ml Clostridum perfringens
neuraminidase type X (Sigma) and incubated at 37°C for 4 hours. To
stop the reactions an equal volume of 2× concentrated SDS sample
buffer was added, the samples boiled for 2 minutes and resolved by
SDS-PAGE. Gels were exposed to X-ray film for 6 days.
minutes, centrifuged at 14,000 g in an Eppendorf microfuge, the
pellets washed twice with ethanol/ether (1:1), vacuum dried,
resuspended in 40 µl of sample buffer, resolved on 10% SDSpolyacrylamide gels and analysed by western blotting.
RESULTS
Endo180 is a member of the macrophage mannose
receptor family
A polyclonal anti-Endo180 antiserum was used to screen a
human placental λgt11 library and one clone (λ23.2) showed
sequence identity to a number of human ESTs. One of these
ESTs (I.M.A.G.E. clone number 1252230) was used to screen
a human stromal λZAP cDNA library and PCR and restriction
digest analysis suggested that clone λ10.2 contained a full
length insert. The insert from λ10.2 (accession number
AF134838) was fully sequenced and subcloned into the
pcDNA3 eukaryotic expression vector.
Three lines of evidence demonstrate that clone λ10.2 encodes
a full length Endo180 cDNA: (a) the purified Endo180 protein
was subjected to N-terminal sequencing and shows a 22/26 amino
acid match with the sequence of λ10.2 after cleavage of the signal
sequence (Fig. 1A). In addition, protein sequence was obtained
from nine Endo180 tryptic peptides. Sequence matches for 8 of
these peptides are found within the human Endo180 cDNA (data
not shown). (b) Clone λ23.2 isolated by polyclonal antibody
screening is identical to the 3′ end of λ10.2 (bases 3,837 to 5,726:
Fig. 1A), (c) clone λ10.2 was inserted into the pCDNA3 vector,
expressed in MDCK epithelial cells and western blot analysis of
transfected cells with an anti-Endo180 mAb revealed a single 180
kDa immunoreactive band (Fig. 1C). In these transfected cells,
the distribution of Endo180 (data not shown) is identical to that
observed for endogenous Endo180 (see below).
Sugar binding assays
Flow2000 human fibroblasts and HMEC-1 cells were grown to
confluence on 9 cm dishes, rinsed with Tris saline (15 mM Tris-HCl,
pH 7.5, 150 mM NaCl), lysed in 900 µl of detergent extraction
solution (150 mM Tris-HCl, pH 8.0, 10 mM NaCl, 10 mM CaCl2, 1
mM MgCl2 and 0.15% Triton X-100), incubated on ice for 15 minutes
before centrifugation at 14,000 g in an Eppendorf microfuge and the
supernatants collected. 1 ml mannose-, mannan-, fucose-, galactoseor -N-acetylglucosamine-agarose (Sigma) were loaded into PolyPrep
chromatography columns (Bio-Rad) and pre-equilibrated by rinsing
with 5 ml elution buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1
mM MgCl2, 5 mM EDTA and 0.15% Triton X-100) followed by 5 ml
of loading buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1 mM
MgCl2, 25 mM CaCl2 and 0.15% Triton X-100). 300 µl of cell lysate
was loaded with an additional 700 µl of loading buffer and this
fraction was collected as fraction 1. The column was rinsed with 4×
1 ml of loading buffer and washes (fractions 2-5) were collected. The
column was then eluted with 5× 0.5 ml elution buffer (fractions 6-10).
All fractions were precipitated by the addition of 20 µg of BSA and
0.5 volumes of 30% TCA. The samples were placed on ice for 10
A
1000
ss
cys
FN CRD1
2000
CRD2
CRD3
3000
CRD4
CRD5
CRD6
4000
CRD7
5000
CRD8 TM cyto
clone 23.2
10
20
30
40
50
|
|
|
|
|
MGPGRPAPAPWPRHLLRCVLLLGCLHLGRPGAPGDAALPEPNIFLIFSHGLQGCLE
*SPGDAALPEPNXFLIFSHGLQG*LE
B
clone λ10.2
N-protein sequence
5 kb
exon 2
32 kb
exon 30
114 kb
Fig. 1. Structure and expression of the human Endo180 receptor. (A) Domain structure of the human Endo180 receptor; ss, putative signal
sequence; cys, cysteine-rich domain; FNII, fibronectin type II repeat; CRD, C-type carbohydrate recognition domain; TM, transmembrane
domain; cyto, cytoplasmic domain. Indicated are (a) the sites for the 29 introns (upper lines) where exon 2 encodes the cysteine-rich domain
and exon 30 the transmembrane and cytoplasmic domains and 3′ untranslated region, (b) position of clone λ23.2 isolated by expression cloning
using an anti-Endo180 polyclonal antibody and (c) a comparison of the clone λ10.2 sequence (predicted signal sequence underlined) with the
N-terminal peptide sequence obtained from purified Endo180 protein (* represents amino acids that could not be assigned, X represent amino
acid 43 which was assigned as Val or Ile). (B) Genomic structure of human Endo180. Exon 2 to exon 30 are indicated as black boxes. The
intron-exon boundaries were assigned by comparison with a 174 kb human genomic sequence (accession number AC005821). (C) MDCK cells
transfected with vector alone (−) or pcDNA3 containing the λ10.2 insert (+) were lysed and blotted with A5/158 anti-Endo180 mAb. Blots were
developed using the ECL reagent for 30 seconds. Molecular size markers are in kDa.
1024 H. Sheikh and others
C
40
Endo 180
30
20
10
0
10
0
10
1
10
2
10
3
10
4
40
CD14
30
Relative Cell Number
20
10
0
10
0
10
1
10
2
10
3
10
4
40
CD11c
30
20
10
0
10
0
10
1
10
2
10
3
10
4
40
HLA-DR
30
Fig. 2. Expression of Endo180 in endothelial cells and macrophages.
20
(A) 10 µg of cultured cell lysates were immunoblotted using mAb
A5/158. Blots were developed using the ECL reagent and exposed to
10
X-ray film for 20 seconds. Molecular size markers are in kDa. Lane
1, RPM-MC (human melanoma cell line); 2, DMEC (human dermal
0
1
0
2
3
4
microvascular endothelial cells); 3, HUVEC (human umbilical vein
10
10
10
10
10
endothelial cells); 4, HMEC-1 (human dermal microvascular
Mean Fluorescence Intensity (log)
endothelial cell line); 5, HepG2 (human hepatoma cell line); 6,
MRC-5 (human fibroblasts). (B) The cell surface expression of Endo180 on resting endothelial cells was assessed by flow cytometry using
mAb A5/158. The figure shows expression on HUVEC and HMEC-1 with background fluorescence (isotype matched irrelevant mAb; dotted
line) and specific antigen expression (solid line). (C) The cell surface expression of Endo180 on human monocyte-derived macrophages was
assessed by flow cytometry using anti-Endo180 mAb A5/158 followed by FITC-labelled anti-mouse Ig. In parallel cells were stained for
expression of three macrophage maturation markers, CD14, CD11c and HLA-DR. Background fluorescence (irrelevant isotype matched mAb)
is indicated by the dotted line.
Analysis of the human Endo180 cDNA sequence reveals a
number of striking features:
(a) The cDNA clone encodes for a protein with a large
extracellular domain containing an N-terminal signal
sequence followed by a cysteine-rich domain, a fibronectin
type II domain and 8 C-type carbohydrate recognition
domains (CRDs). The single transmembrane domain is
followed by a short 43 amino acid cytoplasmic domain (Fig.
1A). This arrangement is unusual due to the presence of
multiple CRDs within a single polypeptide backbone and has
only been found in three other related proteins; the
macrophage mannose receptor (Ezekowitz et al., 1990; Taylor
et al., 1990; Harris et al., 1992), the phospholipase A2
receptor (Higashino et al., 1994; Ancian et al., 1995) and the
DEC-205/MR6 receptor (Jiang et al., 1995; McKay et al.,
1998).
(b) Sequence analysis indicates that Endo180 is the probable
human orthologue of the recently described mouse cDNA
clone identified in a screen for novel proteins containing Ctype lectin domains (Wu et al., 1996; accession number
MMU56734). Overall, the human Endo180 has 87%
nucleotide identity with its mouse counterpart and greater than
Endo180, a novel endocytic lectin receptor 1025
Fig. 3. In vivo expression of Endo180. Cryosections of human placenta were double stained with biotinylated UEA and anti-Endo180 mAb
E1/183 followed by Cy3-conjugated anti-mouse Ig and FITC-avidin and images were collected separately to show (a) UEA staining and (b)
Endo180 staining. (c) Merged image where arrows indicate UEA-positive vessels expressing Endo180 and arrowheads indicate UEA-positive
vessels with negligible Endo180 expression. Cryosections of human skin were stained with anti-Endo180 mAb E1/183 followed by Cy3conjugated anti-mouse Ig, and then biotinylated anti-CD14 mAb followed by FITC-avidin and images were collected separately to show (d)
CD14 staining and (e) Endo180 staining. (f) Merged image where arrows indicated Endo180-positive/CD14-positive cells. Bar, 50 µm.
90% identity in the fibronectin type II repeat, CRD1, 2, 3 and
6 and the transmembrane domain.
(c) ESTs derived from the ENDO180 gene map to the
D17S791-D17S794 interval on human chromosome 17q
(Deloukas et al., 1998). Database searching identified a
174,428 bp genomic sequence from human chromosome 17
(accession number AC005821) which contains 29 exons and
28 introns of Endo180 covering a 29 kb stretch (Fig. 1A and
1B). From the similarity of this intron-exon boundary with the
30 exons of the human and mouse macrophage mannose
receptors (Kim et al., 1992; Harris et al., 1994), it is predicted
that the 29 exons in the AC005821 sequence represent exons
2-30 of human Endo180 and that exon 1 is separated from exon
2 by a large intron of greater than 32 kb.
(d) The sequence of human Endo180 shows a high degree
of similarity to a cDNA clone recently isolated in a strategy to
clone large cDNAs from human brain (Ishikawa et al., 1998;
accession number AB014609). Comparison of the λ10.2 and
AB014609 sequences show that the former has 8 bp and 72 bp
5′ and 3′ extensions, respectively, and the latter contains an
open reading frame disrupted by a 5 bp deletion at position
3341-3345. The presence of these 5 bp in clone λ10.2 suggests
that the deletion in the AB014609 cDNA results from a cloning
artefact rather than representing an alternative splicing event.
Additionally, clone λ10.2 shows two base changes and these
changes are also found in the AC005821 genomic chromosome
17 sequence. The first is a G to A change at position 243 which
would result in an amino acid change from Val to Ile at amino
acid 43. Interestingly, when this region was subjected to protein
sequence analysis, both Val and Ile were assigned at this
position (Fig. 1A) suggesting that this base change represents
a natural amino acid polymorphism. The second (position
2,717) is an A to G change which does not alter the amino acid
sequence and may also be a polymorphism.
Together the cDNA and genomic sequence data strongly
suggest that human Endo180 and the novel murine C-type
lectin identified by Wu et al. (1996) represent the human and
mouse counterparts of a fourth family member related to the
macrophage mannose receptor, the phospholipase A2 receptor
and the DEC-205/MR6 receptor (Taylor, 1997; Stahl and
Ezekowitz, 1998).
Endo180 is expressed by endothelial cells and
macrophages in vivo
We have previously demonstrated that Endo180 has a restricted
tissue and cell type distribution with high levels found in
fibroblastic cells both in vivo and in vitro and little or no
expression on epithelial cells or haematopoietic cell lines
(Isacke et al., 1990). Subsequently, by northern blot analysis,
Wu et al. (1996) demonstrated a similar expression pattern of
murine Endo180 transcripts with high levels found in lung and
kidney but little or no expression in brain, thymus or adult liver.
In order to gain clues as to the functional role of Endo180 it
was important to further characterize its distribution. As
1026 H. Sheikh and others
described here, Endo180 is structurally and functionally (see
preparations. In all of the distribution studies described here,
below) related to the macrophage mannose receptor. These two
identical results were obtained with the different anti-Endo180
receptors clearly have a distinct distribution. For example,
mAbs (data not shown).
unlike Endo180, the macrophage mannose receptor is
Endo180 is an endocytic receptor
expressed both in the liver and thymus (Magnusson and Berg,
1993; Fiete et al., 1997; Linehan et al., 1999) but it is not
The ability of the macrophage mannose receptor, the
known whether there is any overlap in expression in other cell
phospholipase A2 receptor and the DEC-205/MR6 receptor to
types such as macrophages. In addition, by in situ
be endocytosed from the cell surface suggests that this family
hybridization, Wu et al. (1996) detected the presence of
of receptors have a role in the internalization of extracellular
Endo180 transcripts at highly endothelialized sites such as
components (Kruskal et al., 1992; Jiang et al., 1995; Zvaritch
those in the choroid plexus and kidney glomerulai. However,
et al., 1996). Although cell surface Endo180 expression was
the limitation in resolution of this type of analysis precludes a
detected on endothelial cells and macrophages (Figs 2 and 4)
definitive localization of Endo180 protein to endothelial cells.
it was suspected that this represented only a proportion of the
To address these particular issues we have examined the
total Endo180 as flow cytometric analysis of permeabilized
expression of Endo180 on endothelial cells and macrophages
cells demonstrated that the majority of Endo180 is intracellular
both in vivo and in vitro using anti-Endo180 mAbs.
(data not shown). To investigate the cell surface and
Western blot analysis of cultured cells revealed a high level
intracellular localization of Endo180, microvascular
of Endo180 expression in fibroblasts, large vessel endothelial
endothelial cells and fibroblasts were examined by confocal
cells (HUVEC) and both primary (DMEC) and immortalized
microscopy (Fig. 4). Consistent with the flow cytometry data,
permeabilized endothelial cells exhibited much stronger
(HMEC-1) small vessel endothelial cells. By contrast no
Endo180 staining with the protein being localized throughout
expression was detected in a hepatoma cell line (HepG2) in
the cytoplasm to intracellular vesicles (Fig. 4D). In the nonagreement with the lack of detection of Endo180 protein
permeabilized cells, Endo180 is localized on the cell surface
(Isacke et al., 1990) and transcripts (Wu et al., 1996) in the
in a fine punctate distribution (Fig. 4C). This distribution
adult liver (Fig. 2A). Flow cytometric analysis demonstrated
that Endo180 is expressed at the plasma
membrane of both large and microvascular
endothelial cells with a unimodal distribution
(Fig. 2B). To determine whether this
expression of Endo180 by cultured
endothelial cells reflected expression in vivo,
the distribution of Endo180 was examined in
sections of human term placenta
counterstained with an endothelial specific
lectin, UEA (Fig. 3a-c). In these sections it
was found that Endo180 was expressed on a
subset of microvascular vessels. Strong colocalization with UEA was observed on the
larger microvessels but Endo180 was not
detected on the smaller microvessels. In
addition, dispersed Endo180-positive/UEAnegative cells were identified in placenta
mesenchyme
which
morphologically
resembled Hofbauer cells, the macrophagelike placental scavenger cells.
The expression of Endo180 by
macrophages in vivo was confirmed by
staining human skin sections with mAbs
directed against Endo180 mAb and CD14
(Fig. 3d-f). Dispersed throughout the dermis,
double labelled cells are clearly visible, by
contrast the epidermis is Endo180-negative.
To determine whether the expression was
restricted to dermal macrophages, human
peripheral
blood
monocytes
were
differentiated in vitro and their maturation to
macrophages confirmed by their expression
of CD14, CD11c and HLA-DR (Fig. 2C). On
Fig. 4. Subcellular distribution of Endo180. Flow2000 fibroblasts (a and b) or HMEC-1
these monocyte-derived macrophages, a
cells (c and d) were cultured overnight on gelatin-coated coverslips, stained with antiunimodal cell surface distribution of
Endo180 mAb E1/183 followed by rhodamine-conjugated anti-mouse Ig and viewed by
Endo180 was detected and this expression
confocal microscopy. (a and c) Fixed cells, (b and d) cells fixed and then permeablised
with 0.2% saponin. Bar, 25 µm.
was consistent in different macrophage
Endo180, a novel endocytic lectin receptor 1027
Fig. 5. Location of putative endocytosis
motifs within the Endo180 cytoplasmic
domain. The sequences of the human (h-)
and mouse (m-) Endo180, mouse
macrophage mannose receptor (MMR),
phospholipase A2 receptor (PLA2) and
DEC-205/MR6 receptor (DEC-205)
cytoplasmic domains are shown. Amino
acid numbering refers to human Endo180.
Putative endocytosis motifs are shown
below and the conserved tyrosine residue is
in bold.
h-Endo180
1440
1450
1460
1470
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RRRQNIERGAFEGARYSRSSSS-PTEATEKNILVSDMEMNEQQE
m-Endo180
RRRQSAERGSFEFARYSRSSHSGPAEATEKNILVSDMEMNEQQE
h-MMR
m-MMR
h-PLA2
m-PLA2
h-DEC-205
m-DEC-205
RRVHLPQEGAFENTLYFNSQSSFPGTS-DMKDLVGNIEQNEHSVI
HALHIPQEATFENTLYFNS-NLSPGTS-DTKDLMGNIEQNEHAII
KHNGGFFRRLAGFRNPYYPATNFSTVY--LEENILISDLEKSDQ
KQKSDIFQRLTGSRGSYYPTLNFSTAH--LEENILISDLEKNTNDEEVRDAPATESKRGHKGRPICISP
RRLAGFRNPYYP-ATNFSTVY-LEENILISDLEKSDQ
QRSHI-RWTGFSSVRYEHGTN-------EDEVMLPSFHD
putative
endocytosis motifs
pattern is identical to that observed in human fibroblasts (Fig.
4C,D) where it has previously been demonstrated that Endo180
co-localizes with clathrin to plasma membrane coated pits and
with the transferrin receptor to intracellular endosomes (Isacke
et al., 1990).
The ability of receptors to be recruited into clathrin coated
pits and subsequently endocytosed, requires that their
cytoplasmic domains can interact with intracellular adaptin
complexes. An examination of the Endo180 cytoplasmic
domain revealed that although this domain is highly conserved
between human and mouse Endo180, there is only 24%, 30%
and 12% identity to the cytoplasmic sequences of the
macrophage mannose receptor, phospholipase A2 receptor and
DEC-205/MR6 receptor, respectively (Fig. 5). However, two
putative endocytosis motifs are present in the Endo180
sequence. The first of these is based on Tyr1452. Tyrosinebased endocytosis motifs have been extensively characterized
and optimally have the sequence FxNxxY forming a tight turn
structure (Trowbridge et al., 1993). Although Tyr1452 is
conserved between family members, human Endo180 has a
glycine rather than asparagine three residues upstream and
murine Endo180 has a phenylalanine residue in the equivalent
position. The second putative endocytosis motif centres on the
di-hydrophobic Leu1468/Val1469 residues which have a
glutamic acid four amino acids upstream. It is known from
studies on other receptors that di-hydrophobic motifs can
mediate intracellular trafficking (Sandoval and Bakke, 1994)
and the presence of an upstream acidic residue has been shown
to be important for targeting to endocytic vesicles (Pond et al.,
1995). Comparison of the cytoplasmic domains between
family members reveals a second interesting issue. Both of the
putative endocytosis motifs in Endo180 are upstream from an
adjacent serine residue, a feature that is not found in the
macrophage mannose receptor. [32P]orthophosphate labelling
showed a low level of constitutive Endo180 phosphorylation
but interestingly phosphorylation was dramatically and rapidly
enhanced in response to activation of protein kinase C.
Moreover, phosphoamino acid analysis demonstrated that this
protein kinase C mediated phosphorylation is restricted to
serine residues (Fig. 6).
One issue that is not addressed by these studies is whether
endocytosis of Endo180 from the plasma membrane accounts
for all of the intracellular receptor detected by confocal
microscopy. It is known that other lectins such as the
mannose-binding ERGIC-53 protein function intracellularly
to sort immature or incorrectly processed glycoproteins in the
biosynthetic pathway (Arar et al., 1995). To examine the posttranslational processing, Endo180 was immunoprecipitated
FxNxxY
E---LV
from [35S]methionine pulse-chased cells and then treated with
different endoglycosidases. After 15 minutes of
[35S]methionine labelling a single 170 kDa immature form is
detected, which matures into the 180 kDa form with
increasing chase times (Fig. 7). Treatment of this immature
170 kDa form with endo H or endo F results in the removal
of approximately 15 kDa and 10 kDa, respectively, of
Fig. 6. Phosphorylation and phosphoamino acid analysis of
Endo180. (A) 32P-labelled AG1523 human fibroblasts were treated
for 10 minutes without (−) or with (+) TPA. Lysates were
immunoprecipitated with anti-Endo180 mAb E1/183, resolved on a
10% polyacrylamide gel and exposed to film for 16 hours. Molecular
size markers are in kDa. (B) Labelled Endo180 protein was eluted
from the gel shown in A and subjected to two-dimensional
phosphoamino acid analysis. Thin layer plates were exposed to X-ray
film for 6 days with intensifying screens. x marks the point of sample
application.
1028 H. Sheikh and others
Fig. 7. Post-translational processing of Endo180. Flow2000 human fibroblasts were labelled with 300 µCi/ml [35S]methionine in methioninefree DME for 15 minutes and then chased for 0, 1, 2, 4 or 24 hours in complete medium. Lysates were immunoprecipitated with mAb E1/183
and then treated with glycosidases as described in Materials and Methods. Samples were resolved on 7.5% gels and exposed to film for 6 days.
(A) Endo H. Lysates from cells chased for 0, 1, 2 and 24 hours treated with (+) or without (−) 2.5 mU endo H for 3 hours at 37°C. (B) Endo F.
Lysates from cells chased for 0, 1, 2, and 24 hours treated with (+) or without (−) 0.5 U endo F for 3 hour at 37°C. (C) Neuraminidase. Lysates
from cells chased for 0 or 4 hours were treated with (+) or without (−) 0.125 U neuraminidase for 4 hours at 37°C.
carbohydrate, but no change in mobility was detected after
Endo180 is a carbohydrate-binding receptor
neuraminidase treatment. The mature 180 kDa form of
As described above, a key characteristic of the 4 macrophage
Endo180 immunoprecipitated after a 4 or 24 hour chase is
mannose receptor-related proteins is the presence of multiple
resistant to endo H treatment,
but approximately 10 kDa of
carbohydrate is removed by
conservation
x
o Ø
C
Ø Ø
E xØ
r-MBP(a)
SGKKFFVTN-----HERMPFSKVKALCSELRGRVAIPRNAEENKAIQEVAKT
endo F and approximately 5
h-MMR(4)
RTSLCFKLYAKGK-HEKKTWFESRDFCRALGGDLASINNKEEQQTIWRLITA
kDa by neuraminidase. Endo F
h-Endo180(2)
PSWQPFQGHCYRLQAEKRSWQESKKACLRGGGDLVSIHSMAELEFITKQIKQ
hydrolyses the glycosidic bond
m-Endo180(2)
PSWQPFQGHCYRLQAEKRSWQESKRACLRGGGDLLSIHSMAELEFITKQIKQ
of both complex and high
h-Endo180(1)
DKDQLTDSCYQFNFQSTLSWREAWASCEQQGADLLSITEIHEQTYINGLLTG
mannose oligosaccharides Nm-Endo180(1)
DKDQLTDSCYQFNFQSTLSWREAWASCEQQGADLLSITEIHEQTYINGLLTG
linked to asparagine residues,
while endo H only hydrolyses
high mannose structures.
calcium sites
1
1
2 21
21
22
conservation
oØGØ *
* o x G x x W
ZPBB
E*CØ x
G WND C
x C
These data demonstrate that
r-MBP(a)
------SAFLGITDE-VTEGQFMYVTGGRLTYSNWKKDEPNDH-GSGEDCVTI-VDN-GLWNDISCQASHTAVC
maturation of Endo180 is
h-MMR(4)
SGSTHKLFWLGLTYGSPSEG-FTWSDGSPVSYENWAYGEPNNY-QNVEYCGELKGDPTMSWNDINCEHLNNWIC
accompanied by modification
h-Endo180(2)
---EVEELWIGLND-LKLQMNFEWSDGSLVSFTHWHPFEPNNFRDSLEDCVTIWGPE-GRWNDSPCNQSLPSIC
of N-linked carbohydrates to
m-Endo180(2)
---EVEELWIGLND-LKLQMNFEWSDGSLVSFTHWHPFEPNNFRDSLEDCVTIWGPE-GRWNDSPCNQSLPSIC
complex structures and the
h-Endo180(1)
YSSTLWIGLND-LDTSGGWQWSDNSPLKYLNWESDQPDNP--SEENCGVIRTESSGGWQNRDCSIALPYVC
addition of neuraminidasem-Endo180(1)
YSSTLWIGLND-LDTSGGWQWSDNSPLKYLNWESDQPDNP--GEENCGVIRTESSGGWQNHDCSIALPYVC
sensitive terminal sialic acid
residues. As all of the Fig. 8. Comparison of CRD sequences. Comparison of CRD protein sequences between rat serum
Endo180 is fully processed, mannose binding protein A [r-MBP (a)], human macrophage mannose receptor CRD4 [h-MMR(4)],
human and mouse Endo180 CRD2 [h-Endo180(2) and m-Endo180(2)] and human and mouse Endo180
these studies indicate that CRD1 [h-Endo180(1) and m-Endo180(1)]. Symbols above sequence represent conserved residues found
intracellular Endo180 does in functional C-type lectin CRDs (Weis et al., 1991; Mullin et al., 1994;). x = aliphatic or aromatic
not
represent
immature (FWYHLIV), Ø = aliphatic (LIV), o = aromatic (FWYH), * = side chain with carbonyl oxygen (DNEQ),
glycoprotein that has been Z = E or Q, B = D or N. Invariant amino acids are shown in single-letter code. Underlined symbols
retained in the biosynthetic represent residues which are not conserved in Endo180 CRD2. Calcium sites indicates Ca2+ site (1 or 2)
to which a side chain contributes oxygen ligands.
pathway.
Endo180, a novel endocytic lectin receptor 1029
CRDs within their extracellular domains. However, it is
important to note that these domains are defined on the basis
of size and the presence of certain conserved amino acids
resulting in an overall structural homology and that the
presence of a CRD in a protein does not necessarily confer
carbohydrate binding capacity. Moreover, despite the presence
of 8 CRDs within the macrophage mannose receptor, detailed
analysis has revealed that the binding of glycoconjugates
terminating in mannose, N-acetylglucosamine or fucose is
primarily mediated by CRD4 (Weis et al., 1998). Within
CRD4, carbohydrate binding requires four cysteine residues to
create nested disulphide bonds plus other key residues for the
formation of two Ca2+ binding sites and the packing and
formation of the hydrophobic cores (Weis et al., 1991; Mullin
et al., 1994). An examination of the Endo180 sequence reveals
that the Endo180 CRD4 has retained the four cysteine residues
at equivalent spacing but key residues required for Ca2+ and
sugar co-ordination are not conserved. However, as shown in
Fig. 8, CRD2 in both mouse and human Endo180 has a strong
consensus binding site including the critical Ca2+/sugar
binding site residues, correctly spaced cysteine residues and
26/28 of the remaining conserved amino acids found in
functional CRDs. This sequence analysis suggests that if
Endo180 has lectin activity, ligand binding will be primarily
mediated by this domain. Interestingly, many of these residues
are also conserved in Endo180 CRD1 although there is a loss
of conservation in Ca2+ site 2.
To determine whether Endo180 exhibits Ca2+-dependent
carbohydrate binding, cell lysates were passed over mannose,
mannan, fucose, galactose or N-acetylglucosamine columns
and wash fractions and EDTA eluted fractions were assayed
for Endo180 content. By this type of analysis, Endo180
extracted from Flow2000 fibroblasts did not bind to mannose,
fucose (Fig. 9A,B), galactose or mannan (data not shown). By
contrast, Endo180 was retained on N-acetylglucosamine
columns and eluted in the presence of EDTA (Fig. 9C). This
was not a cell line specific phenomenon as identical binding
was observed with Endo180 extracted from AG1523
fibroblasts (data not shown). To determine whether this
carbohydrate binding was restricted to fibroblast Endo180,
experiments were repeated with lysates from the microvascular
endothelial cell line, HMEC-1. As shown in Fig. 9D,
endothelial Endo180 did not bind mannose but was retained
on N-acetylglucosamine columns and eluted after chelation of
the Ca2+ ions (Fig. 9E). The same pattern of binding was
also observed with Endo180 extracted from the large vessel
endothelial cells, HUVEC (data not shown). These
experiments directly demonstrate that Endo180 has lectin
activity and that its ligand specificity is distinct from that of
the macrophage mannose receptor.
DISCUSSION
In this manuscript we describe the cloning and characterization
of human Endo180. Sequence analysis demonstrates that this
receptor and the novel murine C-type lectin identified by Wu
et al. (1996) represent the human and mouse counterparts of a
fourth family member related to the macrophage mannose
receptor, the phospholipase A2 receptor and the DEC-205/MR6
receptor (Stahl and Ezekowitz, 1998).
Fig. 9. Endo180 binds immobilized N-acetylglucosamine. Flow2000
cell lysates were loaded onto 1 ml mannose-agarose (A), fucoseagarose (B) or N-acetylglucosamine-agarose (C) columns. HMEC-1
lysates were loaded onto 1 ml mannose-agarose (D) or Nacetylglucosamine-agarose (E) columns. 5 ml of loading buffer was
added and 1 ml fractions collected (fraction 1-5). 2.5 ml of elution
buffer was then added and 0.5 ml fractions collected (fractions 6-10).
All fractions were TCA precipitated, resuspended in 40 µl of sample
buffer and analysed by western blotting using mAb A5/158. Blots
were developed using the ECL reagent and exposed to X-ray film for
15 seconds. Molecular size markers are in kDa.
1030 H. Sheikh and others
Despite the overall structural similarity between these family
members, there are important functional distinctions, most
notably in their carbohydrate binding abilities. By mutational
analysis it has been shown that CRD5 is the critical domain
involved in the binding of phospholipase A2 to its receptor with
CRDs 3, 4 and 6 being required but of less importance
(Higashino et al., 1994; Nicolas et al., 1995). However,
phospholipase A2 is a non-glycosylated protein and this
binding is via a protein:protein interaction and is Ca2+
independent. Similarly, the DEC-205/MR6 receptor has been
suggested to play a role in antigen presentation of internalized
glycoproteins in dendritic cells and thymic epithelium but like
the phospholipase A2 receptor, none of its CRDs has conserved
the key amino acids involved in carbohydrate and Ca2+
binding. Consequently it is unlikely that the CRDs in either of
these two receptors has functional lectin activity. By contrast,
these key amino acids are conserved in CRD2 and CRD4 of
Endo180 and the macrophage mannose receptor, respectively
(Fig. 8), and both these receptors have been demonstrated to
mediate Ca2+-dependent carbohydrate binding (Fig. 9; Weis et
al., 1998). However, Endo180 and the macrophage mannose
receptor display two important differences in their sugar
binding. First, they have distinct ligand specificities. The
macrophage mannose receptor binds mannose, fucose and Nacetylglucosamine (Stahl and Ezekowitz, 1998; Weis et al.,
1998) whereas Endo180 does not bind mannose or fucose but
does bind N-acetylglucosamine (Fig. 9). Second, although
Ca2+ site 2 is conserved within the macrophage mannose
receptor CRD4, Ca2+ site 1 is not and instead CRD4 has a
distinct mechanism for binding the second cation (Mullin et
al., 1994, 1997). CRD2 within Endo180 has conserved the
Ca2+ site 1 indicating that it is more similar in structure to other
mannose binding proteins (Weis et al., 1991) than the
macrophage mannose receptor. Interestingly, the N-terminal
cysteine-rich domain of the macrophage mannose receptor can
mediate Ca2+-independent binding of oligosaccharides with
terminal SO4-4-GalNAcβ1,4GlcNAcβ1,2Manα structures
(Fiete et al., 1997, 1998). Moreover, Martínez-Pomares et al.
(1996, 1999) have demonstrated that this domain can bind cell
surface ligands such as sialoadhesin and CD45 independently
of the CRDs. Although the cysteine-rich region is the least well
conserved of all the different domains within the receptor
family, it will be important to determine if this region in
Endo180 can mediate Ca2+-independent carbohydrate binding.
Despite the differences in mechanisms of ligand binding, a
feature in common to the macrophage mannose receptor, the
phospholipase A2 receptor and the DEC-205/MR6 receptor is
their ability to function as endocytic receptors (Kruskal et al.,
1992; Jiang et al., 1995; Zvaritch et al., 1996). In previous
biochemical and morphological studies, we have demonstrated
that in fibroblasts Endo180 is localized to clathrin coated pits
on the plasma membrane from where it is rapidly and
constitutively internalized into endosomal compartments and
then recycled back to the cell surface (Isacke et al., 1990). In
this manuscript we have extended these morphological studies
to demonstrate that the endocytic capacity of Endo180 is not a
cell type specific phenomenon as an identical subcellular
distribution is found in endothelial cells. In addition, we have
undertaken biochemical analysis to further characterize the
biosynthesis and trafficking of Endo180. The majority of
Endo180 is found in its mature 180 kDa form within 2 hours
of synthesis and this maturation is accompanied by a resistance
to endo H digestion and a sensitivity to neuraminidase
digestion (Fig. 7). The formation of endo H resistant complex
oligosaccharides and the addition of sialic acid takes place in
the medial and trans-Golgi stacks. Together with the
morphological data (Fig. 4) and results obtained using Fab′
fragments to monitor receptor trafficking (Isacke et al., 1990),
this indicates that all of the intracellular Endo180 protein has
been fully post-translationally modified and subsequently
internalized from the plasma membrane.
The endocytosis of proteins via clathrin-coated pits requires
that they have a ‘positive’ signal in their cytoplasmic domains
for association with intracellular adaptin complexes
(Trowbridge et al., 1993; Kirchhausen et al., 1997; Marsh and
McMahon, 1999). As with the other family members,
examination of the Endo180 cytoplasmic domain sequence
reveals the presence of two putative endocytosis motifs (Fig.
5). However, a distinct feature in Endo180 is the presence of a
C-terminal serine residue adjacent to both putative endocytosis
motifs. We demonstrate here that phosphorylation of Endo180
is dramatically and rapidly upregulated after treatment of cells
with phorbol esters and that this phosphorylation occurs on
serine residues. Evidence that this phosphorylation is directly
mediated by protein kinase C comes from studies showing that
immunopurified Endo180 can be phosphorylated by this kinase
in vitro (Isacke et al., 1990). Receptor phosphorylation can be
an important modulator of intracellular trafficking (Trowbridge
et al., 1993). For example, phosphorylation is required for
efficient endocytosis of the polymeric immunoglobulin
receptor (pIgR; Casanova et al., 1990) and the B2 adrenergic
receptor (Pizard et al., 1999). In the case of CD4, protein kinase
C mediated phosphorylation results in an increased rate of
endocytosis from the cell surface and the diversion of CD4
from the recycling pathway to a pre-lysosomal compartment
(Pelchen-Matthews et al., 1993).
An examination of the cell type distribution of Endo180 in
vivo and in vitro has revealed high levels of expression on
fibroblasts, microvascular endothelial cells and macrophages.
The data presented here strongly suggests that in these cells
Endo180 will function to internalized glycosylated ligands with
terminal N-acetylglucosamine residues via clathrin-coated pit
mediated endocytosis. Like mannose, N-acetylglucosamine is
not a common terminal sugar on mammalian oligosaccharides
(Drickamer and Taylor, 1998) suggesting that Endo180 and the
macrophage mannose receptor have complementary roles in
glycoconjugate clearance. An unusual aspect of the macrophage
mannose receptor is its ability to mediate both clathrin-coated
pit endocytosis of glycoconjugates and phagocytosis of
microorganisms (Ezekowitz et al., 1990; Kruskal et al., 1992).
Despite these internalization events utilizing distinct cellular
machinery (Aderem and Underhill, 1999; Marsh and
McMahon, 1999) both are dependent on the macrophage
mannose receptor cytoplasmic domain and are similarly
impaired by mutation of the conserved tyrosine residue
(Kruskal et al., 1992). As demonstrated here, Endo180 is
expressed on macrophages and therefore it also could
potentially function in host defence in the recognition and
clearance of non-opsonized microorganisms. If this is the case,
then given the differences in ligand specificity between the two
receptors it would be expected that they would mediate the
uptake of different pathogens. Finally, if Endo180 can mediate
Endo180, a novel endocytic lectin receptor 1031
phagocytosis it is possible that it may additionally be an
important receptor in fibroblasts and endothelial cells which are
known to function as non-professional phagocytes in vivo
(Rabinovitch, 1995) and utilise lectin receptors to mediate the
internalization of apoptotic cells (Hall et al., 1994; Oka et al.,
1998).
We thank Dr Peter van der Geer (UCSD, California) for the
Endo180 glycosylation analysis, Dr Kurt Drickamer (Oxford) for his
help and advice with the sugar binding assays, Dr Ian Dransfield
(Edinburgh) for his analysis of the human monocyte-derived
macrophages, Dr Jim Boulter (UCLA, California) for generously
providing the human stromal λZAP library, Dr Peter Clark (Imperial
School of Medicine) for his help and advice with the
immunohistochemistry and James Legg (Imperial College) for
invaluable assistance with the image analysis. Dr Tony Hunter and Dr
Ian Trowbridge (Salk Institute, California), Professor Colin Hopkins
(University College London) and Dr Stephen Neame (Eisai London
Research) provided essential support and contributions to the early
stages of this project. EST clones were provided by the UK HGMP
Resource Centre. This research was supported by the Medical
Research Council, The Wellcome Trust and the Cancer Research
Campaign.
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