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J. Biol. Chem., Vol. 277, Issue 35, 32320-32331, August 30, 2002
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From the Breakthrough Toby Robins Breast Cancer Research Centre,
Institute of Cancer Research, Mary-Jean Mitchell Green Building,
Chester Beatty Laboratories, 237 Fulham Rd.,
London SW3 6JB, United Kingdom
Received for publication, April 15, 2002, and in revised form, June 6, 2002
Endo180/urokinase plasminogen activator
receptor-associated protein together with the mannose receptor,
the phospholipase A2 receptor, and DEC-205/MR6-gp200
comprise the four members of the mannose receptor family. These
receptors have a unique structural composition due to the presence of
multiple C-type lectin-like domains within a single polypeptide
backbone. In addition, they are all constitutively internalized from
the plasma membrane via clathrin-mediated endocytosis and recycled back
to the cell surface. Endo180 is a multifunctional receptor displaying
Ca2+-dependent lectin activity, collagen
binding, and association with the urokinase plasminogen activator
receptor, and it has a proposed role in extracellular matrix
degradation and remodeling. Within their short cytoplasmic domains, all
four receptors contain both a conserved tyrosine-based and
dihydrophobic-based putative endocytosis motif. Unexpectedly, Endo180
was found to be distinct within the family in that the tyrosine-based
motif is not required for efficient delivery to and recycling from
early endosomes. By contrast, receptor internalization is completely
dependent on the dihydrophobic motif and modulated by a conserved
upstream acidic residue. Furthermore, unlike the mannose receptor,
Endo180 does not function as a phagocytic receptor in
vitro. These findings demonstrate that despite an overall
structural similarity, members of this receptor family employ distinct
trafficking mechanisms that may reflect important differences in their
physiological functions.
Endo180 was first identified as a recycling endocytic receptor
expressed in fibroblastic cells (1). Based on peptide sequences obtained from purified protein, a full-length human cDNA clone was
isolated, and Endo180 was demonstrated to be the fourth and last member
of the mannose receptor family (2-4). In addition to Endo180, the
mannose receptor family comprises the mannose receptor, the M-type
phospholipase A2 receptor
(PLA2R),1 and
DEC-205/MR6-gp200. This family grouping is based on an overall structural conservation with the four receptors containing a large extracellular domain comprising an N-terminal signal sequence followed
by a cysteine-rich domain, a fibronectin type II domain, and 8 or 10 C-type lectin-like domains (CTLDs). The single pass transmembrane
domains are followed by a short cytoplasmic domain. As a family, these
receptors have two striking features. First, although they belong to
the large C-type lectin superfamily (information available on the World
Wide Web at ctld.glycob.ox.ac.uk), they uniquely contain multiple CTLDs
within a single polypeptide backbone (4-9). Second, they share the
ability to be recycled between the plasma membrane and intracellular
compartments of the cell (1, 8, 10, 11).
As a consequence of their structural and recycling characteristics, it
was initially assumed that these receptors would commonly function to
internalize glycosylated ligands for intracellular delivery. However,
further characterization has revealed the following. (a)
Although the receptors contain multiple CTLDS, only CTLDs 4 and 5 of
the mannose receptor and CTLDs 1 and 2 of Endo180 contain the conserved
amino acids found in functional C-type lectins, and accordingly only
these two receptors have been demonstrated to exhibit
Ca2+-dependent sugar binding (2, 12).
(b) At least some of the CTLDs in this receptor family have
evolved to mediate protein/protein interactions rather than
protein/sugar interactions. This is exemplified in the
PLA2R, which binds nonglycosylated secretory phospholipase A2 in a Ca2+-independent manner via CTLD5 (13).
(c) Domains in addition to the CTLDs can mediate ligand
interactions. The cysteine-rich domain of the mannose receptor has been
demonstrated to bind sulfated sugars, and structural studies and
sequence analysis have suggested that this feature is not shared with
other family members (4, 14). The fibronectin type II domain of the
PLA2R has been demonstrated to bind collagen, and similarly
structural and sequence analysis predicts that this feature will be
shared with other family members with the possible exception of
DEC-205. In the case of Endo180, collagen binding both in
vitro and in vivo has been demonstrated (3).2 (d) In
addition to binding soluble ligands, members of this receptor family
can bind transmembrane ligands, and this can occur both in
cis and trans. For example, the mannose receptor
on lymphatic endothelia interacts with leukocyte L-selectin
(15), whereas Endo180 was identified independently as part of a
trimolecular complex with the urokinase plasminogen activator receptor
and pro-urokinase plasminogen activator, hence its alternative name, urokinase plasminogen activator receptor-associated protein, or uPARAP
(3). (e) In addition to variation in ligand binding properties, these four family members do not share a common
intracellular destination. Recently, it has been demonstrated that
whereas the mannose receptor is predominantly localized to early
endosomes, DEC-205 is targeted to late endosome/lysosomal compartments
(16). Moreover, the mannose receptor is unusual in that, in addition to
its ability to be internalized via the clathrin-mediated endocytic pathways, it can also mediate phagocytosis of nonopsonized
microorganisms or synthetic large particular ligands (17, 18).
Together, these data demonstrate that rather than representing a group
of related lectin receptors, the mannose receptor family is a group of
multidomain receptors with distinct ligand binding and trafficking properties.
In situ hybridization analysis together with
immunohistochemistry has revealed that most tissues have Endo180
expression but that this is generally restricted to stromal cells,
macrophages, and a subset of endothelial cells (1, 2, 19). In addition, high levels of expression are found in the embryo and neonate in
chondrocytes at areas of active cartilage deposition and in tissues
undergoing primary ossification and on chondrocyte (data not shown) and
osteoblast (22) cell lines. This distribution pattern together with its
C-type lectin activity (2), collagen binding ability, and interaction
with urokinase plasminogen activator receptor (3) suggests a role for
Endo180 in regulating extracellular matrix degradation and remodeling.
In support of this hypothesis is the observed up-regulation of Endo180
on angiogenic endothelial cells (23) and on the stromal fibroblasts and
myoepithelial cells in breast tumors (19), where increased expression
may be required for the dissolution of basement membranes lining the blood vessels or epithelial sheets and/or for degradation of
extracellular matrix components associated with the tumor. Finally, the
observed C-type lectin activity of Endo180 and the demonstration that
it is expressed on macrophages (2) raises the possibility that like the
mannose receptor, Endo180 could function both as an endocytic receptor
and as a phagocytic receptor. To understand the physiological role of
Endo180, we have undertaken experiments to determine the mechanism by
which Endo180 is internalized from the plasma membrane, the
intracellular destination of this receptor, and the potential that
Endo180 can mediate phagocytosis in addition to endocytosis.
Generation of Endo180 Cytoplasmic Domain Constructs--
Cloning
and generation of the pcDNA3-Endo180 construct has been described
elsewhere (2). In addition, Endo180 was inserted into the
NotI and XhoI sites of a pcDNA3 vector in
which the HindIII site had been removed
(pcDNA3 Antibodies--
Anti-human Endo180 mAbs E1/183 and A5/158 and
the rabbit anti-Endo180 polyclonal antisera have been described
previously (1, 2). Anti-human transferrin receptor mAb B3/25,
anti-mouse transferrin receptor mAb R17, and anti-human LAMP1 mAb H4A3
were obtained from Prof. C. Hopkins (Imperial College).
RPE-conjugated anti-Fc Cells--
MG-63 human osteosarcoma cells were cultured in
Dulbecco's modified Eagle's medium, 10% fetal calf serum; NIH-3T3
cells were cultured in Dulbecco's modified Eagle's medium, 10% donor
calf serum. NIH-3T3 cells were transfected with 1 µg of plasmid using LipofectAMINE (Invitrogen) and selected for 10-14 days in 0.6 mg/ml G418. Resistant populations were enriched for cells expressing Endo180 by DynaBead selection using the anti-Endo180 mAb A5/158. For
confocal microscopy, cells cultured on glass coverslips were stained for cell surface Endo180 by incubating the coverslips for 60 min on ice with 10 µg/ml mAb A5/158. Cells were then fixed for 10 min
with 3% paraformaldehyde at room temperature, permeabilized with 0.2%
saponin, and incubated for 60 min at room temperature with 2 µg/ml
Alexa 488-conjugated anti-mouse Ig. For intracellular staining, cells
were fixed with 3% paraformaldehyde and permeabilized with 0.2%
saponin prior to incubation with primary and secondary antibodies.
Nuclei were counterstained with TOPRO-3 (Molecular Probes, Inc.,
Eugene, OR), and images were collected sequentially in three channels
on a Leica TCS SP2 confocal microscope. For fluorescence-activated cell
sorting (FACS) analysis, cells were stained on ice with 10 µg/ml mAb
A5/158 followed by 2 µg/ml Alexa 488 anti-mouse Ig, washed twice,
fixed in 1% paraformaldehyde, and analyzed on a Becton Dickinson
FACScan flow cytometer.
Endocytosis Assays--
MG-63 cells or NIH-3T3 cells expressing
Endo180 were cultured for 24-48 h in 35-mm dishes. Cells were washed
three times in cold binding buffer (Dulbecco's modified Eagle's
medium, 10 mM HEPES, 2 mg/ml BSA, pH 7.4), incubated on ice
for 60 min with 100,000 cpm 125I-E1/183 in 1 ml of binding
buffer, washed extensively, and then incubated for 0-30 min at
37 °C with 2 ml of prewarmed binding buffer. At defined time points,
medium was collected, cell surface 125I-E1/183 was removed
by incubating cells with 1 ml of acid strip buffer (Dulbecco's
modified Eagle's medium, 10 mM MES, 2 mg/ml BSA, pH 2.5),
and the cell-associated radioactivity collected by detaching the cells
with trypsin. To monitor the steady state distribution of Endo180,
cells were incubated for 90 min with 100,000 cpm of
125I-E1/183 in 2 ml of binding buffer, pH 7.4. Cells were
then washed extensively, and the cell surface and intracellular
125I-E1/183 were monitored as described above.
Phagocytosis Assays--
Transfected NIH-3T3 cells or MG-63
cells were incubated for 1 h in serum-free medium and then for
4 h in serum-free medium with mAb A5/158- or BSA-coated
FITC-polystyrene beads. For FACS analysis, cells were washed
extensively in PBS, acid-stripped for 5 min on ice using pH 2.5 acid
buffer to remove cell surface-associated beads, and fixed for 10 min in
1% paraformaldehyde. Cells were then monitored by single-color FACS or
stained with RPE-anti-CD32 antibody and monitored by two-color FACS.
For confocal analysis, cells were fixed in 3% paraformaldehyde without
acid stripping, stained with 10 µg/ml anti-CD44 mAb IM7 followed by 2 µg/ml Alexa 568 anti-rat Ig, and the nuclei were counterstained with
TOPRO-3.
Generation and Expression of Endo180 Cytoplasmic Domain
Mutants--
Examination of the mannose receptor family cytoplasmic
domains reveals that all members contain two putative endocytosis
motifs, the first based on a conserved tyrosine residue and the second based on a conserved dihydrophobic motif (Fig.
1a). In the mannose receptor,
PLA2R, and DEC-205, mutational analysis has indicated that
it is the tyrosine-based motif that primarily mediates the internalization of these receptors into the endocytic pathway (10, 11,
16, 25). To determine whether the equivalent motif was responsible for
Endo180 internalization, the conserved tyrosine was mutated,
Endo180(Ala1452). In addition, alanine substitutions were
made in the conserved Leu-Val dihydrophobic motif,
Endo180(Ala1468/Ala1469) and in the glutamic
acid lying in the Distribution and Trafficking of Wild Type Endo180 and Endo180
Cytoplasmic Domain Mutants--
To assess the subcellular localization
of Endo180, cells were either stained prior to fixing to determine the
cell surface distribution or fixed and then stained to assess the total
cellular distribution (Fig. 3). Wild type
human Endo180 expressed in murine NIH-3T3 cells showed a distribution
indistinguishable from endogenous Endo180 in MG-63 cells. A distinct
punctate plasma membrane distribution was observed, which has
previously been demonstrated to represent clustering of receptors in
clathrin-coated pits (1). Intracellularly, a strong vesicular staining
was observed, which was concentrated in the perinuclear region. In
agreement with the FACS analysis and immunoblotting results (Fig. 2),
NIH-3T3 cells transfected with vector alone showed no staining with the
anti-human Endo180 mAb (data not shown). Analysis of the Endo180
mutants gave unexpected results. Substitution of the conserved tyrosine
residue with an alanine residue, Endo180(Ala1452), resulted
in a receptor with a distribution identical to that of wild type
Endo180. In contrast, mutation of the dihydrophobic motif,
Endo180(Ala1468/Ala1469), resulted in a highly
diffuse Endo180 staining over the whole plasma membrane, and this
staining pattern did not substantially change when cells were
permeabilized prior to staining. This increased cell surface expression
is in keeping with the higher fluorescence intensity observed in the
FACS staining of this mutant (Fig. 2a). It has been reported
that the presence of an acidic acid residue in the
MG-63 cells or NIH-3T3 cells expressing the different constructs were
incubated on ice with 125I-anti-Endo180 mAb, washed, and
then warmed to 37 °C for 0-30 min. At each time point, the cells
were incubated with an acid strip pH 2.5 buffer to determine the amount
of cell surface-associated radioactivity, and the cells were then
trypsinized and counted to assess the amount of internalized antibody
(Fig. 4). In control experiments, it was
determined that acid stripping removed >95% of cell
surface-associated antibody, that vector alone-transfected cells showed
background binding of the 125I-anti-Endo180 mAb that was
<10% of that bound to cells expressing wild type Endo180, and that
the binding and internalization of 125I-E1/183 was
identical to that of 125I-E1/183 Fab' fragments (1) (and
data not shown). A comparison of MG-63 cells and NIH-3T3 cells
transfected with wild type human Endo180 revealed a similar
internalization profile. 125I-anti-Endo180 mAb was very
rapidly internalized from the cell surface upon warming to 37 °C,
with 64 and 74% of cell surface-bound antibody found cell-associated
within 2 min, respectively (Fig. 4f). After 10 min, only
10-15% of receptor was detected on the cell surface (Fig. 4). An
essentially identical profile was obtained with cells expressing the
Endo180(Ala1452) mutant, again indicating that mutation of
the conserved tyrosine residue does not impair interaction with the
endocytic internalization machinery. In cells expressing
Endo180(Ala1468/Ala1469), antibody
internalization was drastically impaired, with less than 15% of
receptor internalized within the first 2 min and 78% of the receptor
still detected at the cell surface after 30 min of incubation at
37 °C. Mutation of the acidic residue upstream of the dihydrophobic
motif, Endo180(Ala1464), resulted in a partial phenotype in
that antibody could be internalized but at a much reduced rate compared
with wild type Endo180 or Endo180(Ala1452) with 28%
internalized within the first 2 min after warming. In addition to this
slower rate of internalization, the total amount of receptor
internalized was reduced such that after a 30-min incubation at
37 °C, the amount of receptor within the cells was less then
50%.
To assess the steady state distribution of Endo180 between the plasma
and intracellular membranes, cells were incubated with 125I-anti-Endo180 mAb at 37 °C for 1.5 h, and then
the proportion of cell surface and intracellular antibody was assessed
(Fig. 5). In MG-63 cells, 16% of the
counts were detected on the cell surface and 84% intracellularly.
Again a similar profile was observed with NIH-3T3 cells expressing wild
type Endo180 and Endo180(Ala1452). In contrast, in cells
expressing Endo180(Ala1468/Ala1469), only 33%
was detected inside the cell, whereas 67% was retained on the plasma
membrane. In cells expressing Endo180(Ala1464), some
variability in distribution was found between experiments, but an
increase in cell surface expression was observed compared with
endogenously expressed and transfected wild type Endo180.
Together these data indicate that Endo180 is unique among the mannose
receptor family in that mutation of the conserved cytoplasmic tyrosine
residue does not affect its distribution or trafficking. In contrast,
substitution of the dihydrophobic Leu-Val motif at position 1468/1469
results in a near total restriction of Endo180 to the cell surface,
thus effectively removing it from the endocytic system. As has been
demonstrated for other receptors that utilize a dihydrophobic
endocytosis motif, the presence of an upstream acidic acid residues
plays a role in modulating Endo180 trafficking. Substitution of this
residue results in a receptor that has a more diffuse distribution at
the cell surface and a reduced rate of internalization. However, after
90 min of incubation with 125I-anti-Endo180 mAb, a
substantial proportion of receptor is found intracellularly.
Endo180 Is Localized to the Early Endosomes--
In previous
immunoelectron microscopy and immunofluorescence analyses, Endo180 was
demonstrated to be clustered on the cell surface in clathrin-coated
pits (1). In permeabilized cells, it was noted that the distribution of
Endo180 was similar to that of the transferrin receptor, although in
these studies direct colocalization was not assessed. Recently, it has
been demonstrated that other members of the mannose receptor family
target to distinct intracellular destinations; in particular, the
mannose receptor has been shown to predominantly localize to early
endosomes, whereas DEC-205 is found in the late endosomes/lysosomes
(see "Discussion"). Consequently, it was important to better
determine the intracellular localization of wild type Endo180 and to
assess whether Endo180(Ala1464), which showed a partial
impairment in internalization, was nevertheless able to be delivered to
the correct intracellular compartment. To address these issues, MG-63
cells were double-labeled with antibodies directed against Endo180 and
either the transferrin receptor as a marker of early endosomes or
LAMP-1 as a marker of late endosomes/lysosomes (Fig.
6). As previously described for
fibroblasts and endothelial cells (1, 2), in MG-63 cells, Endo180 is
seen distributed in a vesicular pattern throughout the cytoplasm. A
similar staining is observed with the anti-transferrin receptor
antibody (Fig. 6b). Examination of the merged images demonstrates substantial colocalization of Endo180 and transferrin receptor. However, it should be noted that a minor population of
vesicles with predominantly Endo180 staining or, conversely, predominantly transferrin receptor staining are observed. This suggests
that these two receptors can be independently internalized from the
cell surface, but both are targeted to the early endosomes. Compared
with Endo180 and the transferrin receptor, LAMP-1 was observed in
larger, less abundant, more irregularly shaped vesicles that were
concentrated to the perinuclear region. No colocalization of Endo180
and LAMP-1 was detected in merged images, demonstrating that Endo180 is
not delivered to the late endosomes/lysosomes. To better determine the
intracellular destination of mutant receptors, transfected NIH-3T3
cells were double-labeled for Endo180 and the transferrin receptor
(Fig. 7). As expected, both wild type Endo180 and Endo180(Ala1452) showed a strong colocalization
with the transferrin receptor, again indicating that the trafficking of
transfected receptor was identical to that of endogenous receptor.
Given the predominant cell surface localization of the
Endo180(Ala1468/Ala1469) receptor, essentially
no overlap between this mutant and the transferrin receptor was
observed. Of particular interest was the intracellular localization of
the Endo180(Ala1464) mutant, since this mutant has a
diffuse plasma membrane distribution but can be internalized albeit
inefficiently. A considerable overlap in the localization of this
intracellular population with the transferrin receptor was observed,
suggesting that mutation of the acidic residues results in a defect in
recruitment into the clathrin-coated pits rather than in delivery to or
recycling from the early endosomes.
Endo180 as a Phagocytic Receptor--
A striking feature of the
mannose receptor is its ability to mediate both
clathrin-dependent internalization of ligands into the
endosomal system and phagocytic internalization of nonopsonized microorganisms or ligand-coated synthetic particles (17, 18). Moreover,
the efficiency of both of these internalization processes is severely
impaired by mutation of the conserved cytoplasmic tyrosine residue
(10). To investigate whether Endo180 functioned as a phagocytic
receptor and, if so, whether this was dependent on a key motif(s)
within the cytoplasmic domain, phagocytic assays were performed with
NIH-3T3 cells expressing human Endo180. Fibroblasts are not
professional phagocytes, but, like the COS cells used for the mannose
receptor studies, they can act as nonprofessional phagocytes (27)
(e.g. to internalize collagen (28) and apoptotic cells
(29)). To investigate the suitability of these cells for phagocytic
assays, they were transiently transfected with the Fc
To monitor phagocytic uptake by Endo180, the same anti-human Endo180
mAb-coated FITC beads were employed, since these will specifically
associate with cells expressing transfected human Endo180 but not with
endogenous murine Endo180. As expected, a similar FACS profile was
obtained for Endo180 expressing NIH-3T3 cells and parental NIH-3T3
cells incubated with BSA-coated FITC beads (Fig.
10a). The RFI values for
these two cell types were 4.68 and 4.71, respectively, giving a
phagocytic index of 0.99. In contrast to cells transiently transfected
with the Fc Clathrin-mediated Endocytosis--
A characteristic feature of the
four mannose receptor family members is that they are all subject to
constitutive clathrin-mediated endocytosis. For Endo180, examination of
the kinetics has revealed this to be a rapid process, with 64-70% of
cell surface receptor being internalized within 2 min (Fig. 4).
Clathrin-mediated internalization requires a "signal" within the
cytoplasmic domain that allows association of the transmembrane
proteins with adaptor complexes, in particular with the plasma
membrane-localized AP-2 complexes (34-36). An examination of the
cytoplasmic domains of the mannose receptor family demonstrates two
potential endocytosis motifs (Fig. 1). The first is based on a
conserved tyrosine within a low density lipoprotein receptor type
motif,
In other members of the mannose receptor family, the role of the
dihydrophobic motif has not been so extensively examined. In the
PLA2R, mutation of the leucine within the Leu-Ile motif to
a glycine had no effect on receptor internalization (11). In DEC-205,
generation of a truncated cytoplasmic domain that retains the conserved
tyrosine motif but removes the dihydrophobic motif does not affect the
rate of receptor internalization (16). Both tyrosine-based and
dihydrophobic motifs interact with the adaptor complex AP2 although
most likely at separate binding sites/subunits within the complex (35,
36), which raises the interesting issue of why members of this receptor
family that are efficiently endocytosed from the cell surface contain
two putative endocytosis motifs but only utilize one, and why different
family members utilize different motifs.
Trafficking of Receptor from the Early Endosomes--
Despite the
rapid rate of Endo180 endocytosis, at steady state 15-25% of the
total receptor population is located at the cell surface (Fig. 5), and
the receptor has a long half-life of ~24 h (1), indicating that
Endo180 is efficiently recycled back to the cell surface from
intracellular compartments. Once recruited into clathrin-coated pits,
receptors are internalized into early endosomes. At this stage,
segregation occurs, targeting some proteins into recycling vesicles
destined for return to the plasma membrane, whereas others are destined
for vesicular transport to the late endosomes/lysosomes (34, 40).
Recent detailed examination of the mannose receptor family has revealed
that the mannose receptor (16, 41) and Endo180 (Figs. 6 and 7) are
predominantly found in early endosomes, raising the question of whether
recycling from this compartment is a signal-dependent or
signal-independent mechanism. For the mannose receptor, this has been
addressed by generating receptor chimeras with an additional C-terminal
endocytosis motif that can rescue the internalization defect associated
with mutation of the conserved tyrosine residue (25). However, in these
chimeric proteins, mutation of this conserved tyrosine residue together
with the adjacent phenylalanine results in a receptor that is
efficiently internalized but mistargeted to the late
endosomes/lysosomes, indicating that the diaromatic Tyr-Phe motif is
required for recycling from the early endosome to the plasma membrane.
A diaromatic residue Tyr-Tyr is present in the PLA2R, but
neither the intracellular destination of this receptor nor the ability
of this motif to mediate recycling has yet been investigated. DEC-205
does not contain an equivalent diaromatic motif, and this is in keeping with the recent demonstration that this receptor is not recycled from
the early endosomes but rather is localized to the late
endosome/lysosomes (16). As discussed above, truncation of DEC-205
C-terminal to the conserved tyrosine residue results in a receptor that
is efficiently internalized. However, this truncated DEC-205, instead
of being delivered to the late endosomes/lysosomes, is colocalized with the transferrin receptor in the early endosomes. Moreover, these receptors showed inefficient antigen presentation, demonstrating a
functional requirement for DEC-205 to be delivered to the degradative compartments. Further mutagenesis identified three acidic residues, EDE, as a late endosome/lysosomal targeting motif (Fig. 1) (16). Unlike
the mannose receptor and the PLA2R, Endo180 does not
contain a diaromatic motif, yet it is efficiently recycled from the
early endosome. It may be that since Endo180 does not utilize a
tyrosine-based motif for recruitment into the clathrin-coated pits, it
employs a separate recycling motif. In addition, it has been suggested that association of recycling receptors with specific membrane lipid
domains has a role to play in segregation from the late endosomal/lysosomal pathway (40). For the mannose receptor family, association with membrane lipids either directly or via linker proteins
has not been investigated. However, swapping of the mannose receptor
transmembrane domain does impair receptor internalization (10),
indicating that this domain may have a role to play in regulating
receptor trafficking.
Phagocytosis--
In mammals, phagocytosis is the process
primarily used to engulf microorganisms and apoptotic or unwanted cells
and plays a critical role in innate immunity and tissue remodeling (31, 42-44). In addition to the size of the material internalized, the processes of clathrin-mediated endocytosis and phagocytosis are mechanistically distinct. Engagement of phagocytic receptors such as
the Fc and complement receptors results in localized polymerization of
actin and extension of the membrane to engulf the particle into an
intracellular phagosome, which rapidly fuses with the endosomes and/or
lysosomes to expose the contents to the hydrolytic enzymes. In this
respect, the mannose receptor is unusual in that it can mediate the
endocytic clearance of glycosylated ligands and the phagocytic uptake
of a wide variety of microorganisms (18).
It would seem unlikely that the PLA2R and DEC-205 would
function as phagocytic receptors, given their defined functions in the
uptake of macromolecules and their lack of C-type lectin activity. In
contrast, Endo180 is expressed both on professional phagocytic cells
such as macrophages and nonprofessional phagocytic cells in the stroma
and is a functional C-type lectin (2), leading to the suggestion that
Endo180 may play a complementary or overlapping phagocytic role to the
mannose receptor. However, as demonstrated here, although cells
expressing Endo180 can bind to 1-µm anti-Endo180 mAb coated beads,
but not control beads, no phagocytic uptake was observed in a system
where phagocytic uptake of the same beads by the Fc
In conclusion, the mannose receptor and Endo180 share a common
structural organization and bind a discrete but overlapping set of
ligands. In addition, they both constitutively recycle between the
plasma membrane and the early endosomes, indicating a major role in
transporting ligands into the cell for release in the low pH
environment. However, despite these similarities, we show here that
Endo180 employs a distinct mechanism for interacting with the cellular
trafficking machinery. These results provide a basis with which to
investigate the role of transport between subcellular compartments in
the biological function of Endo180.
We thank Annegret Pelchen-Matthews and Mark
Marsh for advice with the endocytosis assays, Laura Machesky and Robin
May for advice and reagents for the phagocytosis assays, Helen Yarwood for help with the FACS analysis, David Robertson for confocal microscopy expertise, and Dirk Wienke and Lucy East for comments on the manuscript.
*
This work was supported by grants from the Wellcome Trust
and Breakthrough Breast Cancer Research.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 44-20-7970-6106;
Fax: 44-20-7878-3858; E-mail: c.isacke@icr.ac.uk.
Published, JBC Papers in Press, June 14, 2002, DOI 10.1074/jbc.M203631200
2
D. Wienke and C. M. Isacke, unpublished observations.
The abbreviations used are:
PLA2R, phospholipase A2 receptor;
CTLD, C-type lectin-like domain;
mAb, monoclonal antibody;
FITC, fluorescein isothiocyanate;
BSA, bovine
serum albumin;
FACS, fluorescence-activated cell sorting;
MES, 4-morpholineethanesulfonic acid;
PBS, phosphate-buffered saline;
RFI, relative fluorescence intensity;
RPE, R-phycoerythrin.
The C-type Lectin Receptor Endo180 Displays Internalization and
Recycling Properties Distinct from Other Members of the Mannose
Receptor Family*
and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
HindIII). For generation of the cytoplasmic
domain mutants, pcDNA3-Endo180 or
pcDNA3HindIII-Endo180 was subject to site-directed
mutagenesis using the QuikChange XL kit (Stratagene). The
pcDNA3-Endo180(Ala1452),
pcDNA3HindIII-Endo180(Ala1468/ Ala1469),
and pcDNA3-Endo180(Ala1464) mutant constructs were
confirmed by sequencing. A human Fc
IIA receptor cDNA construct in
the pRK5 vector was obtained from Dr. L. Machesky (University of Birmingham).
IIA receptor antibody (RPE-anti-CD32
antibody) was purchased from Serotec. mAb E1/183 was
125I-labeled using the Bolton-Hunter reagent (Amersham
Biosciences) according to the method of Pelchen-Matthews et
al. (24). 1-µm diameter FITC-labeled polystyrene beads
(Polysciences) were conjugated with mAb A5/158 or BSA as follows. 5 µl of a 2.5% polystyrene bead solution was washed twice in 1 ml of
0.1 M borate buffer, pH 8.5, and then incubated overnight
at room temperature with 0.7 mg of purified mAb A5/158 predialyzed into
0.5 ml of 0.1 M borate buffer, pH 8.5, or 10% BSA in 0.1 M borate buffer, pH 8.5. Beads were subsequently blocked by
incubating for 2 h in 20% BSA, 0.1 M borate buffer,
washed twice in PBS, and resuspended in 0.5 ml of PBS.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4 position to the dihydrophobic motif,
Endo180(Ala1464) (Fig. 1b). Many investigations
on receptor trafficking have employed chimeric constructs; however,
since it was demonstrated for the mannose receptor that different
results can be obtained with different chimeras (10), these mutations
were made in the context of full-length human Endo180 and Endo180
monitored using a human-specific anti-Endo180 mAb. Wild type and mutant
constructs were transfected into murine NIH-3T3 cells as these cells
express endogenous Endo180 and therefore will contain the correct
trafficking machinery. As has been described by others (26), it was
noted in preliminary studies that overexpression of Endo180 in cells in
transient transfections resulted in an abnormally high percentage of
receptor localized to the plasma membrane (data not shown). Consequently, permanently expressing populations were generated by
selecting transfected cells in G418 and enriching for receptor-positive cells by magnetic immunobead sorting. To assess receptor expression, resulting populations were subject to FACS (Fig.
2a). As expected, human MG-63
osteosarcoma cells that express endogenous Endo180 have homogenous cell
surface receptor expression. 70-95% of the transfected NIH-3T3
populations were receptor-positive and no reactivity of the anti-human
Endo180 mAb A5/158 was detected on NIH-3T3 cells transfected with
vector alone. By immunoblot analysis (Fig. 2b), a single
immunoreactive band of ~180 kDa was observed in the MG-63 cells
corresponding to expression of endogenous Endo180. A similar sized band
was detected in lysates from NIH-3T3 cells transfected with wild type
and mutant Endo180 constructs. Together, these data demonstrate that
mutation of the cytoplasmic domain did not alter Endo180
posttranslational processing or prevent delivery to the cell
surface.
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Fig. 1.
Cytoplasmic domain endocytosis motifs.
a, comparison of the cytoplasmic domains of the mannose
receptor family. MR, mannose receptor. The putative
tyrosine-based endocytosis motif is boxed in
black, and the putative dihydrophobic endocytosis motif and
upstream acidic residue are boxed in gray.
b, the alanine substitutions in the human Endo180
cytoplasmic domain are shown boxed in light
gray.
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Fig. 2.
Expression of Endo180 cytoplasmic domain
mutants. a, MG-63 cells or NIH-3T3 cells expressing
wild type (WT) Endo180, Endo180(Ala1452),
Endo(Ala1468/Ala1469),
Endo180(Ala1464), or vector alone were stained with
anti-Endo180 mAb A5/158 followed by Alexa 488 anti-mouse Ig antibody
and analyzed by flow cytometry (solid profiles).
Open profiles indicate cells stained with Alexa
488 anti-mouse Ig alone. b, cells were lysed and resolved by
10% SDS-PAGE. Gels were blotted onto nitrocellulose and probed with
anti-Endo180 mAb A5/158 followed by horseradish peroxidase-conjugated
anti-mouse Ig. Blots were developed with ECL reagent and exposed to
film for 30 s. Molecular size markers are in kDa.
3 to 4 position
to a dihydrophobic motif play a role in the internalization of
receptors from the cell surface and/or in regulating their
intracellular trafficking (see "Discussion"). Consequently, the
distribution of Endo180 containing an alanine substitution for the
conserved glutamic acid, Endo180(Ala1464), was examined.
Interestingly, this receptor showed a partial phenotype with a
stronger, more evenly distributed cell surface staining compared with
wild type Endo180 or Endo180(Ala1452) but with stronger
intracellular staining than was observed with Endo180(Ala1468/Ala1469). To determine whether
this intracellular staining observed with Endo180(Ala1464)
and the other mutants resulted from internalization of receptor from
the cell surface, the trafficking of the receptor was monitored using
125I-anti-Endo180 mAb.
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Fig. 3.
Subcellular distribution of Endo180
cytoplasmic domain mutants. MG-63 cells or NIH-3T3 cells
expressing wild type (WT) Endo180,
Endo180(Ala1452),
Endo180(Ala1468/Ala1469), or
Endo180(Ala1464) were either cell surface-stained by
incubating for 1 h at 4 °C with anti-Endo180 mAb A5/158;
fixed, permeabilized, and stained with Alexa 488 anti-mouse Ig ( saponin); or fixed, permeabilized with 0.2% saponin, and
stained with mAb A5/158 followed by Alexa 488-anti-mouse Ig and nuclei
counterstained with TOPRO-3 (+ saponin). Scale
bar, 20 µm.
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Fig. 4.
Trafficking of Endo180 cytoplasmic domain
mutants. MG-63 cells (a) or NIH-3T3 cells expressing
wild type (WT) Endo180 (b),
Endo180(Ala1452) (c),
Endo(Ala1468/Ala1469) (d), or
Endo180(Ala1464) (e) were incubated on ice for
1 h with 125I-anti-Endo180 mAb E1/183, washed, and
then incubated in binding buffer at 37 °C for 0-30 min. At each
time point, the amount of 125I-E1/183 in the incubation
medium (not shown), acid-dissociable from the cell surface
(black lines and circles), or
remaining cell-associated (gray lines and
squares) was collected and counted. f, the
percentage of 125I-E1/183 internalized after a 2-min
incubation at 37 °C. Values given are for duplicate samples, with
error bars showing S.D. values. Similar results
were obtained on three separate occasions.
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Fig. 5.
Equilibrium distribution of Endo180.
Cells plated overnight in 35-mm dishes were incubated with
125I-anti-Endo180 mAb E1/183 for 90 min at 37 °C. Cells
were then washed extensively, and the amount of 125I-E1/183
dissociable from the cell surface by acid stripping (dark
bars) and remaining cell-associated (pale
bars) was assessed. Values given are for duplicate samples,
with error bars showing S.D. values. Similar
results were obtained on three separate occasions. WT, wild
type.
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Fig. 6.
Endo180 is targeted to the early
endosomes. MG-63 cells were fixed, permeabilized, and
double-stained with anti-Endo180 polyclonal antiserum followed by Alexa
488 anti-rabbit Ig and either anti-transferrin receptor
(TfR) mAb B3/25 or anti-LAMP1 mAb H4A3 followed by Alexa 568 anti-mouse Ig. Nuclei were counterstained with TOPRO-3, and cells were
analyzed by confocal microscopy, collecting sequential
images in three channels. Right panels show
merged images. Scale bar, 20 µm.
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Fig. 7.
Intracellular targeting of Endo180
cytoplasmic domain mutants. NIH-3T3 cells expressing wild type
(WT) Endo180, Endo180(Ala1452),
Endo180(Ala1468/Ala1469), or
Endo180(Ala1464) were permeabilized and double-stained with
anti-Endo180 mAb A5/158 followed by Alexa 488 anti-mouse Ig
(left panels) and with anti-transferrin receptor
mAb R17 followed by Alexa 568 anti-rat Ig (middle
panels). Nuclei were counterstained with TOPRO-3, and cells
were analyzed by confocal microscopy collecting sequential images in
three channels. Right panels show merged images.
Scale bar, 20 µm.
IIA (CD32)
receptor and tested for their ability to internalize anti-Endo180
mAb-coated 1-µm polystyrene FITC beads. FcIIA receptors bind to
the Fc domain of immunoglobulins to mediate phagocytic uptake of
opsonized microorganisms (30, 31), and this can be monitored by FACS
analysis (32) and confocal microscopy. For FACS analysis (Fig.
8a), cells that had been
incubated with mAb- or BSA-coated FITC beads for 4 h at 37 °C
were washed, and cell surface-associated beads were removed by acid
stripping as described for the endocytosis assays. A comparison of
FcIIA receptor or mock-transfected cells revealed that
(a) as expected for a transient transfection, only a
proportion of the FcIIA receptor-transfected cells were associated
with FITC beads, (b) in receptor-transfected cells, cells
that were associated with beads were observed as a wide peak,
suggesting that variable numbers of beads had been internalized,
(c) the fraction of bead-positive cells and the fluorescent
intensity of positive cells was much reduced when FcIIA receptor
cells were incubated with BSA beads and similarly when mock-transfected
cells were incubated with either mAb beads or BSA beads. To facilitate
further analysis, a reference system was required so that the
phagocytic ability of both transiently and permanently transfected
cells expressing different receptors could be compared. For this
purpose, the ratios of the mean fluorescence intensities of cells
incubated with beads to cells incubated without beads were calculated
to give a relative fluorescence intensity (RFI). A phagocytic index for
the receptors under investigation was generated by dividing the RFI
values for cells expressing receptor with nonexpressing or
mock-transfected cells. The RFI values for cells transfected with
FcIIA receptor or mock-transfected and incubated with mAb-coated
FITC beads were 13.35 and 2.45, respectively, giving a phagocytic index
of 5.26. The RFI values of the same cells incubated with BSA beads were
2.57 and 3.60, respectively, giving a phagocytic index of 0.71. To
confirm that within the transiently transfected populations, mAb-coated
beads specifically associated with FcIIA receptor expressing cells, parallel experiments were undertaken with cells stained prior to FACS
analysis with RPE-conjugated anti-FcIIA receptor (RPE-anti-CD32) antibody (Fig. 8b). By comparing cells incubated with and
without anti-CD32 mAb and with or without either mAb- or BSA-coated
FITC beads, it was demonstrated that cells expressing FcIIA receptor were selectively associated with mAb-coated FITC beads. Finally, although FACS analysis demonstrates a specific association of mAb beads
with FcIIA receptor-expressing cells, it cannot distinguish between
beads that have been internalized and cell surface-associated beads
that have not been removed by acid stripping. Consequently, confocal
microscopy studies were undertaken on cells in which the acid strip to
remove cell surface-associated beads was omitted, and cells were
stained after fixation with mAb IM7 and the nuclei were counterstained
with TOPRO-3 (Fig. 9). mAb IM7 recognizes murine CD44, an abundant transmembrane protein that is predominantly restricted to the plasma membrane (33). Analysis of FCIIA
receptor-transfected cells incubated with mAb-coated FITC beads
revealed the presence of multiple FITC beads associated with the cells
that by z sectional analysis were clearly located
intracellularly. By contrast, few BSA-coated FITC beads were associated
with the cells, and these were predominantly cell surface-associated.
Similarly, few mAb-coated or BSA-coated FITC beads were associated with
mock-transfected cells. Together, these data demonstrate that NIH-3T3
cells are capable of mediating uptake of ligand coated synthetic beads, and, as a consequence, they were judged a suitable in vitro
system for analyzing the phagocytic properties of Endo180.
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Fig. 8.
Fc IIA receptor can
mediate phagocytosis in NIH-3T3 cells. NIH-3T3 cells were
transiently transfected with the pRK5-FcIIA plasmid or
mock-transfected. 24 h later, cells were incubated in serum-free
medium for 1 h and then for 4 h with mAb-coated FITC beads,
BSA-coated FITC beads, or no beads. Cells were washed in PBS, incubated
in ice-cold pH 2.5 acid wash for 5 min to remove cell
surface-associated beads, washed in PBS, detached with trypsin, and
fixed in 1% paraformaldehyde. a, cells incubated with
(solid profiles) or without (open
profiles) FITC beads were subject to single-channel FACS
analysis. b, cells treated as described above were incubated
with or without RPE-conjugated anti-CD32 (anti-FcIIA receptor)
antibody and subjected to two-color FACS analysis. Quadrants were set
on cells incubated without FITC beads and without RPE-anti-CD32
antibody.
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Fig. 9.
Confocal analysis of phagocytosis mediated by
the Fc IIA receptor. NIH-3T3 cells were
transiently transfected with the pRK5-FcIIA plasmid or
mock-transfected. 24 h later, cells were incubated in serum-free
medium for 1 h and then for 4 h with mAb-coated FITC beads or
BSA-coated FITC beads. Cells were then washed in PBS, fixed, and
stained with anti-CD44 mAb IM7 followed by Alex 568 anti-rat Ig and
counterstained with the nuclear marker TOPRO-3. For confocal analysis,
15 1-µm horizontal xy sections were collected sequentially
in three channels (main panel). xz and
yz vertical sections are shown to the right and
below, respectively. Scale bar, 20 µm.
IIA receptor, the FACS profile of NIH-3T3 cells
expressing Endo180 incubated with mAb-coated FITC beads was similar to
that of parental NIH-3T3 cells, with RFI values of 2.55 and 4.71, respectively, giving a phagocytic index of 0.54. Confocal microscopy
analysis (Fig. 10b) revealed that in the absence of acid
stripping, there was an increased number of mAb-coated beads, compared
with BSA-coated beads, associated with wild type Endo180-transfected
cells. However, by z section analysis, these beads were
found predominantly associated with the cell surface, and few were
detected intracellularly. As previously described for mock-transfected
cells (Fig. 9), few mAb- or BSA-coated beads were found associated with
parental cells (data not shown). To confirm that this result was not
due to an intrinsic defect in the transfected NIH-3T3 cells,
phagocytosis experiments were repeated with MG-63 cells. Essentially
identical results were obtained. Transfection with the FcIIA
receptor resulted in uptake of mAb beads but not BSA beads with
phagocytic indices of 2.85 and 0.41, respectively (data not shown). In
nontransfected cells, there was no uptake of either mAb or BSA beads
(Fig. 10a; RFI values of 2.69 and 2.83, respectively)
despite endogenous expression of human Endo180 (Fig. 2). These data
indicate that the FcIIA receptor, but not the Endo180 receptor, is
capable of efficient phagocytic internalization of ligand-coated
synthetic particles.
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Fig. 10.
Endo180 does not mediate mAb-coated bead
phagocytosis. NIH-3T3 cells expressing wild type (WT)
human Endo180, parental NIH-3T3 cells, or MG-63 cells were incubated in
serum-free medium for 1 h and then for 4 h with mAb-coated
FITC beads, BSA-coated FITC beads, or no beads. Cells were then washed
in PBS. a, cells were then incubated in ice-cold pH 2.5 acid
wash for 5 min to remove cell surface-associated beads, washed in PBS,
detached with trypsin, and fixed in 1% paraformaldehyde. Cells
incubated with (solid profiles) or without
(open profiles) FITC beads were subject to
single-channel FACS analysis. b, NIH-3T3 cells expressing
Endo180 were fixed, permeabilized, and stained with anti-CD44 mAb IM7
followed by Alexa 568 anti-rat Ig, and the nuclei were counterstained
with TOPRO-3. Confocal microscopy analysis was undertaken as described
in the legend to Fig. 9. Main panels show the
xy horizontal images; xz and yz
vertical sections are shown to the right and
below, respectively. Scale bar, 20 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
XNXXY, in which represents a bulky
hydrophobic residue (34). This motif is well conserved within the
mannose receptor and, interestingly, conserved in some
PLA2R and DEC-205 species but not others. Despite these
differences, mutation of the conserved tyrosine residue within the
mannose receptor, PLA2R, and DEC-205 severely impairs receptor internalization. For PLA2R, this mutant is
essentially internalization-deficient (11), whereas in DEC-205
internalization is reduced to 50% of wild type values (16). Initially,
it was reported that a mannose receptor tyrosine mutant had a similar phenotype to DEC-205 (10). However, more recently it has been reported
that this conserved tyrosine is essential for mannose receptor
endocytosis (25). In contrast to these other family members, we show
here that mutation of the conserved tyrosine in Endo180 has no effect
on receptor internalization or intracellular destination. In addition
to the tyrosine-based motif, all members of the mannose receptor family
have a dihydrophobic sequence of LV, LM, LI, or ML (Fig. 1).
Dihydrophobic based endocytosis motifs have been identified in a number
of receptors including the Fc receptor, major histocompatibility
complex class II-associated invariant chains, mannose 6-phosphate
receptors, and IF receptor (34-37). Mutation of the dihydrophobic
Leu-Val amino acids in Endo180 essentially blocks receptor
internalization. Among constitutively endocytosed receptors with
dihydrophobic motifs, Endo180 is somewhat unusual in that it is
targeted from the plasma membrane to the recycling endosomes rather
than from the cell surface or trans-Golgi network to a late
endosome/lysosome compartment, suggesting that other motifs or the
sequence context of the dihydrophobic motif provides additional
targeting specificity. One such candidate is the upstream acidic
residue, Asp1464, which is conserved in all mannose
receptor family members (Fig. 1a). An acidic acid residue in
the 3 or 4 position has been subjected to close scrutiny in other
receptors (35, 38, 39) and has been shown to be required for
intracellular trafficking from the early endosome and in some cases to
modulate internalization. However, mutation of this residue in Endo180,
although resulting in impaired internalization, does not result in
receptor mistargeting (see below).
IIA receptor was
readily apparent. These data indicate that Endo180 does not function as
a primary phagocytic receptor and, unlike the mannose receptor, cannot
mediate uptake of ligand-coated synthetic particles. It remains to be
determined whether Endo180, like some integrins, the lipopolysaccharide
receptor, and scavenger receptors, may alone not be competent to
mediate phagocytosis but may have a role in professional phagocytes in
tethering particles and engaging a classical phagocytic receptor to
drive the phagocytic machinery (45).
ACKNOWLEDGEMENTS
FOOTNOTES
Supported by a Medical Research Council studentship.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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