From the Institute of Biochemistry, University of Lausanne, BIL Biomedical Research Center, 155 Ch. des Boveresses, 1066 Epalinges, Switzerland
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ABSTRACT |
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Transfer of passive immunity from the mother to
the fetus or newborn involves the transport of IgG across several
epithelia. Depending on the species, IgG is transported prenatally
across the placenta and yolk sac or is absorbed from colostrum and milk by the small intestine of the suckling newborn. In both cases apical to
basolateral transepithelial transport of IgG is thought to be mediated
by FcRn, an IgG Fc receptor with homology to major histocompatibility
class I antigens. Here, we analyzed the intracellular routing of
chimera encoding the rat FcRn tail fused to the ecto- and transmembrane
domain of the macrophage Fc Simple epithelia form barriers that allow the selective exchange
of molecules between the lumen of an organ and the underlying tissue.
One mechanism by which molecules can cross the epithelial barrier
involves vesicular transepithelial transport (transcytosis). Our
current knowledge about transcytosis of proteins is almost exclusively
based on studies of the
pIgR,1 which mediates the
transport of serosal polymeric IgA and IgM into mucosal secretions (for
review, see Refs. 1 and 2). Transcytosis of the pIgR can be faithfully
reproduced in polarized kidney MDCK cells transfected with the receptor
cDNA (3). Briefly, the pIgR is transported from the trans-Golgi
network to the basolateral surface where it binds ligand and is
internalized. Basolateral targeting and internalization require signals
in the form of short amino acid sequences located in the cytosolic
receptor domain. Transcytosis occurs via basolateral early endosomes
and an apically located endosomal recycling compartment (4, 5) and
requires microtubules (6, 7) and probably BFA-sensitive coat proteins (5, 8, 9). Although free receptors transcytose, binding of ligand
results in the transduction of signals (10) that stimulate the rate of
transcytosis (11). It is therefore not surprising that transcytosis is
regulated by a number of molecules involved in signal transduction,
including CaM (12, 13).
In contrast to basolateral to apical transcytosis, little is known
about transport in the opposite direction, and the scant information
available is derived from proteins that are not physiologically involved in transcytosis (14-16). These studies indicate that apical to basolateral transcytosis may be independent of microtubules (6) and
may also be regulated differently from transport in the opposite
direction (15). In analogy to the pIgR, however, apical to basolateral
transcytosis of a bona fide transcytotic receptor may be subject to
additional regulatory mechanisms.
One of the few known receptors to mediate the physiologically relevant
apical to basolateral transcellular transport of a ligand is FcRn.
FcRn, an IgG FcR related to major histocompatibility complex class I
molecules (17) was initially identified in the neonatal small intestine
where it mediates the uptake of maternal IgG present in colostrum and
milk (18-26). The receptor has also been implicated in the transport
of IgG from the maternal circulation across the placental
syncytiotrophoblast (27-30) or the yolk sac splanchnopleur (31) into
the fetal circulation. Like in the intestine, net transplacental IgG
transport occurs in an apical to basolateral direction. More recently,
a more general role of FcRn in the maintenance of IgG homeostasis by
recycling internalized IgG and thus preventing its degradation in
lysosomes has been proposed (32-34). Because binding of IgG to FcRn
occurs preferentially at a slightly acidic pH of ~6.0 (for review,
see Ref. 35), FcRn can bind ligand either at lumenal surfaces exposed
to an acidic environment such as in the intestine (24, 25), or in
acidic endosomal compartments following the fluid phase internalization of IgG (31). Exposure of the receptor-ligand complex to a neutral pH
following transcytosis or recycling may then lead to the
dissociation of IgG into the extracellular milieu.
Characterization of the intracellular transport of human and rat FcRn
in transfected epithelial MDCK cells indicates that newly synthesized
receptors are transported in a nonvectorial fashion to the apical and
basolateral cell surface and that they transcytose in both
directions.2 To analyze the
role of the cytosolic tail in intracellular sorting, we generated MDCK
cell lines stably expressing chimera consisting of the ecto- and
transmembrane domains of the macrophage Fc Materials--
Brefeldin A (Epicentre Technologies, Madison,
WI), nocodazole (Sigma, Buchs, Switzerland) and W-7
(N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide; Bio-Science Products) were prepared and used as described previously (6, 8, 13). Protease inhibitor mixture contained 10 mg/ml chymostatin,
antipain, leupeptin, and pepstatin A (all from Sigma) in
Me2SO and was used at a 1:1,000 dilution.
125I-NaI was from Amersham Corp. (Little Chalfont, UK),
Easytag Express protein labeling mix from NEN Life Science Products(Du
Pont de Nemours, Switzerland). Immunopure sulfo-NHS-biotin was
purchased from Pierce Europe (Oud Beijerland, The Netherlands),
prepared as a stock of 200 mg/ml Me2SO and used at a final
concentration of 1.5 mg/ml. Fixed Staphylococcus aureus
cells and protein A-negative S. aureus strain (Wood 46 strain) were obtained from Zymed Laboratories Inc.
(San Francisco, CA) and used as a 10% suspension in phosphate-buffered saline + 0.5% Triton X-100 containing 0.5% bovine serum albumin. Streptavidin-agarose was from Sigma. Fixed S. aureus cells
and agarose beads were washed with phosphate-buffered saline + 0.5% Triton X-100 containing 0.5% bovine serum albumin before use. Mowiol
4-88 (Calbiochem-Novabiochem Corp.) was used at 0.1 g/ml supplemented
with 0.2% (w/v) diazabicyclo(2.2.2)octane (Sigma).
Antibodies and Ligands--
Fab fragments of the monoclonal rat
anti-mouse Fc Cell Culture and Transfection of MDCK Cells--
Cell culture
and stable transfection of MDCK strain II cells was carried out as
described (14). Two or three clones expressing each construct were
analyzed. In some experiments, cells were incubated overnight with
5-10 mM butyrate (38) to induce expression. Butyrate
treatment did not significantly alter the intracellular transport of
the chimera under the conditions used. 125I-canine
transferrin bound in a polarized fashion to the basolateral side (39)
and gp80 was secreted apically (40), indicating that the cell
monolayers were fully polarized.
Construction of FcRII/FcRn Chimera--
Deletion and
substitution mutants of the FcRn tail were generated by PCR using rat
FcRn cloned into Bluescript or pCB6 as a template. A sense primer
covering the 5' region of FcRn was combined with the different
mutagenic antisense primers carrying XbaI sites in the 3'
noncoding region. PCR fragments were cut with
BglII/XbaI and used to replace the
BglII/XbaI fragment containing the tail domain of
FcRn in Bluescript. The complete FcRn coding fragment was then excised
from Bluescript with KpnI/XbaI and subcloned into
the expression vector pCB6. To generate chimera between the macrophage
Fc Binding, Internalization, and Transcytosis of 2.4G2 Fab
Fragments--
Endocytosis and transcytosis of FcRII/FcRn was analyzed
by measuring the uptake and translocation of prebound iodinated 2.4G2 Fab fragments as described previously (6, 14), except that Leibowitz 15 medium (L-15) was used instead of Dulbecco's modified Eagle's medium.
Binding of 125I-2.4G2 Fab fragments (2-3 × 105 cpm) was specific because the addition of 100-fold
excess unlabeled Fab fragments inhibited binding by 80-90%. Treatment
of cells with nocodazole (6), BFA (8), or W7 (13) was carried out as
described. Transcytosis was defined as the fraction of total initially
bound Fab fragments present on the surface and released into the
compartment opposite that from which Fab fragments had initially been
allowed to bind. More than 80% of the transcytosed Fab fragments
represented surface-associated material. Experiments were done several
times using duplicate or triplicate filters.
Immunofluorescence--
Staining of cells with a polyclonal
anti-FcR serum or with 2.4G2 Fab fragments and internalization of
prebound antibodies was carried out as described previously (14). To
measure reinternalization of transcytosed receptor-Fab complexes, cells
grown on Transwell filters were incubated in the presence of 2.4G2-Fab
(2 µg/ml in L-15) in the apical or basolateral chamber for 45 min at
37 °C. After cooling the cells on ice and washing with ice-cold
phosphate-buffered saline+, labeled second antibody (2 µg/ml) was
allowed to bind to transcytosed molecules for 1 h. Thereafter,
cells were either fixed or incubated at 37 °C for another 15 min to
allow reinternalization of bound fluorescent antibodies. Following
internalization, cells were washed with L-15, pH 2.5, on ice to remove
surface bound antibody, fixed with 3% paraformaldehyde and mounted.
Colocalization of internalized (60 min at 37 °C) biotinylated
2.4G2-Fab (1 µg/ml in L-15) with lysosomes was done using the
monoclonal antibody AC17 (kindly provided by A. Le Bivic, Marseille) as
described (41).
Polarized Membrane Insertion--
Insertion of newly synthesized
receptors into the apical or basolateral cell surface was analyzed as
described Hunziker et al. (8, 14, 43).
Expression of Chimera Encoding Wild-type and Mutant Cytosolic Tails
of FcRn (FcRII/FcRn)--
To determine the role of the cytosolic tail
of FcRn in intracellular transport, we generated chimera in which the
ecto- and transmembrane domains of Fc
A first indication as to the subcellular distribution of FcRII/FcRn was
obtained by permeabilizing cells grown on coverslips and staining with
the anti-Fc
These results demonstrate that the FcRII/FcRn chimera is present
intracellularly and on the plasma membrane and that chimera expressed
on the cell surface can be internalized.
The Di-leucine Motif in the FcRn Tail Is Critical for Efficient
Endocytosis--
To determine the kinetics of endocytosis by wild-type
and mutant FcRII/FcRn, we followed the internalization of prebound
125I-labeled 2.4G2 Fab fragments. Fab fragments were
allowed to bind on ice and the cells were then transferred to 37 °C
for different periods of time to allow for internalization to occur.
After returning the cells on ice, noninternalized Fab fragments were
removed by washing with acid, and the fraction that had been
internalized was determined.
As shown in Fig. 3, chimera encoding the
wild-type tail rapidly and efficiently endocytosed Fab fragments and
after 10 min of warming the cells, up to 50% of the Fab fragments had
been internalized. Although deletion of the C-terminal half of the tail
in CT24 led to a reduction in the rate and extent of endocytosis when
compared with FcRII/FcRn, this mutant was still capable of significant
internalization. In contrast, substitutions of either Leu-22, Leu-23,
or both to alanines led to a dramatic reduction in endocytosis of CT24.
Nevertheless, internalization by the leucine substitutions was still
above that observed for the tail-minus construct. Similar results were
obtained for the different mutants if endocytosis from the apical or
basolateral cell surface was analyzed in cells grown as polarized
monolayers (data not shown). These results indicate that the di-leucine
motif in the cytosolic tail plays a critical role for rapid and
efficient endocytosis of FcRn.
FcRII/FcRn Is Present on the Apical and Basolateral Surface of
Polarized MDCK Cells--
We next determined the steady state
distribution of the chimeric proteins to the apical and basolateral
domain of polarized MDCK cell monolayers. Cells expressing the
different constructs were grown on Transwell filters and allowed to
bind 125I-labeled 2.4G2 Fab fragments added from the apical
or basolateral compartment at 4 °C, and the radioactivity
specifically bound to one or the other surface was quantitated.
As shown in Fig. 4A, 70-80%
of the total surface receptors were present on the apical domain of
cells expressing the wild-type as well as the CT24 and CT24 L22A,L23A
mutants. As expected, also the tail-minus CT4 construct was enriched on
the apical surface (14). Binding experiments using
125I-canine transferrin confirmed the basolateral
localization of the endogenous transferrin receptor and gp80 was
predominantly secreted into the apical medium (data not shown), showing
that the predominant apical localization of the different chimeric receptors was not because of a lack of polarization of the different clones.
Thus, FcRII/FcRn preferentially localized to the apical surface at
equilibrium, and the localization was not significantly altered for the
different tail mutants analyzed.
Newly Synthesized FcRII/FcRn Are Transported in a Nonvectorial
Fashion to the Apical and Basolateral Domain--
To determine whether
the apical steady state localization of FcRII/FcRn reflected the direct
delivery of newly synthesized receptors to the apical domain, we
monitored the appearance of a cohort of newly synthesized receptors on
the apical and basolateral cell surface. Cells grown on Transwell units
were pulse labeled for 15 min with
[35S]methionine/cysteine, and labeled proteins were
chased to the cell surface for 30 or 45 min. Cells were then cooled on
ice, and the apical or basolateral surface was biotinylated. Following cell lysis, total receptors were immunoprecipitated. An aliquot of the
immunoprecipitated receptors was used to determine the total amount of
receptor labeled, the rest was precipitated with streptavidin-agarose
to isolate molecules that had appeared on the cell surface. The samples
were analyzed by SDS-polyacrylamide gel electrophoresis and
autoradiography and the amount of biotinylated surface receptors was
normalized to the amount of total labeled receptors for each filter.
As shown in Fig. 4B, an equal fraction of pulse-labeled
FcRII/FcRn was biotinylated from the apical and basolateral side, indicating that cell surface transport was not vectorial. Also CT24 and
CT24 L22A,L23A were inserted to similar extents into the apical and
basolateral surface, indicating that the mutations did not alter the
nonvectorial transport of FcRII/FcRn to the plasma membrane. In
contrast, however, the CT4 tail-minus construct was preferentially
transported to the apical domain, consistent with previous studies (15,
42-44). Similar results were obtained if cells were chased for 30 min
in the continuous presence of the anti-Fc
These experiments thus show that regardless of the predominant apical
localization at equilibrium, FcRII/FcRn and the mutants are
nonvectorially transported from the trans-Golgi network to the apical
and basolateral surface.
FcRII/FcRn Transcytoses in Both Directions--
Despite the
nonpolarized membrane insertion, FcRII/FcRn was enriched on the apical
domain at steady state, indicating that transcytosis may play a role in
receptor distribution. We therefore analyzed transcytosis of
125I-2.4G2 Fab fragments prebound at 4 °C from the
apical or basolateral surface of MDCK cell monolayers. After washing
nonbound Fab fragments, the cells were incubated at 37 °C for
different periods of time and returned on ice. The fraction of Fab
fragments released into the apical or basolateral compartment or
present on the apical and basolateral plasma membrane or inside the
cells was quantitated. Fab fragments present on the membrane or in the
medium opposite the compartment of addition at a given time were
defined as having undergone transcytosis.
Fab fragments prebound to the basolateral cell surface were
internalized by FcRII/FcRn as evidenced by their removal from the
apical surface (Fig. 5A) and
their intracellular appearance (Fig. 5B). After 20-30 min,
Fab fragments started to appear on the opposite cell surface and by 60 min more than 30% of the basolaterally prebound Fab fragments had
transcytosed (Fig. 5D). In contrast, cells expressing CT4
transcytosed significantly less prebound Fab fragments. Of the
transcytosed Fab fragments, more than 80% remained membrane associated
(i.e. were removed by the acid wash, not shown). A small
fraction of Fab fragments was released basolaterally (Fig.
5C) and less than 20% was still on the basolateral surface after 60 min (Fig. 5A). Because transcytosed receptors are
able to reinternalize (see below) and our biochemical transcytosis assay only detects Fab fragments present on the opposite surface at any
given time, the actual extent of transcytosis is most likely underestimated.
Although Fab fragments internalized from the basolateral side
efficiently transcytosed, only 15-20% of the Fab fragments
internalized from the apical side transcytosed (Fig.
6D) and ~60% were released into the apical medium or still on the apical surface after 60 min
(Fig. 6, A and C). Transcytosis by FcRII/FcRn,
however, was still significantly higher than that by the tail-minus
mutant, where less than 5% of the Fab fragments transcytosed (Fig.
6D). As discussed above, also transcytosis in the apical to
basolateral direction is probably underestimated, because translocated
receptors can reinternalize from the basolateral cell surface (see
below).
More than 90% of the Fab fragments recovered in the medium after 60 min at 37 °C were trichloroacetic acid precipitable and thus
represented intact Fab fragments that had either dissociated after
warming the cells or following recycling or transcytosis. Consistent
with the lack of Fab degradation, little if any colocalization of
internalized Fab fragments with late endosomes and lysosomes (labeled
with the anti-canine lamp-2 antibody AC17, see Ref. 45) was observed
(not shown).
To visualize transcytosis, cells expressing FcRII/FcRn were incubated
for 45 min at 37 °C in the presence of 2.4G2 Fab fragments added to
the apical or basolateral compartment. Cells were then cooled on ice,
and a labeled goat anti-rat antibody was added to the opposite chamber
for 1 h at 4 °C. If Fab fragments were transcytosed via
FcRII/FcRn, then they should be able to bind the labeled second
antibody on the opposite cell surface. As shown in Fig.
7, A and D, cells
incubated with Fab fragments in the apical chamber showed the typical
labeling of the basolateral surface, whereas addition of Fab fragments
to the basolateral chamber resulted in apical plasma membrane staining,
indicating that Fab fragments had transcytosed in both directions. As
expected for plasma membrane labeling, the staining was sensitive to
acid pH wash (not shown), and no labeling was observed in the absence of Fab fragments (not shown), if cells were incubated with Fab fragments at 4 °C (Fig. 7,
C and F) or if nontransfected cells were used
(not shown). If cells carrying labeled second antibody bound to
transcytosed Fab fragments (as shown in Fig. 7, A and D) were warmed for 15 min at 37 °C, labeled antibodies
were internalized into a vesicular compartment (Fig. 7, B
and E), showing that transcytosed FcRII/FcRn could indeed
reinternalize from the opposite cell surface.
In summary, these experiments show that Fab fragments bound to apical
or basolateral FcRII/FcRn are able to transcytose to the opposite
plasma membrane domain where they can reinternalize.
Basolateral to Apical and Apical to Basolateral Transcytosis Are
Differently Regulated--
Depending on the protein or pathway
analyzed, BFA, calmodulin antagonists, and microtubule-disrupting drugs
have been shown to have different effects on basolateral to apical and
on apical to basolateral transcytosis. To analyze how these compounds
affect bidirectional transcytosis of FcRII/FcRn, 125I-2.4G2
Fab was bound to the apical or basolateral surface, and the fraction of
Fab fragments that transcytosed in the presence or absence of the drugs
was determined following the incubation of the cells at 37 °C for
60 min.
As shown in Fig. 8, BFA had no significant effect on transcytosis
in either direction. Apical to basolateral transport was also not
affected if microtubules were depolymerized or if calmodulin function
was impaired using W-7. In contrast, W-7 and nocodazole severely
interfered with basolateral to apical transport and reduced transcytosis to 40 and 60% of control values, respectively. These results show different requirements for calmodulin activity and microtubules in apical to basolateral and basolateral to apical transcytosis of the same protein.
FcRn is implicated in the lumenal to serosal transepithelial
transport of IgG in the small intestine, the placental
syncytiotrophoblast and the yolk sac (2, 46). When expressed in MDCK
cells, newly synthesized human and rat FcRn are delivered in a
nonpolarized manner to the apical and basolateral surface from where
they transcytose to the opposite domain.2 Using chimera
encoding the cytosolic tail of rat FcRn fused to the ecto- and
transmembrane domain of Fc Biosynthetic Surface Transport--
Newly synthesized FcRII/FcRn
was delivered to the cell surface in a nonpolarized fashion. Because
FcRn can shuttle between the apical and basolateral surfaces following
endocytosis, efficient sorting to one or the other domain during
biosynthesis may not be required. In contrast, the pIgR, which
transcytoses in the basolateral to apical direction and is then
proteolytically cleaved upon arrival on the apical domain (3), requires
efficient basolateral sorting to ensure that all receptor molecules are
available for transcytosis. The nonvectorial transport of FcRII/FcRn
may be because of a weak basolateral sorting signal in the cytosolic domain of FcRn in conjunction with a putative recessive apical determinant in the transmembrane and/or ectodomain of Fc Endocytosis and Transcytosis--
In contrast to basolateral
sorting, efficient internalization of FcRII/FcRn requires the presence
of the di-leucine motif. As observed for Fc
FcRII/FcRn transcytosed in both directions. Although receptors
internalized from the apical domain mostly recycled back to the apical
domain, 15-20% of apically prebound Fab fragments transcytosed to the
basolateral surface after 60 min. Fab fragments internalized from the
basolateral surface transcytosed more efficiently (30%) and a smaller
fraction recycled. The amount of transcytosis is likely underestimated
because transcytosed Fab fragments are rapidly reinternalized and the
biochemical assay only detects Fab fragments present on the surface at
any particular time point. For comparison, 20 and 40% of antibodies
prebound to a mutant low density lipoprotein receptor (15) or
Fc
Signals for polarized sorting are not only decoded at the level of the
trans-Golgi network but are also recognized in endosomes. Thus, the
nonvectorial surface transport of FcRn in the biosynthetic pathway
correlates well with the observation that receptors internalized from
one or the other cell surface could both recycle and transcytose and is
consistent with FcRn encoding a "weak" basolateral signal. Indeed,
routing of FcRII/FcRn is very similar to that of low density lipoprotein receptor mutants, which still encode the weak
membrane-proximal basolateral sorting signal but lack the strong distal
determinant (15). Because serine phosphorylation is implicated in
transcytosis of the pIgR (58, 59), phosphorylation of the CKII site may also regulate transcytosis of the rat FcRn, either by acting as an
independent signal or by activating or inactivating a basolateral sorting activity. Because the human FcRn lacks the CKII site but still
undergoes bidirectional transcytosis, a putative regulatory role of
phosphorylation on receptor traffic may be restricted to the rodent
receptor. Internalized Fab fragments were not transferred to lysosomes,
consistent with a second function of FcRn in addition to transcytosis,
namely recycling IgG internalized in the fluid phase, thereby
preventing lysosomal degradation of endocytosed IgG (2, 46).
BFA-sensitive coat proteins, microtubules, and the activity of
calmodulin have been implicated in transcytosis. BFA inhibits basolateral to apical transcytosis of ligand-loaded but not of empty
pIgR (8, 60). Although BFA did not affect FcRII/FcRn mediated
transcytosis of Fab fragments, it will be interesting to determine
whether transcytosis of IgG by the native FcRn is affected by the drug.
Nocodazole selectively inhibited basolateral to apical transcytosis of
FcRII/FcRn. This result is consistent with the inhibition of
basolateral to apical transport of the pIgR (6, 7) and the lack of an
effect on apical to basolateral transport of Fc
MDCK and other cell lines expressing FcRn and the chimera will provide
in vitro systems to unravel the molecular mechanisms underlying IgG transcytosis and maintenance of IgG homeostasis. Because
transcytosis of the chimera in the apical to basolateral direction was
not very efficient, it will be interesting to determine whether IgG
binding stimulates the transcytotic activity we found associated with
the FcRn tail. Specific stimulation of apical to basolateral
transcytosis by ligand may ensure efficient transfer of IgG across the
enterocyte and syncytiotrophoblast. The observed basolateral to apical
transport may then serve to recycle empty FcRn molecules for reuse. The
presence of a CKII phosphorylation site in the cytosolic tail of the
rat and not the human FcRn raises the question as to the functional
role, if any, of FcRn phosphorylation and whether the intracellular
traffic of the rodent and human isoforms differ with respect to
transport kinetics, regulation of transport by ligand, or a particular
cell type (i.e. enterocyte versus placental syncytiotrophoblast).
RIIb. Newly synthesized chimera were
delivered in a nonvectorial manner to the apical and basolateral cell
surface, from where the chimera were able to internalize and
transcytose. Apical to basolateral and basolateral to apical
transcytosis were differently regulated. This intracellular routing of
the chimera is similar to that of the native FcRn, indicating that the
cytosolic tail of the receptor is necessary and sufficient to endow an
unrelated FcR with the intracellular transport behavior of FcRn.
Furthermore, the di-leucine motif in the cytosolic domain of FcRn was
required for rapid and efficient endocytosis but not for basolateral
sorting of the chimera.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RIIb and wild-type or
mutant cytosolic domains of rFcRn. Newly synthesized FcRII/FcRn
receptors were delivered in a nonpolarized manner to the apical and
basolateral surface of MDCK cells. A di-leucine motif in the cytosolic
domain of FcRn was critical for efficient endocytosis but not for
basolateral sorting. Following internalization, the chimeric receptors
mediated bidirectional transcytosis. Trafficking of the FcRII/FcRn
chimera closely resembles that of FcRn, consistent with a critical role
of the cytosolic tail in FcRn traffic. Transcytosis of the chimera was
not affected by BFA and only basolateral to apical transport required
calmodulin and microtubule function, showing that transcytosis in the
two directions is subject to different regulatory mechanisms.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RIIb antibody 2.4G2 (36) and a rabbit polyclonal anti
Fc
RIIb serum (kindly provided by I. Mellman, Yale University, New
Haven, CT) were used to detect chimeric receptors. Affinity purified
labeled second antibodies were purchased from Jackson ImmunoResarch
Laboratories, Inc. (West Grove, PA). 2.4G2 Fab fragments were
radioiodinated to specific activities of 2-7 × 106
cpm/µg using Iodogen (Pierce), unincorporated 125I was
removed by ion exchange chromatography on Dowex-1 (Sigma) as described
(37).
RIIb2 ecto- and transmembrane domain and the wild-type or mutated
FcRn cytosolic tail, a 5' PCR primer was used to introduce an in frame
AflII site just adjacent to the C-terminal end of the
transmembrane domain of FcRn. PCR products encoding wild-type and
mutant FcRn tails were then appended to the lumenal and transmembrane domains of a tail-less Fc
RIIb2 construct carrying a unique
AflII site following the transmembrane domain (38),
resulting in the in frame fusion to the ecto- and transmembrane region
of Fc
RIIb2 (see Fig. 1). All fragments synthesized by PCR were
verified by sequencing. The sequence of the primers is available upon request.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RIIb2 were fused to wild-type
or mutant cytosolic domains of FcRn (Fig.
1). Tail-less Fc
RIIb2 accumulates on
the apical surface of MDCK cells because it lacks the cytosolic di-leucine motif required for basolateral sorting and endocytosis (15,
42-44), making it an attractive tag to analyze the role of cytosolic
tails in targeting to different intracellular compartments or the
basolateral plasma membrane (15, 38, 41). In addition to a chimera
encoding the wild-type FcRn tail (FcRII/FcRn), we generated constructs
encoding a deletion of the C-terminal third (CT24) or the complete tail
(CT4). Because the FcRn tail encodes a di-leucine motif similar to that
responsible for basolateral sorting and endocytosis of Fc
RIIb2 (43,
44), we also constructed chimera encoding alanine substitutions of
Leu-22 and/or Leu-23 in the context of the CT24 mutant (CT24 L22A, CT24
L23A, and CT24 L22A,L23A). The different constructs were transfected
into MDCK cells, and several clones stably expressing the different
receptor chimera were selected and further analyzed.
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Fig. 1.
Amino acid sequences of the cytosolic domains
of wild type and mutant FcRII/FcRn chimera. Sequences are shown in
the single-letter code, and alanine substitutions in the wild-type
sequence are in bold. The CKII phosphorylation consensus
sequence is underlined. Residues are numbered from left to
right, with position 1 corresponding to the presumed first amino acid
in the cytosolic domain.
RII ectodomain monoclonal antibody 2.4G2. FcRII/FcRn
predominantly localized to intracellular compartments (Fig.
2A), whereas the tail minus
construct was mostly observed on the plasma membrane (Fig.
2B). If cells expressing FcRII/FcRn were incubated with the
antibody on ice and then fixed, plasma membrane staining was readily
detectable (Fig. 2C), indicating that a fraction of
FcRII/FcRn was present on the plasma membrane at steady state. Antibody
was internalized into a vesicular compartment if cells carrying
prebound antibody were warmed to 37 °C (Fig. 2E), showing
that the chimeric receptors were able to internalize. In contrast,
antibody prebound to cells expressing the tail minus construct (Fig.
2D) was not internalized and could be removed from the cell
surface by washing the cells with acid (Fig. 2F).
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Fig. 2.
Steady state distribution, surface expression
and internalization of FcRII/FcRn and a tail-minus mutant. Cells
expressing FcRII/FcRn (A) or CT4 (B) were fixed,
permeabilized, and stained with Fab fragments of the mouse monoclonal
anti-Fc RIIb antibody 2.4G2 followed by a labeled second antibody. In
panels C-F, 2.4G2 was bound to cells expressing
FcRII/FcRn (C and E) or CT4 (D and
F) at 4 °C. Cells were then washed and either fixed
(C and D) or incubated at 37 °C for 30 min to
allow for the internalization of bound antibody (E and
F). After internalization, 2.4G2 remaining on the cell
surface was removed by washing with acid (pH 2.5) before fixation,
permeabilization, and staining with the labeled second antibody.
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Fig. 3.
A di-leucine containing cytosolic region of
FcRn is critical for efficient endocytosis. MDCK cells expressing
the indicated chimera were allowed to internalize prebound
radioiodinated 2.4G2 Fab fragments for the periods of time shown.
After cooling the cells on ice, Fab fragments present in the medium, on
the cell surface (pH 2.5 wash) or inside the cells was determined. The
fraction of the initially bound Fab fragments that have been
internalized at the different time points is shown. Values were
determined in triplicate and differed by less than 15%.
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Fig. 4.
Polarized steady state distribution and
insertion of newly synthesized chimeric receptor. A,
steady state distribution of wild-type and mutant FcRII/FcRn. Cells
expressing the indicated chimera were incubated with radiolabeled 2.4G2
Fab added either from the apical or the basolateral compartment on ice.
Quantitation of the binding for each clone was obtained from five to
nine independent experiments, each carried out using duplicate or
triplicate filters. Values differed by less than 5% and similar
distributions were obtained for several clones expressing different
levels of the chimeric proteins. B, cell surface transport
of newly synthesized chimeric receptors. MDCK cells expressing the
indicated chimera were metabolically labeled for 15 min. Labeled
proteins were chased for 30 or 45 min and the apical (ap) or
basolateral (bl) cell surface was then biotinylated. Cells
were lysed and total chimeric receptors were immunoprecipitated. An
aliquot of the immunoprecipitate was used to determine total labeled
receptor (Total); the rest was incubated with immobilized
streptavidin to isolate biotinylated cell surface receptors
(Surface). Precipitates were analyzed by SDS-polyacrylamide
gel electrophoresis and autoradiography. The bands on the
autoradiograph were quantitated by densitometry, and the amount of
surface receptor was normalized to the total amount of labeled chimera.
The fraction of each mutant inserted into the apical or basolateral
domain is shown for a typical experiment. Similar results were obtained
if cells were chased for 30 min in the continuous presence of the
anti-FcRII antiserum to detect labeled receptors that would only
transiently be exposed on the cell surface (not shown).
RII antiserum (42) to
detect labeled receptors, which may only transiently be exposed on the
cell surface (data not shown).
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Fig. 5.
Analysis of Fab fragments internalized from
the basolateral surface. Radiolabeled Fab fragments were prebound
to the basolateral surface of MDCK cells expressing FcRII/FcRn or CT4,
and cells were then transferred to 37 °C. The fraction of Fab
fragments transcytosed (released into the apical medium and present on
the apical surface; panel D), present on the basolateral
surface (acid sensitive; panel A), intracellular (acid
resistant; panel B), or released into the basolateral medium
(panel C) were determined at the indicated periods of time
and plotted as percent of initially bound Fab fragments. Shown is a
typical experiment carried out in triplicates, and the individual
values differed by less than 5%.
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Fig. 6.
Analysis of Fab fragments internalized from
the apical surface. Radiolabeled Fab fragments were prebound to
the apical surface of MDCK cells expressing FcRII/FcRn or CT4, and
cells were then transferred to 37 °C. The fraction of Fab fragments
transcytosed (released into the basolateral medium and present on the
basolateral surface; panel D), present on the apical surface
(acid sensitive; panel A), intracellular (acid resistant;
panel B), or released into the apical medium (panel
C) were determined at the indicated periods of time and plotted as
percent of initially bound Fab fragments. Shown is a typical experiment
carried out in triplicate, and the individual values differed by less
than 5%.
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Fig. 7.
Transcytosed Fab fragments are reinternalized
from the opposite cell surface. MDCK cells expressing FcRII/FcRn
were incubated for 45 min at 37 °C (A, B,
D, and E) or 4 °C (C and
F) with 2.4G2 Fab fragments present in the basolateral
(A-C) or apical (D-F)
compartment and then cooled on ice. A labeled second antibody was
allowed to bind from the opposite compartment for 60 min on ice. Cells
were then either fixed and stained (A, D,
C, and F), or transferred to 37 °C for 15 min
to allow for endocytosis of bound labeled antibodies (B and
E). Membrane staining (A and D)
indicates transcytosis of Fab fragments and vesicular labeling
(B and E) shows reinternalization of transcytosed
Fab fragments. No binding of second antibody was seen if cells were
incubated with Fab fragments at 4 °C (C and
F).
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Fig. 8.
Differential regulation of apical to
basolateral and basolateral to apical transcytosis. Radiolabeled
Fab fragments were prebound to the apical or basolateral surface of
MDCK cells expressing FcRII/FcRn. Cells were then transferred to
37 °C for 60 min in the presence or absence of BFA, nocodazole, or
W-7. Fab fragments that had transcytosed were determined in triplicates
and are plotted as percent of initially bound Fab fragments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RIIb2, we analyzed the contribution of the
cytosolic tail of FcRn to its intracellular routing. Interestingly, the
overall intracellular traffic of the chimera was very similar to that
of native FcRn, indicating that the cytosolic domain of FcRn can confer
the intracellular transport behavior of FcRn to an unrelated FcR.
Furthermore, a di-leucine motif in the tail of FcRn was identified that
is critical for rapid and efficient endocytosis but not for basolateral sorting.
RIIb2. The
presence of a weak basolateral sorting activity in the FcRn tail is
implicated by the larger fraction of FcRII/FcRn transported basolaterally as compared with the tail-minus construct (50%
versus 20%). Di-leucine-based signals can mediate
basolateral sorting of several proteins, including Fc
RIIb2 (43, 44).
Although FcRn encodes a di-leucine motif at a similar position in the
tail as Fc
RIIb2, cell surface insertion of FcRII/FcRn was not
affected by inactivating the di-leucine motif in the context of a
truncated tail construct (CT24 L22A,L23A), suggesting that an unknown
feature in the membrane proximal half of the tail is responsible for
the more pronounced basolateral surface transport of FcRII/FcRn as compared with CT4. Alternatively, because the di-leucine motif is
located upstream from a serine residue within a CKII site (see Fig. 1)
that is subject to
phosphorylation,3 its
basolateral sorting activity may be subject to regulation. CKII
phosphorylation of serines has been implicated in modulating the
traffic of several proteins (47-50). Also the pIgR encodes a
phosphorylated serine (Ser-664) in the vicinity of the basolateral sorting signal and substitution of Ser-664 by an aspartic acid to mimic
the negative charge of a phosphoserine results in the nonpolarized
transport of the normally basolaterally sorted receptor (51). Although
it is conceivable that CKII phosphorylation plays a direct role in
basolateral sorting or indirectly regulates the activity of a
basolateral signal, it has to be noted that the human FcRn lacks the
CKII phosphorylation site, but it is also transported in a nonvectorial
fashion in the biosynthetic route.2
RIIb2 (43, 44, 52),
endocytosis of FcRII/FcRn was only slightly reduced by deleting the
C-terminal half of the tail (CT24) but was dramatically inhibited if
one or both leucines were mutated to alanine. Nevertheless, CT24
L22A,L23A retained some residual internalization activity when compared
with a tail-minus construct. A CKII site plays a role in
internalization of furin (53-55), and efficient endocytosis of the
pIgR requires, in addition to two tyrosine determinants (56), a serine
within a CKII consensus sequence (57). Thus, a possible involvement of
the CKII site in the cytosolic domain of rat FcRn in regulating the
endocytic activity of the di-leucine signal remains to be analyzed.
RIIb2 (6) are transcytosed in the apical to basolateral direction,
but less that 5% of prebound dIgA is translocated basolaterally by the
pIgR (6). In the opposite direction, 25-50% (depending on the nature
of the ligand or the assay) of the pIgR transcytoses (3, 6, 11), but
less than 5% of the low density lipoprotein receptor, Fc
RIIb2, or
the transferrin receptor is translocated to the apical surface (14, 15,
39).
RIIb2 (6, 7) and
suggests that microtubules are either not required for apical to
basolateral transcytosis or that a subset of nocodazole-insensitive
microtubules is involved. Also similar to results obtained for the pIgR
(12, 13), the CaM antagonist W-7 selectively interfered with
basolateral to apical transcytosis of FcRII/FcRn. CaM binds to a region
in the cytosolic domain of the pIgR that overlaps with the basolateral sorting determinant (61). It is not known whether CaM binds to the FcRn
tail, but the effect of CaM antagonists on recycling and lysosomal
transport (12, 13), as well as that on endosome morphology (12),
indicate that CaM may play a general role in endocytic membrane traffic.
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ACKNOWLEDGEMENTS |
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We thank Catherine Fumey for excellent technical expertise in the construction of the chimeric receptors, Thierry Laroche for help with the confocal microscope, and Thomas Simmen, Isabelle Baribaud, and Renate Fuchs for fruitful discussions. We thank Ira Mellman, Andre Le Bivic, and Claudia Koch-Brandt for antibodies.
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FOOTNOTES |
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* This work was funded by a grant and a career development award from the Swiss National Science Foundation (to W. H.) and supported by the Canton de Vaud.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.
Supported in part by the Austrian Science Research Fund
(P-12084Med). Present address: Allgemeines Kraukenhaus, Dept. of
General and Experimental Pathology, University of Vienna, Vienna,
Austria 1090.
§ To whom correspondence should be addressed. Tel.: 41-21-692-5737; Fax: 41-21-692-5705; E-mail: Walter.Hunziker{at}ib.unil.ch.
2 A. Praetor, I. Stefaner, and W. Hunziker, manuscript in preparation.
3 I. Stefaner and W. Hunziker, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are:
pIgR, polymeric
immunoglobulin receptor;
BFA, brefeldin A;
CaM, calmodulin;
FcR, Fc
receptor;
FcRn, neonatal IgG FcR;
rFcRn, rat FcRn;
FcRII/FcRn, chimera
between FcRIIb2 ecto- and transmembrane domain and FcRn cytosolic
tail;
CKII, casein kinase II;
MDCK, Madin-Darby canine kidney;
PCR, polymerase chain reaction.
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REFERENCES |
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