Institute of Molecular and Cell Biology, Epithelial Cell Biology Laboratory, 30 Medical Drive, Singapore 117609, Republic of Singapore
* Author for correspondence (e-mail: hunziker{at}imcb.nus.edu.sg )
Accepted 19 March 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Human immunoglobulin G, Major histocompatibility complex, Endoplasmic reticulum, Oligomerization
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The transcytotic and protective functions of FcRn are intimately linked to its pH-dependent IgG-binding properties. FcRn binds IgG at a mildly acidic pH, as found in the intestinal lumen or in endosomes, but not at neutral pH. In the gut, FcRn is therefore thought to bind IgG on the lumenal surface and, following transcytosis, to release the IgG into the circulation upon exposure to the neutral serosal pH. In the absence of a pH gradient, IgG may be internalized in the fluid phase and bind FcRn in the acidic milieu of endosomes, from where it could either be transcytosed or recycled.
FcRn, like MHC class I, consists of an -chain and
ß2-microglobulin (ß2m). Indications that an
association of the FcRn
-chain with ß2m is important
for the assembly of a functional receptor come from
ß2m-knockout mice
(Zijlstra et al., 1990
), which
show defects in several functions associated with FcRn. Newborn
ß2m-deficient pups show lower IgG serum levels at birth and
accumulate less IgG before weaning than normal littermates
(Israel et al., 1995
;
Zijlstra et al., 1990
).
Furthermore, adult mice lacking ß2m have a higher IgG
turnover, resulting in lower serum IgG levels
(Ghetie et al., 1996
;
Israel et al., 1996
;
Junghans and Anderson,
1996
).
While the importance of ß2m for a functional FcRn is well
recognized, the precise role of ß2m in FcRn function is not
known. IgG transport in mice lacking ß2m could reflect a
failure of FcRn to either reach the cell surface or to bind IgG. In the case
of major histocompatibility complex (MHC) class I molecules, assembly of the
-chain with ß2m and loading of antigenic peptide in the
endoplasmic reticulum (ER) are required for cell surface transport of
functional class I molecules (Pamer and
Cresswell, 1998
). However, CD1d, an MHC class I-like CD1 molecule,
is efficiently expressed on the cell surface even in the absence of
ß2m (Hyun et al.,
1999
). Thus, assembly with ß2m does not appear to
be a general requirement for surface transport of MHC class I-like proteins.
The absence of ß2m could also interfere with other
intracellular transport steps such as recycling from endosomes to the cell
surface. Alternatively, binding of IgG by FcRn may depend or the presence of
ß2m.
To determine whether ß2m is required for cell surface
expression of FcRn, IgG binding or both, we characterized intracellular
transport and ligand-binding properties of human FcRn (hFcRn) expressed in
ß2m-deficient FO-1 cells (FO-1) or in FO-1 cells stably
expressing human ß2m (FO-1 ß2m)
(D'Urso et al., 1991). We show
that in cells lacking ß2m, the FcRn
-chain fails to
reach the cell surface and is retained in the ER. Furthermore, binding of
human IgG (hIgG) to the FcRn
-chain is reduced at acidic pH in the
absence of ß2m. Thus, assembly of the FcRn
-chain with
ß2m is important for transport of the receptor from the ER to
the cell surface as well as for efficient pH-dependent binding of IgG.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies
For western blotting, hybridoma supernatants containing 9E10 anti-Myc
antibodies (kindly provided by R. Iggo, Epalinges, Switzerland), polyclonal
anti-ß2m antibodies (Sigma) and a polyclonal rabbit anti-FcRn
peptide serum (Praetor et al.,
1999) were used. Polyclonal anti-Myc (Santa Cruz Biotechnology)
and polyclonal anti-ß2m antibodies (Abcam) were used for
co-immunoprecipitation experiments. For immunofluorescence, polyclonal
anti-Myc (Upstate Biotechnology), polyclonal anti-ß2m (Abcam),
monoclonal anti-EEA1 (Transduction Laboratories) and monoclonal
anti-ribophorin II (kindly provided by D. Meyer, Heidelberg, via A. Helenius,
Zurich) antibodies were used. HRP-coupled secondary antibodies were purchased
from Research Biolabs. Affinity purified fluorescently labeled secondary
antibodies were from Molecular Probes.
Plasmids
In analogy to a Flag-tagged hFcRn
(Praetor et al., 1999), a cDNA
carrying the Ig
leader sequence and an N-terminal Myc-epitope tag
(Myc-hFcRn) was generated and cloned into a modified pLNCX expression vector
(Clontech) carrying a hygromycin resistance gene
(Reichert et al., 2000
).
Details on the construction of the G418-resistant plasmid carrying the human
ß2m cDNA can be found elsewhere
(D'Urso et al., 1991
).
Cell culture and transfection
FO-1 and G418-resistant FO-1 cells stably expressing human
ß2m (FO-1ß2m) (D'Urso
et al., 1991) were generously provided by Patrizio Giacomini
(Rome, Italy). Cells were cultured in DMEM (low glucose) supplemented with 10%
FCS, 50 µg/ml penicillin and 50 µg/ml streptomycin and glutamine. Cells
were transfected with a Myc-hFcRn plasmid using the Transfast transfection kit
(Promega) and selected in 0.5 mg/ml hygromycin. Resistant clones were analyzed
for expression by immunofluorescence and immunoblotting and two clones for
each transfection were used in further experiments. Stably transfected cells
were maintained in G-418 (0.25 mg/ml) and/or hygromycin (0.5 mg/ml).
Western blot analysis
To detect FcRn and ß2m, cells were lysed in 0.5% Triton
X-100 in PBS containing protease inhibitors. Equal amounts of total protein,
as determined by Bradford, were separated on 10% Tris-tricine gels. Proteins
were transferred onto PVDF membranes by wet blotting at 200 mA for 5 hours.
After blocking the membranes with 5% milk in PBS, they were probed with mouse
monoclonal 9E10 antibodies (1:1000), polyclonal anti-FcRn serum (1:500) or
polyclonal anti-ß2m (1:500), followed by secondary HRP-labeled
anti-mouse or anti-rabbit antibodies (1 µg/ml) and visualized by
chemiluminescence.
Co-immunoprecipitation
Cells expressing the Myc epitope-tagged human FcRn alone or in combination
with ß2m were lysed in 5 mg/ml CHAPS in 50 mM phosphate buffer
pH 7.4 containing protease inhibitors. Cell lysates (equal amounts of total
protein) were precleared and incubated for 2 hours at 4°C with either 9E10
prebound to protein G sepharose (1 µl/100 µg lysate) or polyclonal
anti-ß2m prebound to protein G sepharose (0.5 µg/100 µg
lysate). Immune complexes were washed three times with CHAPS lysis buffer and
bound proteins were eluted by heating in unreducing sample buffer for 30
minutes at 40°C. SDS-PAGE and blotting was carried out as described above.
Membranes were probed with polyclonal anti-Myc (2.5 µg/ml) or polyclonal
anti-ß2m (0.2 µg/ml) antibodies.
Immunofluorescence
Cells grown on coverslips were processed for immunofluorescence as
described (Stefaner et al.,
1999). Briefly, cells were fixed and labeled with polyclonal
anti-Myc antibodies (5 µg/ml). To monitor cell surface expression or
internalization, cells were incubated in the presence of polyclonal anti-Myc
antibodies (5 µg/ml in L-15 medium, pH 7.4) at 4°C for 45 minutes or at
37°C for 60 minutes, respectively. For co-localization experiments, cells
grown on coverslips were fixed and stained with polyclonal anti-Myc (5
µg/ml), polyclonal anti-ß2m (5 µg/ml), monoclonal
anti-EEA1 (12 µg/ml) or monoclonal anti-ribophorin II (1:100) antibodies.
Fluorescently labeled anti-mouse (Alexa 488) or anti-rabbit (Alexa 568)
secondary antibodies were used at 2 µg/ml. IgG internalization was
performed as described (Praetor et al.,
1999
). Briefly, cells were allowed to internalize hIgG (1
µg/ml) for 30 minutes at 37°C and then washed on ice with PBS.
Internalized IgG was visualized with fluorescently labeled goat anti-human
(Alexa 488; 2 µg/ml) secondary antibodies.
Surface biotinylation
Cell surface biotinylation was carried out as described
(Praetor et al., 1999).
Briefly, precleared cell lysates were incubated with streptavidin-agarose (5
µl/100 µg lysate) or 9E10 prebound to protein G sepharose (1 µl/100
µg lysate) for 2 hours at 4°C. The precipitates were washed three times
with RIPA buffer, proteins eluted either by boiling for 10 minutes
(streptavidin precipitate) or heating for 30 minutes to 40°C (9E10
precipitate) in reducing sample buffer and analyzed by SDS-PAGE as described
above. Streptavidin precipitates were probed with 9E10 antibodies as
described. In the case of 9E10 precipitates, membranes were blocked with 1%
BSA in PBS and probed with streptavidin-HRP (0.1 µg/ml).
EndoH and glycosidase F digestion
30 µl cell lysate was incubated with EndoH (5 mU) or glycosidase F (1 U)
at 37°C for 3.5 hours in the presence of PMSF. Cell lysates were adjusted
to pH 5-6 by the addition of 0.5 µl 50 mM sodium acetate pH 5.2 for the
EndoH digest. Control lysates were incubated at 37°C for 3.5 hours in the
absence of the enzyme. The reaction was terminated by the addition of reducing
SDS-PAGE sample buffer.
IgG precipitation
Binding and precipitation of FcRn from cell lysates using IgG-agarose was
carried out as outlined (Praetor et al.,
1999).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As shown in Fig. 1A, a 47
kDa protein corresponding to hFcRn was detected on blots probed with either
anti-Myc (-Myc) or anti-FcRn (
-FcRn) antibodies in transfected
(lanes 3,4) but not in control (lanes 1,2) FO-1 cells. As expected,
ß2m was not present in FO-1 cells
(
-ß2m; lanes 1,3) but readily detected in FO-1 ß2m
cells stably expressing human ß2m
(
-ß2m; lanes 2,4).
|
Immunofluorescence experiments confirmed the co-localization of
ß2m with the FcRn -chain in FO-1 ß2m cells. As
shown in Fig. 1B, the FcRn
-chain (
-Myc) and ß2m
(
-ß2m) were detected by indirect immunofluorescence in
transfected FO-1 ß2m cells (Fig.
1Bb,d), but not in control FO-1 cells
(Fig. 1Ba,c). Merging the
staining for the
-chain (green) and ß2m (red) showed
extensive co-localization of the two proteins
(Fig. 1Be, yellow).
To demonstrate directly that ß2m associates with the FcRn
-chain in FO-1 ß2m cells, cell lysates were immunoprecipitated
with anti-Myc antibodies and immunoprecipitates blotted with
anti-ß2m antibodies (Fig.
1C, lanes 1-4). Alternatively, ß2m
immunoprecipitates were blotted with anti-Myc antibodies to detect the FcRn
-chain (Fig. 1C, lanes
5-8). ß2m was specifically co-precipitated with the
-chain from cells expressing both proteins
(Fig. 1C, lane 4) but not from
control FO-1 cells (Fig. 1C,
lane 1) or from cells expressing the
-chain alone
(Fig. 1C, lane 2) or
ß2m alone (Fig.
1C, lane 3). Similarly, FcRn was co-precipitated with
ß2m only from FO-1 ß2m cells expressing the receptor
-chain (Fig. 1C, lane
8).
Thus, FO-1 cells expressing the FcRn -chain either alone or in
combination with ß2m were obtained and, in cells co-expressing
the two proteins, the
-chain and ß2m assembled with
each other.
ß2m is important for efficient pH-dependent binding
of IgG by FcRn
We next determined whether FO-1 and FO-1 ß2m cells expressing the FcRn
-chain were able to bind and internalize IgG. Cells were allowed to
endocytose hIgG (1 µg/ml) at 37°C, either at pH 6.5 or pH 7.4, and
internalized IgG was detected with a labeled secondary antibody. As shown in
Fig. 2A, FO-1 ß2m cells
expressing the FcRn
-chain internalized IgG at pH 6.5
(Fig. 2Ad) but not at pH 7.4
(Fig. 2Ah), consistent with the
known pH-dependence of ligand binding by FcRn. FO-1 cells expressing the
-chain alone failed to internalize hIgG at either pH
(Fig. 2Ac,g). Similarly,
control FO-1 and FO-1 ß2m cells not expressing the
-chain did not
internalize IgG (Fig.
2Aa,b,e,f), showing that, where detected
(Fig. 2A,d), internalization
was receptor mediated and not due to fluid phase endocytosis. Similar results
were obtained with 10 µg/ml IgG (data not shown).
|
The above results indicate that in the absence of ß2m, FcRn
does not bind IgG or, alternatively, it is not expressed on the cell surface.
To test whether ß2m was required for IgG binding, we analyzed
whether FcRn present in cell lysates of transfected FO-1 or FO-1ß2m cells
was able to bind to IgG agarose. As shown in
Fig. 2B, FcRn from FO-1ß2m
cells efficiently bound to IgG agarose at pH 6.5
(Fig. 2B, top panel, lane 8,
-Myc) but no binding was detected at pH 7.4
(Fig. 2B, bottom panel, lane 8,
-Myc). ß2m was present in the bound fraction
(Fig. 2B, top panel, lane 8,
-ß2m), consistent with the binding of an
-chain-ß2m heterodimer to the IgG-agarose. In contrast,
binding of the
-chain from lysates of FO-1 cells lacking
ß2m, although still detectable, was reduced at pH 6.5
(Fig. 2B, top panel, lane 6,
-Myc). Interestingly, while FcRn in FO-1ß2m lysates failed to bind
to IgG agarose at pH 7.4, binding of the
-chain was reproducibly
observed in the absence of ß2m
(Fig. 2B, lower panel, lane 6,
-Myc). Immunoblots of aliquots of the FO-1 and FO-1ß2m cell
lysates used for the binding experiments confirmed the presence of similar
amounts of the FcRn
-chain, ruling out the possibility that differences
in binding were due to different expression levels of the
-chain (lanes
1-4).
In conclusion, binding of IgG to FcRn was significantly reduced in the absence of ß2m. However, since IgG binding was not completely abolished in the absence of ß2m, the reduced ligand binding alone is unlikely to account for the lack of IgG internalization in FO-1 cells.
ß2m is important for surface expression of FcRn
We next analyzed the subcellular distribution of the FcRn -chain in
FO-1 and FO-1ß2m cells by indirect immunofluorescence to determine
whether ß2m was required for cell surface expression of FcRn.
Cells were fixed, permeabilized and labeled with anti-Myc antibodies to
visualize the steady state distribution of the FcRn
-chain
(Fig. 3a-d). As expected, the
-chain was detected only in transfected FO-1 or FO-1ß2m cells
(Fig. 3c,d) but not in
untransfected control cells (Fig.
3a,b). In FO-1ß2m cells, the FcRn
-chain showed a
discrete punctate distribution (Fig.
3d), whereas the labeling in parental FO-1 cells was rather
diffuse and reticular (Fig.
3c). To determine whether the FcRn
-chain was expressed on
the cell surface, anti-Myc antibodies were allowed to bind to live cells on
ice and visualized by indirect immunoflurescence
(Fig. 3e-h). Surface staining
for the FcRn
-chain was observed only in transfected FO-1ß2m cells
(Fig. 3h) but not in FO-1 cells
lacking ß2m (Fig.
3g) or in untransfected FO-1 or FO-1ß2m cells
(Fig. 3e,f).
|
Since the FcRn cycles between the plasma membrane and endosomes
(Praetor et al., 1999), it is
conceivable that the FcRn
-chain shows a predominant intracellular
equilibrium distribution in FO-1 cells but nevertheless transiently appears on
the cell surface. We therefore incubated cells in the presence of anti-Myc
antibodies in culture media (pH 7.4) at 37°C for 60 minutes, a sensitive
assay to measure the transient surface appearance of a membrane protein since
proteins cycling through the plasma membrane will bind and internalize
antibodies in the media (Höning and
Hunziker, 1995
). As shown in
Fig. 3, FO-1ß2m cells
expressing the FcRn
-chain efficiently accumulated anti-Myc antibodies
in endocytic vesicles (Fig. 3l)
but no antibody uptake was observed in FO-1 cells expressing hFcR alone
(Fig. 3k). Antibody uptake in
FO-1ß2m cells was receptor mediated since control cells showed no
internalization (Fig. 3i,j).
This data therefore indicates that the FcRn
-chain is not delivered to
the cell surface of ß2m-deficient cells and is in agreement
with the lack of IgG internalization in these cells
(Fig. 2A).
To confirm biochemically that the FcRn -chain was absent from the
surface of FO-1 cells, we carried out surface biotinylation experiments.
Following modification of cell surface proteins with a membrane-impermeable
biotinylation reagent, cells were lysed, FcRn was immunoprecipitated with
anti-Myc antibodies and precipitates were blotted with streptavidin-HRP.
Alternatively, biotinylated surface receptors were first precipitated with
streptavidin-agarose and blots probed with anti-Myc antibodies. As shown in
Fig. 4, biotinylated surface
FcRn was readily detected in FO-1ß2m cells
(Fig. 4, lane 6, SA-HRP and
-Myc) but was absent or strongly reduced in FO-1 cells
(Fig. 4, lane 4, SA-HRP and
-Myc). Immunoblotting of cell lysates
(Fig. 4, lanes 1,2) confirmed
that FO-1 cells expressed similar or larger amounts of the FcRn
-chain
than FO-1ß2m cells, showing that the absence of the
-chain on the
cell surface in these cells was not due to lower expression levels.
|
Thus, cell surface expression of the FcRn -chain is greatly reduced
in the absence of ß2m.
ß2m is important for FcRn to exit the endoplasmic
reticulum
Since the FcRn -chain was not detected on the surface of cells
lacking ß2m, we next determined whether the
-chain is
retained in an intracellular compartment in these cells by analyzing whether
the FcRn
-chain co-localizes with markers for early endosomes (i.e.
EEA1, Fig. 5 a-f), lysosomes
(i.e. lamp-1, Fig. 5g-1) and
the ER (i.e. ribophorin II, Fig.
5m-r). Antibodies to the endosomal marker EEA1 labeled a vesicular
compartment in FO-1 and FO-1ß2m cells. As observed above
(Fig. 3), the
-chain was
present in a vesicular compartment in FO-1ß2m cells but showed a more
diffuse reticular labeling in FO-1 cells. While the
-chain (red) showed
extensive but incomplete co-localization with EEA1 (green) in FO-1ß2m
cells, no co-localization was observed in FO-1 cells. Thus, a fraction of FcRn
was present in early endosomes in FO-1ß2m but not in FO-1 cells. The FcRn
-chain (red) did not co-localize with the lysosomal marker lamp-1
(green) in either FO-1 or FO-1ß2m cells
(Fig. 5g-1), consistent with
previous results obtained in MDCK cells
(Praetor et al., 1999
).
|
To determine whether the reticular staining for the FcRn -chain
observed in FO-1 cells corresponds to the ER, we also determined whether the
FcRn
-chain co-localizes with ribophorin II (m-r). A diffuse reticular
staining characterisitic for the ER was obtained with anti-ribophorin II
antibodies. Merging the staining for the FcRn
-chain (red) with that of
ribophorin II (green), showed extensive co-localization in FO-1 but not in
FO-1ß2m cells. The FcRn
-chain in FO-1 cells also co-localized
with a second ER marker, protein disulfide isomerase (data not show). Thus, in
ß2m-deficient cells the FcRn
-chain was retained in the
ER.
To confirm biochemically that the FcRn -chain did not exit the ER in
cells lacking ß2m, we analyzed whether the single N-linked
carbohydrate in hFcRn (Israel et al.,
1997
) remained EndoH sensitive in FO-1 cells. Cell lysates were
incubated with buffer, EndoH or, to cleave all N-linked carbohydrates,
glycosidase F, and then immunoblotted for detection of the FcRn
-chain
or ß2m. As shown in Fig.
6, EndoH (lane 4) and glycosidase F (lane 6) treatment resulted in
a reduction of the apparent molecular mass of the FcRn
-chain in
lysates from FO-1 cells (lanes 1-6,
-Myc), consistent with the cleavage
of an N-linked carbohydrate. In FO-1ß2m cells
(Fig. 6, lanes 7-12,
-Myc), however, while sensitive to glycosidase F treatment
(Fig. 6, lane 12), the FcRn
-chain was resistant to digestion by EndoH
(Fig. 6, lane 10). Incubation
of lysates with buffer alone (Fig.
6, lanes 3,5,9,11) had no effect, showing that the change in
-chain mobility was due to deglycosylation. As expected from the lack
of N-linked carbohydrates, the mobility of ß2m was unaffected
by EndoH or glycosidase F treatment (Fig.
6, lanes 7-12,
-ß2m).
|
In conclusion, morphological and biochemical data show that the FcRn
-chain is retained in the ER in ß2m-deficient
cells.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Despite the apparent relevance of ß2m, its precise role for
FcRn function has not been established. In the case of the homologous MHC
class I molecules, ß2m is required but not sufficient for the
-chain to exit the ER and a similar role may seem plausible for FcRn
due to its homology to MHC class I. However, the experimental data from
ß2m-deficient mice, could also reflect a requirement of
ß2m for IgG binding. Furthermore, the less efficient IgG
transport in ß2m-null mice may also result from an increased
turnover rate or an altered intracellular trafficking of the
-chain in
the absence of ß2m. For example, since only a minor fraction
of FcRn is on the cell surface at equlibrium
(Berryman and Rodewald, 1995
;
Kristoffersen and Matre, 1996
;
Roberts et al., 1990
), a small
change in the steady-state distribution in the absence of ß2m
may result in a significant decrease in the amount of FcRn present on the cell
surface.
To establish experimentally the precise role of ß2m in FcRn
function, we expressed the -chain in ß2m-deficient FO-1
cells, either alone or in combination with ß2m. Although the
FcRn
-chain could be expressed in cells lacking ß2m, it
failed to efficiently appear on the cell surface. Based on its co-localization
with ER markers and the retention of EndoH-sensitive N-linked carbohydrates in
FO-1 cells, the
-chain was not exported from the ER in
ß2m-deficient cells. Thus, as for MHC class I antigens,
ß2m is important to satisfy ER quality control that allows
FcRn to exit the ER.
In the case of MHC class I antigens, folding and assembly is a multi-step
process and involves several chaperones
(Pamer and Cresswell, 1998).
Assembly with ß2m is essential but not sufficient for surface
transport of MHC class I, which also requires binding of the antigenic
peptide. In contrast, H-2 class I alleles may differ from HLA alleles in this
respect since they can be transported to the cell surface in the absence of
bound peptide (Pamer and Cresswell,
1998
). Since FcRn does not bind peptide, it may rather resemble
H-2 molecules in that binding of ß2m may be sufficient to
conform to ER quality control. However, an additional level of ER quality
control for FcRn may involve the assembly of FcRn homodimers from
-chain-ß2m heterodimers (A.P. and W.H., unpublished).
Since FcRn dimerization does not require ligand (A.P. and W.H., unpublished),
dimerization is likely to occur in the ER and to be integral to the assembly
of a transport competent receptor.
In addition to the importance of ß2m for exit of the
-chain from the ER, ß2m was also required for efficient
pH-dependent binding of IgG by FcRn. In the absence of ß2m,
binding of the
-chain to IgG-agarose was significantly reduced at pH
6.5. The co-crystal structure of FcRn and Fc shows contact points between the
Fc domain of IgG and the N-terminal Ile of ß2m
(Burmeister et al., 1994
;
Martin et al., 2001
) and in
vitro mutagenesis studies corroborate a role for Ile(1) in IgG binding
(Vaughn et al., 1997
). Ile(1),
widely conserved in ß2m from different species (e.g. human,
mouse and rat), may mediate a hydrophobic interaction with IgG near residues
309-311 on the Fc domain (Vaughn et al.,
1997
). Alternatively, Ile(1) may play an indirect role in IgG
binding since the presumably protonated
-NH2
[pKa
8 (Fersht,
1985
)] is positioned to form a hydrogen bond with the backbone
carbonyl group of residue 115 in the
-chain as well as a pH-dependent
salt bridge with Glu(117) in the heavy chain
(Vaughn et al., 1997
). Thus,
the protonated
-NH2 could help to align Glu(117) on the
-chain to form an anionic binding site for His(310) on Fc
(Vaughn et al., 1997
).
Although the critical role of ß2m in FcRn function was
established in non-polarized FO-1 melanoma cells, ß2m is
probably equally important for FcRn surface expression and ligand binding in
other cell types in which the receptor plays a physiological role in IgG
transport. The importance of ß2m for FcRn function suggests
that expression of a transport competent receptor could be regulated
indirectly via ß2m expression levels. Little is known
concerning the developmental regulation of FcRn and ß2m
expression in different organs and tissues. In neonatal rats, -chain
mRNA is present in the intestine at birth and declines within 10 days
(Simister and Mostov, 1989
),
indicating that FcRn expression is regulated at the level of
-chain
transcription or mRNA stability. However, in mammary glands of possum and
bovine, FcRn
-chain mRNA levels remain constant throughout lactation,
but expression of ß2m mRNA increases at the time of active IgG
transfer into milk (Adamski et al.,
2000
). Thus, depending on the tissue, species or developmental
stage, expression of functional FcRn may be controlled by the mRNA levels for
either the
-chain or ß2m. Furthermore, since most cells
express MHC class I antigens and newly synthesized MHC class I and FcRn are
likely to compete for the available ß2m in the ER, the
expression of the MHC class I and FcRn
-chains and ß2m
must be coordinately regulated.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adamski, F. M., King, A. T. and Demmer, J. (2000). Expression of the Fc receptor in the mammary gland during lactation in the marsupial Trichosurus vulpecula (brushtail possum). Mol. Immunol. 37,435 -444.[Medline]
Berryman, M. and Rodewald, R. (1995). Beta
2-microglobulin co-distributes with the heavy chain of the intestinal IgG-Fc
receptor throughout the transepithelial transport pathway of the neonatal rat.
J. Cell Sci. 108,2347
-2360.
Burmeister, W. P., Huber, A. H. and Bjorkman, P. J. (1994). Crystal structure of the complex of rat neonatal Fc receptor with Fc. Nature 372,379 -383.[Medline]
D'Urso, C. M., Wang, Z. G., Cao, Y., Tatake, R., Zeff, R. A. and Ferrone, S. (1991). Lack of HLA class I antigen expression by cultured melanoma cells FO-1 due to a defect in ß2m gene expression. J. Clin. Invest. 87,284 -292.[Medline]
Fersht, A. (1985). Enzyme Structure and Mechanism. New York, USA: W. H. Freeman.
Ghetie, V. and Ward, E. S. (2000). Multiple roles for the major histocompatibility complex class I-related receptor FcRn. Annu. Rev. Immunol. 18,739 -766.[Medline]
Ghetie, V., Hubbard, J. G., Kim, J. K., Tsen, M. F., Lee, Y. and Ward, E. S. (1996). Abnormally short serum half-lives of IgG in beta 2-microglobulin-deficient mice. Eur. J. Immunol. 26,690 -696.[Medline]
Höning, S. and Hunziker, W. (1995). Cytoplasmic determinants involved in direct lysosomal sorting, endocytosis, and basolateral targeting of rat lgp 120 (lamp-1) in MDCK cells. J. Cell Biol. 128,321 -332.[Abstract]
Hunziker, W. and Kraehenbuhl, J. P. (1998). Epithelial transcytosis of immunoglobulins. J. Mammary Gland Biol. Neoplasia 3,287 -302.[Medline]
Hyun, S. K., Garcia, J., Exley, M., Johnson, K. W., Balk, S. P.
and Blumberg, R. S. (1999). Biochemical characterization of
CD1d expression in the absence of ß2-microglobulin. J. Biol.
Chem. 274,9289
-9295.
Israel, E. J., Patel, V. K., Taylor, S. F., Marshakrothstein, A.
and Simister, N. (1995). Requirement for a
beta(2)-microglobulin-associated Fc receptor for acquisition of maternal IgG
by fetal and neonatal mice. J. Immunol.
154,6246
-6251.
Israel, E. J., Wilsker, D. F., Hayes, K. C., Schoenfeld, D. and Simister, N. E. (1996). Increased clearance of IgG in mice that lack beta(2)-microglobulin possible protective role of FcRn. Immunology 89,573 -578.[Medline]
Israel, E. J., Taylor, S., Wu, Z., Mizoguchi, E., Blumberg, R. S., Bhan, A. and Simister, N. E. (1997). Expression of the neonatal fc receptor, FcRn, on human intestinal epithelial cells. Immunology 92,69 -74.[Medline]
Junghans, R. P. and Anderson, C. L. (1996). The
protection receptor for IgG catabolism is the beta(2)-microglobulin-containing
neonatal intestinal transport receptor. Proc. Natl. Acad. Sci.
USA 93,5512
-5516.
Kristoffersen, E. K. and Matre, R. (1996). Co-localization of the neonatal Fc-gamma receptor and IgG in human placental term syncytiotrophoblasts. Eur. J. Immunol. 26,1668 -1671.[Medline]
Martin, W. L., West, A. P., Gan, L. and Bjorkman, P. J. (2001). Crystal structure at 2.8 Å of an FcRn/heterodimeric Fc complex: mechanism of ph-dependent binding. Mol. Cell 7,867 -877.[Medline]
Pamer, E. and Cresswell, P. (1998). Mechanisms of MHC class I-restricted antigen processing. Annu. Rev. Immunol. 16,323 -358.[Medline]
Praetor, A., Ellinger, I. and Hunziker, W.
(1999). Intracellular traffic of the MHC class I-like IgG Fc
receptor, FcRn, expressed in epithelial MDCK cells. J. Cell
Sci. 112,2291
-2299.
Reichert, M., Muller, T. and Hunziker, W.
(2000). The PDZ domains of zonula occludens-1 induce an
epithelial to mesenchymal transition of Madin-Darby canine kidney I cells
evidence for a role of beta-catenin/Tcf/Lef signaling. J.
Biol. Chem. 275,9492
-9500.
Roberts, D. M., Guenthert, M. and Rodewald, R. (1990). Isolation and characterization of the Fc receptor from the fetal yolk sac of the rat. J. Cell Biol. 111,1867 -1876.[Abstract]
Simister, N. E. and Mostov, K. E. (1989). An Fc receptor structurally related to MHC class I antigens. Nature 337,184 -187.[Medline]
Stefaner, I., Praetor, A. and Hunziker, W.
(1999). Nonvectorial surface transport, endocytosis via a
di-leucine-based motif, and bidirectional transcytosis of chimera encoding the
cytosolic tail of rat FcRn expressed in Madin-Darby canine kidney cells.
J. Biol. Chem. 274,8998
-9005.
Vaughn, D. E., Milburn, C. M., Penny, D. M., Martin, W. L., Johnson, J. L. and Bjorkman, P. J. (1997). Identification of critical IgG binding epitopes on the neonatal Fc receptor. J. Mol. Biol. 274,597 -607.[Medline]
Zijlstra, M., Bix, M., Simister, N. E., Loring, J. M., Raulet, D. H. and Jaenisch, R. (1990). Beta2-microglobulin deficient mice lack CD4-8+ cytolytic T cells. Nature 344,742 -746.[Medline]