From the Rosenstiel Center for Basic Biomedical Sciences, W. M. Keck Institute for Cellular Visualization, and the Department of Biology, Brandeis University, Waltham, Massachusetts 02254-9110
Received for publication, July 26, 2000, and in revised form, November 10, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The neonatal Fc receptor, FcRn, transports
immunoglobulin G across intestinal cells in suckling rats. FcRn enters
these cells by endocytosis and is present on the apical and basolateral
surfaces. We investigated the roles of aromatic amino acids and a
dileucine motif in the cytoplasmic domain of rat FcRn. We expressed
mutant FcRn in which alanine replaced Trp-311, Leu-322, and Leu-323, or
Phe-340 in the inner medullary collecting duct cell line IMCD. Individual replacement of the aromatic amino acids or the dileucine motif only partially blocked endocytosis of 125I-Fc,
whereas uptake by FcRn containing alanine residues in place of both
Trp-311 and the dileucine motif was reduced to the level obtained with
the tailless receptor. Leu-314 was required for the function of the
tryptophan-based endocytosis signal, and Asp-317 and Asp-318 were
required for the dileucine-based signal. Nonvectorial delivery of newly
synthesized FcRn to the two cell surfaces was unaffected by loss of the
endocytosis signals. However, the steady-state distribution of
endocytosis mutants was predominantly apical, unlike wild-type FcRn,
which was predominantly basolateral. This shift appeared to arise
because the loss of endocytosis signals inhibited apical to basolateral
transcytosis of FcRn more than basolateral to apical transcytosis.
The major histocompatibility complex class I-related Fc
receptor FcRn1 (1) carries
IgG into a vesicular pathway across epithelia. In this way FcRn
protects from degradation much of the IgG that enters epithelia by
endocytosis. Such protection is necessary for two physiological
processes: the transmission of IgG from mother to offspring, and the
protection from catabolism of IgG entering and leaving the blood
circulation. This necessity is illustrated by the low serum IgG
concentration in the first days after birth (2), the failure to obtain
IgG by suckling (2), and the rapid catabolism of IgG (3-5), in
In intestinal epithelial cells from neonatal rats, less than 10% of
FcRn is on the cell surface; most is intracellular (14). Both FcRn and
IgG enter these cells at coated pits in the plasma membrane and are
delivered to tubular and vacuolar structures that appear to be
endosomes (14-16). At the slightly acidic luminal pH of the neonatal
duodenum and jejunum (17) IgG can bind FcRn (17-20) and may therefore
enter epithelial cells bound to its receptor. IgG can also cross
isolated intestinal segments at neutral pH (21), which is unfavorable
for its binding to FcRn (17-20). Under these conditions IgG may enter
epithelial cells in the fluid phase and bind FcRn only after delivery
to acidified endosomes (21). Likewise, IgG may enter yolk sac endoderm
(7), syncytiotrophoblast (22, 23), and capillary endothelium (3-5) in
the fluid phase at neutral pH before binding FcRn in endosomes. It is
probable that an endocytic pathway analogous to that in the neonatal
intestinal epithelium delivers FcRn to endosomes even in tissues in
which IgG does not bind the receptor at the plasma membrane (although there may also be a direct route from the trans Golgi network). Consistent with this, there is some FcRn on the plasma membrane of the
yolk sac endoderm (R. Rodewald personal communication cited in Ref.
24), although almost all is intracellular (7).
The signals known to direct receptors and other membrane proteins to
undergo endocytosis at coated pits are almost all in the cytoplasmic
domains (for review, see Ref. 25). The majority of these signals are
tyrosine-based motifs and dileucine-based motifs (for review, see Refs.
25 and 26). A few endocytosis signals contain phenylalanine residues
(e.g. in bovine cation-dependent mannose
6-phosphate receptor (27)) or tolerate substitution of phenylalanine
for tyrosine (e.g. in bovine cation-independent mannose
6-phosphate receptor (28)); none is reported to contain tryptophan,
although tyrosine residues in the endocytosis signals of the human low
density lipoprotein receptor (29) and human transferrin receptor (30)
can be replaced by tryptophan without loss of function. Amino acid
sequences overlapping some but not all endocytosis signals are used
additionally to target membrane proteins to the basolateral surfaces of
polarized cells (for review, see Ref. 31).
The Cell Culture--
Rat inner medullary collecting duct (IMCD)
cells and their transfected derivatives were cultured as described
previously (33). Except where noted, cells were plated on 24-mm
Transwells (polycarbonate, 3-µm pores; Corning Costar, Acton, MA) and
grown for 3-6 days before experiments to allow a polarized monolayer
with resistance greater than 300 Mutagenesis and Expression--
Codons were substituted for
those encoding Leu-307, Pro-308, Ala-309, Pro-310, Trp-311, Leu-312,
Ser-313, Leu-314, Ser-315, Asp-317, Asp-318, Ser-319, Gly-320, Asp-321,
Leu-322, Leu-323, and Phe-340 individually or in combination using
overlap exchange polymerase chain reaction (34, 35) with Pfu
DNA polymerase (Stratagene, La Jolla, CA), to make DNA encoding rat
FcRn W311A, W311A/S315A, W311A/D317A/D318A, W311A/S319A, W311A/G320A,
W311A/G320L, W311A/D321A, W311A/L322A/L323A, W311A/F340A, L322A/L323A,
L307A/L322A/L323A, P308A/L322A/L323A, A309G/L322A/L323A,
P310A/L322A/L323A, W311Y/L322A/L323A, W311F/L322A/L323A,
L312A/L322A/L323A, S313A/L322A/L323A, L314A/L322A/L323A, L314I/L322A/L323A, S315A/L322A/L323A, F340A, and L322A/L323A/F340A (Fig. 1). All sequences were verified.
DNAs that coded for mutant and wild-type rat FcRn Western Blots--
Western blots were used to look for FcRn
expression in clones, essentially as described (9). Briefly, cells were
lysed in 1% SDS. Lysates containing 15 µg of protein were resolved
on 8% polyacrylamide denaturing gels and electroblotted onto
polyvinylidene difluoride membranes (Novex, San Diego). Blots were
probed with purified rabbit antibodies against amino acids 176-190 of
rat FcRn (33), diluted 1:150 (~5 µg/ml). Bound antibodies were
detected with horseradish peroxidase-conjugated goat anti-rabbit IgG
(Bio-Rad) and Renaissance Chemiluminescence Reagent (PerkinElmer Life Sciences).
Surface-specific Fc Binding Assay--
The Fc fragment of human
IgG (Jackson Immunoresearch, West Grove, PA) was labeled with
Na125I (PerkinElmer Life Sciences) using IODO-GEN (Pierce
Chemical Co.). The Fc fragment of human IgG binds rat FcRn as
effectively as rat Fc (37). Cells were cooled on ice and washed twice
with ice-cold Dulbecco's modified Eagle's medium (DMEM) pH 6.0 at the loading surface (apical or basolateral) and pH 8.0 at the nonloading surface. 125I-Fc (100 ng/ml, 2 × 10 Fc Uptake Assay on Nonpolarized Cells--
Cells were grown on
12-well plates until nearly confluent but still fibroblast-like in
appearance. The cells were cooled on ice for 1 h, washed twice
with ice-cold DMEM pH 6.0, and allowed to bind 125I-Fc (100 ng/ml) in DMEM-KIGH pH 6.0 for 6 h. After the cells were washed
four times with ice-cold DMEM pH 6.0, they were incubated at 37 °C
with prewarmed DMEM-KIGH pH 6.0 for 0, 2, 5, 15, and 30 min. Then the
cells were cooled on ice, and the medium was removed and counted
(cpmmed). The cells were washed with ice-cold DMEM pH 6.0 and then incubated on ice for 45 min with chymotrypsin and proteinase
K, 50 µg/ml each in phosphate-buffered saline pH 8.0 to digest
125I-Fc from the cell surface (cpmsurf).
Finally, the cells were washed with ice-cold DMEM pH 8.0 and then
dissolved in 0.1 M NaOH and counted (cpmint).
For each time point of each cell line, the percentage of
125I-Fc internalized was calculated as cpmint × 100/(cpmmed + cpmsurf + cpmint).
Domain-specific Uptake Assay--
Because the signal obtained by
loading at 4 °C was low, uptake of 125I-Fc from the
apical and basolateral surfaces was measured by loading briefly at
37 °C. After each surface was washed twice with DMEM pH 8.0, the
cells were cooled on ice. Cells were pulsed for 5 min at 37 °C with
125I-Fc (100 ng/ml) in DMEM-KIGH pH 6.0 at either the
apical or basolateral (loading) surface, and DMEM pH 6.0 at the
opposite (nonloading) surface. Immediately the cells were washed five
times with ice-cold DMEM pH 6.0 and then proteins were digested at both
cell surfaces as in the uptake assay above. Proteins released from the
loading and nonloading surfaces were collected, and 125I-Fc
was counted (cpmloading and cpmnonloading,
respectively). Each Transwell filter was cut out, and cell-associated
radioactivity was counted (cpmint). The fraction of
125I-Fc internalized was calculated as (cpmint + cpmnonloading)/(cpmloading + cpmnonloading + cpmint) and was expressed as a
percentage of the fraction taken up by wild-type FcRn.
Surface Biotinylation--
To measure the steady-state
distribution of FcRn between the apical and basolateral cell surfaces,
the apical, basolateral, or both surfaces were treated with
sulfosuccinimidyl 2-(biotinamido) ethyl-1,3-dithiopropionate
(NHS-SS-biotin, Pierce), twice for 15 min on ice, essentially as
published (38). After the biotinylation was quenched with DMEM, the
filters were cut from the Transwells (three for each surface). The
cells were lysed and precipitated with rabbit anti-rat FcRn Pulse-Chase Biosynthetic Labeling--
To measure the delivery
of newly synthesized FcRn to the apical and basolateral cell surfaces,
cells were washed twice with Met-, Cys- DMEM,
starved in the same medium for 45 min at 37 °C, and then labeled for
15 min with 0.5 mCi/ml 35S-labeled Met+Cys (PerkinElmer
Life Sciences) in the basolateral compartment. After 0, 30, 90, and 210 min of chase in DMEM containing 10% fetal bovine serum (Hyclone
Laboratories, Logan, UT), the cells were washed twice with ice-cold
phosphate-buffered saline containing 1 mM CaCl2
and 0.5 mM MgCl2, cooled on ice for 1 h, then biotinylated on either the apical or basolateral surface (three
Transwells each) twice for 30 min, as above. The cells were lysed and
precipitated with anti-FcRn and then with streptavidin-agarose as
above. Proteins were eluted, pooled, and subjected to electrophoresis as above. Gels were incubated in 1 M sodium salicylate for
20 min at room temperature and dried. 35S-Labeled proteins
were detected using a Molecular Dynamics PhosphorImager (Sunnyvale, CA).
Transcytosis Assay--
125I-Fc was allowed to bind
the loading surface (apical or basolateral) as in the Fc binding assay,
above. The loading and nonloading surfaces were then washed five times
each with ice-cold DMEM-KIGH pH 6.0 and pH 8.0, respectively. The
nonloading surface medium was counted (cpm0) and replaced
with DMEM-KIGH pH 8.0, prewarmed to 37 °C. Cells were incubated at
37 °C for 0, 2, 7, 22, and 52 min, after which times the medium from
the nonloading surface was removed and counted (cpm2,
cpm7, etc.) and replaced with fresh DMEM pH 8.0. At the
last time point, the loading surface medium was counted
(cpmmedium), and the Transwell membrane was cut out and
cell-associated radioactivity counted (cpmcell). For each well at each time point (t), the cumulative percentage of
125I-Fc transported was calculated as (cpm0 + cpm2 + ... + cpmt) × 100/(cpm0 + cpm2 + cpm7 + cpm22 + cpm52 + cpmmedium + cpmcell).
Expression of FcRn Mutants in IMCD Cells--
To test whether the
dileucine motif and aromatic amino acids in the cytoplasmic domain of
rat FcRn were required for endocytosis and basolateral targeting we
expressed in IMCD cells FcRn Endocytosis of FcRn and Mutants--
We first compared the uptake
of Fc by subconfluent, nonpolarized IMCD cells expressing wild-type and
mutant FcRn
These results suggested that in nonpolarized cells Trp-311 and the
dileucine motif were components of independent endocytosis signals. We
next measured uptake from the apical and basolateral surfaces of
polarized IMCD cells to determine whether either endocytosis signal was
used preferentially at one or the other surface. We allowed cells to
take up 125I-Fc from the apical or basal medium for 5 min
at 37 °C. Disruption of either signal alone reduced endocytosis by
less than 25% (Fig. 3B); disruption of both signals reduced
apical endocytosis by 75% and basolateral endocytosis by 50%.
We explored the sequence requirements of the tryptophan-based
endocytosis signal by alanine-scanning and additional substitutions, in
the context of a disrupted dileucine motif. Again we used subconfluent, nonpolarized IMCD cells. Replacement of Trp-311 with tyrosine (open circles in Fig.
4A) or phenylalanine
(open triangles) allowed endocytosis of Fc at levels similar
to or only slightly below those obtained with wild-type FcRn
(closed triangles). However, the rate of endocytosis by
W311F/L322A/L323A was only 35% of that seen with the wild-type
receptor. Mutations in positions 307, 308, 309, 310, 312, 313, and 315 had little or no effect on the extent of Fc uptake (Fig.
4B), although uptake by S315A/L322A/L323A (open
diamonds) was 35% slower than by wild-type FcRn. Substitution of
isoleucine for Leu-314 was without effect on the amount of Fc taken up
(open circles in Fig. 4C) but decreased the rate
of endocytosis by 65%. In contrast, substitution with alanine
(closed circles) decreased endocytosis to the level and rate
of tailless FcRn.
Similarly, we measured endocytosis by FcRn with mutations in the
vicinity of the dileucine motif in the context of a disrupted tryptophan-based signal. Mutations in positions 315, 319, 320, and 321 had little or no effect on Fc uptake (Fig. 4D), although replacement of Ser-315A reduced the rate of uptake by 25% (open triangles). In contrast, substitution of alanine residues for Asp-317 and Asp-318 (open diamonds) reduced the rate of
uptake by 90% and amount of Fc taken up by 60%, compared with
wild-type FcRn.
Apical/Basolateral Distribution of FcRn and Mutants--
We used
two types of assay to measure the steady-state distribution of FcRn
mutants between the apical and basolateral surfaces of polarized IMCD
cells, one based on ligand binding and the other on biotinylation. In
both assays we used cells grown on Transwell filters for 3 days after
they had become confluent. We have shown previously that these
conditions allow the cells to become polarized (33).
First we measured the binding of 125I-Fc from the apical or
basal compartment at 4 °C. Fig. 5,
which summarizes data from 3-10 experiments on each mutant, shows the
specific binding to the apical and basolateral surfaces (calculated as
the radioligand competed off by unlabeled IgG) as percentages of the
total specific binding. In IMCD cells expressing wild-type FcRn we
detected ~75% of the cell surface FcRn on the basolateral plasma
membrane. We found similar percentages of the FcRn mutants W311A,
L322A/L323A, F340A, W311A/F340A, and L322A/L323A/F340A at the
basolateral surface. Strikingly, the distribution of FcRn with alanine
replacing both Trp-311 and the dileucine motif (W311A/L322A/L323A) was
reversed, with only 30% on the basolateral membrane. The distribution
of the tailless FcRn 304t was intermediate between the wild-type and
W311A/L322A/L323A receptors, with ~45% on the basolateral surface.
We used surface-specific biotinylation as an independent measure of the
steady-state distribution of the W311A/L322A/L323A FcRn mutant. We
found that 65% of the wild-type FcRn was biotinylated at the
basolateral surface (Fig. 6). The
distribution of FcRn with alanine replacing both Trp-311 and the
dileucine motif was again reversed, with less than 5% at the
basolateral plasma membrane. We detected 35% of the tailless FcRn at
the basolateral surface.
Using the domain-specific binding assay we compared the distributions
of FcRn with mutations close to Trp-311 in the context of the
L322A/L323A mutation and mutations near the dileucine motif in the
context of W311A. L314A/L322A/L323A and W311A/D317A/D318A, which were
impaired in endocytosis, were predominantly apical (Fig.
7). Mutants that showed normal
endocytosis were found mostly on the basolateral cell surface.
Apical/Basolateral Delivery of FcRn and Mutants--
We measured
the delivery of newly made FcRn to the apical and basolateral cell
surfaces by pulse labeling the receptor, and biotinylating one surface
or the other after various chase times. Some labeled FcRn Transcytosis by FcRn and Mutants--
We compared the abilities of
the FcRn mutants to transport a cohort of Fc bound at 4 °C. In the
basolateral to apical direction, W311A/L322A/L323A, W311A, and
L322A/L323A all transported Fc at a level similar to that of wild-type
FcRn (Fig. 9B). In the apical to basolateral direction, W311A and L322A/L323A showed slight impairment, transporting, respectively, 90 and 85% as much Fc in 52 min as wild-type FcRn (triangles and circles,
respectively, in Fig. 9A). Transcytosis by W311A/L322A/L323A
(squares) was reduced to 15% of the wild-type level
(diamonds).
Tryptophan- and Dileucine-based Endocytosis Signals in
FcRn--
We set out to test the hypothesis that the dileucine motif
and the two aromatic amino acid residues in the cytoplasmic domain of
rat FcRn are required for rapid endocytosis. Adjacent leucine residues
are used as endocytosis signals in several membrane proteins (39-42).
Similarly, many endocytosis signals contain an essential tyrosine
(43-45) or, less commonly, phenylalanine (27).
We compared endocytosis of Fc by wild-type FcRn, tailless FcRn, and
FcRn in which alanine had replaced Trp-311, Leu-322 and Leu-323, and
Phe-340, singly or in combination. FcRn 304t is truncated after the
four amino acids that immediately follow the hydrophobic predicted
membrane-spanning region of the
The replacement of either aromatic amino acid or of the dileucine motif
caused a modest reduction in the level of endocytosis. Of the possible
combinations of substitutions, only simultaneous replacement of Trp-311
and the dileucine motif had a greater effect on endocytosis, reducing
Fc uptake to a level similar to or less than that seen with the
tailless receptor. These data suggest that Trp-311 and the dileucine
motif are necessary components of independent endocytosis signals.
Our results differ from those of a recent study in which the
dileucine motif alone was critical for efficient endocytosis (32).
Three features of that study could explain this discrepancy. First, the
Fc
The tryptophan-based endocytosis signal we identified is, to our
knowledge, the first of its kind. In mutagenesis studies, tryptophan
can be made to replace tyrosine residues in some endocytosis signals
(30) but not in others (48). We found that Trp-311 could be replaced
functionally by tyrosine or phenylalanine, although the Phe
substitution slowed endocytosis. Tyrosine residues essential for
endocytosis mostly occur in the motif YXXØ (49, 50), where X is any amino acid and Ø has a large and hydrophobic side
chain. Of the amino acids near Trp-311, only Leu-314 was critical to the endocytosis signal. This suggests that in FcRn Trp-311 is part of
an unusual endocytosis signal of the YXXØ type.
The endocytosis signal based upon the dileucine motif at positions 322 and 323 was inactivated by the replacement of Asp-317 and Asp-318. One
or more negatively charged groups are present in positions
The two endocytosis signals in rat FcRn are partly redundant. Several
other receptors contain multiple endocytosis signals that are redundant
to various extents. The purpose of such redundance is not clear.
Because similar signals may direct endocytosis, basolateral targeting
and sorting to endosomes from the trans Golgi network, it is possible
that signals redundant in one function have distinct roles in others
(40).
Nonvectorial Delivery to the Cell Surface--
FcRn was delivered
to the apical and basolateral surfaces of IMCD cells simultaneously.
None of the mutants tested affected this process. The nonvectorial
delivery of FcRn to the surface of IMCD cells is consistent with
reports of the biosynthetic targeting of the Fc
In our pulse-chase studies in IMCD cells, wild-type FcRn first appeared
on the cell surface in a low molecular weight form. We have shown
previously that this form is sensitive to endoglycosidase H.2 Because endoglycosidase H resistance is acquired in the
cis or medial Golgi, this implies either that FcRn is delivered to the plasma membrane directly from the endoplasmic reticulum or that most of
it passes through the Golgi without becoming fully glycosylated. The
low molecular weight form of FcRn was mostly removed from the cell
surface during the chase period and was replaced by the high molecular
weight form. More of the high molecular weight form of wild-type FcRn
was biotinylated at the basolateral surface than the apical surface,
whereas the opposite was true for the endocytosis mutants
W311A/L322A/L323A and W311A/D317A/D318A. In all cases the high
molecular weight form was preferentially biotinylated at the cell
surface where FcRn was more abundant in the steady state (below). These
observations suggest that the low molecular weight form of FcRn
undergoes endocytosis and is further glycosylated during subsequent
trafficking that establishes the steady-state distribution.
The delivery of incompletely glycosylated proteins to the cell surface
is unusual but not unique. The glycosylation of FcRn after its delivery
to the cell surface indicates that it enters the Golgi stack during
recycling or transcytosis. Several other membrane proteins, including
transferrin receptor (54) and mannose 6-phosphate receptors (55),
recycle through the Golgi. A mutant low density lipoprotein receptor
lacking the cytoplasmic domain retains the ability to return to the
Golgi (56), as does tailless FcRn in the present study. The
anti-transferrin receptor antibody OX26 also enters Golgi cisternae
during transcytosis through the blood-brain barrier endothelium (57).
Similar trafficking would allow glycosylation of FcRn after its initial
delivery to the plasma membrane.
Complex-type glycans on the Steady-state Surface Distribution--
In the steady state, there
was approximately twice as much wild-type FcRn at the basolateral
surface as at the apical surface, consistent with previous observations
in IMCD cells (33). Studies with BeWo and Madin-Darby canine kidney
cell lines show a more apical steady-state distribution (32). Whether
this reflects differences in the receptors or the cells remains to be
seen. The basolateral surfaces of polarized lines such as Madin-Darby canine kidney (60) are larger than the apical, and the greater abundance of FcRn at the basolateral surface of IMCD cells might result
in part from nonvectorial delivery to a smaller apical and larger
basolateral surface. For such an initial distribution to be maintained,
apical to basolateral transport of FcRn would have to exceed
basolateral to apical transport, which is consistent with studies of Fc
transport across IMCD cells expressing FcRn (33).
All of the mutants we identified as defective in endocytosis,
FcRn-304t, W311A/L322A/L323A, L314A/L322A/L323A, and W311A/D317A/D318A, showed a reversed steady-state distribution in which there was more
FcRn at the apical surface than the basolateral surface. In all of
these mutants that we have tested (FcRn-304t, W311A/L322A/L323A, and
W311A/D317A/D318A), newly synthesized FcRn is delivered to both
surfaces at approximately similar rates. Therefore the distribution of
the mutant receptors does not arise from altered biosynthetic targeting. Rather, it appears to be attributable to the effects of the
mutations on subsequent trafficking. Specifically, W311A/L322A/L323A was inhibited in apical to basolateral but not basolateral to apical
transcytosis. Likewise we have found that FcRn-304t is inhibited in
apical to basolateral but not basolateral to apical transcytosis.2 Thus mutations that eliminate the
endocytosis signals in FcRn appear to perturb its steady-state
distribution in favor of the apical cell surface by affecting apical to
basolateral transcytosis more than basolateral to apical transcytosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-microglobulin knockout mice, which lack functional FcRn (6).
Materno-fetal transmission of IgG appears to be mediated by FcRn in the
yolk sacs of mice and rats (7, 8) and in the human placental
syncytiotrophoblast (9-11). FcRn expressed at high levels in the small
intestines of suckling mice and rats mediates the uptake of IgG from
milk (for review, see Ref. 12). FcRn expressed at lower levels (3) at
widely dispersed sites including capillary endothelium (13) appears
responsible for protection from catabolism.
chain of rat FcRn has a 44-amino acid cytoplasmic domain (1),
which includes the dileucine motif Leu-322, Leu-323. There is no
tyrosine, but there are two aromatic amino acid residues in this
region, Trp-311 and Phe-340. It has recently been reported that in a
chimeric protein comprising the extracellular and transmembrane regions
of mouse Fc
RII-B2 fused to the membrane-proximal 24 amino acids of
the cytoplasmic domain of rat FcRn, expressed in Madin-Darby canine
kidney cells, the dileucine motif is an endocytosis signal, but not a
basolateral targeting signal (32). Our goal in this study was to
examine the roles in endocytosis of Trp-311 and Phe-340 as well as
Leu-322 and Leu-323, in full-length rat FcRn in a rat kidney cell line
(33). We also examined the effects of these amino acids on the
distribution of FcRn between the apical and basolateral cell surfaces.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cm2 to form (33).
chains were
subcloned into the expression vector pRc/RSV (neomycin resistance,
Invitrogen, San Diego). IMCD cells were transfected with each
construct, using a calcium phosphate method (36). Cells resistant to
G418 were selected, and individual colonies were expanded to establish
cell lines. In some experiments IMCD cells were used which expressed
essentially tailless FcRn, truncated after Arg-304
(304t).2
View larger version (53K):
[in a new window]
Fig. 1.
Mutations in the cytoplasmic domain of
FcRn. Schematic representation of the amino acid sequences of the
cytoplasmic domains of wild-type (WT) and mutant rat FcRn
chains used in this study.
9 M, iodinated to 0.8 mCi/µmol) in ice-cold DMEM, 1 mM KI, 1.5% fish gelatin
(Sigma), 20 mM HEPES (DMEM-KIGH) pH 6.0 was then added to
the loading surface with or without 5 mg/ml unlabeled human IgG
(Jackson Immunoresearch, 3.3 × 10
5
M). The cells were allowed to bind 125I-Fc for
6 h at 4 °C. Radioactivity in the nonloading compartment was
measured in a CliniGamma 1272 gamma counter (LKB Wallac, Piscataway, NJ), and wells in which more than 1% of the applied
125I-Fc had crossed the monolayer were rejected. Cells were
rapidly washed five times with ice-cold DMEM pH 6.0 at the loading
surface and pH 8.0 at the nonloading surface. Lastly, the filters were cut from the Transwells, and cell-associated 125I-Fc was
measured. Specific binding to each surface of each cell line was
calculated as the difference between the cell-associated radioactivity
in the absence and presence of unlabeled IgG, and the mean amounts of
125I-Fc specifically bound at the apical and basolateral
surfaces were represented as percentages of their sum.
-chain
and protein A-trisacryl and then with streptavidin-agarose essentially
as described (33). Proteins were eluted from streptavidin-agarose into
reducing sample buffer, pooled from each set of three Transwells, and
resolved on 8% polyacrylamide denaturing gels. Gels were
electroblotted onto polyvinylidene difluoride membranes, and FcRn was
detected with antipeptide antibodies as above. The FcRn
chain bands
(upper and lower bands in Fig. 6) were quantified using a Gel
Doc 1000 work station and Molecular Analyst 2.1.1 software
(Bio-Rad).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chains with alanine replacing Trp-311,
Leu-322 plus Leu-323, and Phe-340, individually or combination (Fig.
1). Fig. 2 shows a Western blot of
extracts of cell lines selected for high and fairly uniform levels of
expression of the mutant forms FcRn. Purified anti-peptide antibodies
against rat FcRn
chain (33) typically reveal two bands, as here,
when FcRn is expressed in either IMCD cells (33) or Rat1 fibroblasts.
In Rat1 cells, both bands migrate with the same decreased apparent
molecular weight after digestion with peptide N-glycosidase
F; the lower band is also shifted by endoglycosidase H treatment, but
the upper band is resistant.2 These data suggest that the
lower band is the high mannose form of FcRn usually found in the
endoplasmic reticulum, whereas FcRn in the upper band contains
complex-type oligosaccharide chains modified in the Golgi. Consistent
with this interpretation, the upper band is greatly enriched at the
cell surface compared with the lower band (see Fig. 6). We confirmed
that all of the IMCD cell lines expressing these and other mutant forms
of FcRn could bind 125I-Fc (below).
View larger version (28K):
[in a new window]
Fig. 2.
Expression of mutant forms of FcRn in IMCD
cells. Stable cell lines were selected after transfection of IMCD
cells with cDNA encoding the chain of wild-type or mutant FcRn.
Extracts of these lines and of untransfected IMCD cells were analyzed
on Western blots using affinity-purified anti-peptide antibodies
against rat FcRn
chain. The two forms of FcRn detected differ in
their glycosylation (see "Results"). Lane 1,
wild-type; lane 2, W311A/L322A/L323A; lane 3,
304t; lane 4, L322A/L323A; lane 5, W311A;
lane 6, F340A; lane 7, W311A/F340A; lane
8, L322A/L323A/F340A; lane 9, untransfected IMCD.
chains. We allowed cells to bind 125I-Fc at
4 °C and measured uptake when they were warmed to 37 °C. Cells
expressing wild-type FcRn and the tailless truncation 304t were used as
positive and negative controls, respectively. Replacement with alanine
of Trp-311, of Leu-322 and Leu-323, or of Phe-340 (open
squares, open triangles, and open circles,
respectively, in Fig. 3A)
decreased the amount of Fc taken up in 30 min by 5-25% compared with
wild-type FcRn (closed triangles). Likewise, simultaneous substitution of Trp-311 and Phe-340 or of Leu-322, Leu-323 and Phe-340
resulted in only a small impairment of endocytosis. The initial rates
of endocytosis by W311A (open squares) and L322A/L323A/F340A (open diamonds) were, respectively, 55 and 65% lower than
for wild-type FcRn, although both took up near wild-type amounts of Fc
over 30 min. In contrast, endocytosis by FcRn containing alanine residues in place of Trp-311, Leu-322, and Leu-323 was decreased by
80% (closed squares), somewhat more than uptake by the
tailless receptor (closed circles). Furthermore, the rates
of endocytosis by W311A/L322A/L323A and tailless FcRn were both less
than 10% of that obtained with the wild-type receptor. Similar results were obtained with Rat1 cells expressing these mutants (data not shown).
View larger version (33K):
[in a new window]
Fig. 3.
Panel A, endocytosis of Fc by IMCD cells
expressing FcRn and mutants. Cells were allowed to bind
125I-Fc on ice and were then warmed to permit uptake. The
amounts of radioligand released into the medium, taken up
(protease-resistant), and remaining on the cell surface
(protease-sensitive) were measured at the times shown. The percentage
of internalized Fc is shown for cells expressing wild-type
(WT) FcRn (closed triangles), W311A (open
squares), L322A/L323A (open triangles), F340A
(open circles), W311A/L322A/L323A (closed
squares), W311A/F340A (closed diamonds),
L322A/L323A/F340A (open diamonds), and the tailless receptor
304t (closed circles). Each symbol represents the
mean of three to seven measurements. Bars indicate the S.E.
and are omitted when they are smaller than the symbols.
Panel B, endocytosis of Fc from the apical and basolateral
surfaces of IMCD cells. Cells grown on Transwells until confluent were
incubated with 125I-Fc for 5 min at 37 °C at either at
the apical or basolateral surface. The fraction of Fc internalized was
calculated (see "Experimental Procedures") and expressed as
a percentage of the fraction taken up by cells expressing wild-type
FcRn (mean ± S.E., n = 6).
View larger version (28K):
[in a new window]
Fig. 4.
Endocytosis of Fc by IMCD cells expressing
FcRn with multiple mutations. Panel A, effects of
replacement of Trp-311 in wild-type FcRn (closed triangles),
with tyrosine (open circles), phenylalanine (open
triangles), or alanine (closed circles) in the context
of the L322A/L323A mutation (mean ± S.E., n = 12). Panel B, effects of mutations near Trp-311 in the
context of the L322A/L323A mutation: L307A/L322A/L323A (open
triangles), P308A/L322A/L323A (open circles),
A309G/L322A/L323A (closed circles), P310A/L322A/L323A
(closed squares), L312A/L322A/L323A (open
squares), S313A/L322A/L323A (closed diamonds),
S315A/L322A/L323A (open diamonds; mean ± S.E.,
n = 6-12). Panel C, effects of replacement
of Leu-314 with alanine (closed circles) or isoleucine
(open circles) in the context of the L322A/L323A mutation
(mean ± S.E., n = 9-12). Panel D,
effects of mutations near the dileucine motif in the context of the
W311A mutation: W311A/S315A (open triangles),
W311A/D317A/D318A (open diamonds), W311A/S319A (closed
diamonds), W311A/G320A (open squares), W311A/G320L
(closed squares), W311A/D321A (open circles;
mean ± S.E., n = 3-9).
View larger version (23K):
[in a new window]
Fig. 5.
Steady-state distribution of FcRn measured by
Fc binding at the apical and basolateral surfaces of polarized IMCD
cells. Cell monolayers on Transwells were incubated on ice with
125I-Fc at the apical or basolateral surface in the absence
or presence of excess unlabeled IgG. Binding in the presence of
competing IgG was considered nonspecific and was subtracted from the
total. Specific Fc binding at the apical and basolateral surface was
calculated as a percentage of the total specific binding. Columns
represent the mean apical and basolateral percentages ± S.E. from
3-10 measurements. WT, wild-type.
View larger version (32K):
[in a new window]
Fig. 6.
Biotinylation of mutant and wild-type FcRn at
the apical and basolateral surfaces of polarized IMCD cells. Cells
expressing wild-type (WT) FcRn, W311A/L322A/L323A, and the
tailless receptor 304t were grown on Transwells. Proteins at the apical
or basolateral surfaces were labeled with a membrane-impermeant
biotinylating reagent. FcRn was immunoprecipitated from cell lysates
with a rabbit polyclonal antiserum, and the biotinylated fraction was
reprecipitated with streptavidin-agarose. FcRn eluted from
streptavidin-agarose was detected on Western blots using
affinity-purified anti-peptide antibodies against rat FcRn chain.
View larger version (35K):
[in a new window]
Fig. 7.
Steady-state distribution of FcRn with
mutations near Trp-311 in the context of L322A/L323A and mutations near
the dileucine motif in the context of W311A. Fc binding at the
apical and basolateral surfaces of polarized IMCD cells was measured as
in Fig. 5 (mean ± S.E., n = 3-12).
WT, wild-type.
chain was
biotinylated at the zero time point (Fig.
8). This probably represents FcRn that
reached the cell surface during the time the cells cooled on ice
between the chase and biotinylation. It is unlikely that it represents
intracellular FcRn because actin was not biotinylated under these
conditions (data not shown). FcRn became available for biotinylation
simultaneously on the apical and basolateral cell surfaces. At early
time points we detected only the low molecular weight form of FcRn (see
above). Generally, between 90 and 210 min of chase the abundance of
this material decreased. In parallel, the amount of biotinylated high molecular weight form of FcRn increased. In cells expressing wild-type FcRn, the high molecular weight FcRn was biotinylated predominantly at
the basolateral cell surface. In contrast, in cells that expressed W311A/L322A/L323A and W311A/D317A/D318A, high molecular weight FcRn was
biotinylated predominantly at the apical surface (Fig. 8). The high
molecular weight form of tailless FcRn was biotinylated in similar
abundance at either surface.
View larger version (68K):
[in a new window]
Fig. 8.
Surface delivery of newly synthesized
receptors. Cells were pulsed for 15 min with
35S-labeled Met+Cys. After the chase times shown, cells
were cooled on ice, and proteins were biotinylated at either the apical
or basolateral surface. Biotinylated FcRn was detected as in Fig. 6.
WT, wild-type.
View larger version (15K):
[in a new window]
Fig. 9.
Transport of I-Fc across
monolayers of IMCD cells that express wild-type or mutant FcRn.
Cells on ice were loaded with 125I-Fc from either the
apical (panel A) or basolateral (panel B)
compartment, and free ligand was removed. The medium in the loading
compartment was at pH 6, and in the nonloading compartment it was at pH
8. The cells were warmed to 37 °C to stimulate transcytosis, and
medium was collected from the nonloading compartment at the indicated
times. After 52 min, the medium was also collected from the loading
compartment, and the cells were lysed. Media and lysates were
precipitated with trichloroacetic acid and counted for
125I. The cumulative percentage of each cohort transported
to the nonloading compartment was calculated (mean ± S.E.,
n = 6). Panel A, apical to basolateral
transcytosis: wild-type FcRn (open diamonds), W311A
(open triangles), L322A/L323A (open circles),
W311A/L322A/L323A (open squares). Panel B,
basolateral to apical transcytosis: wild-type FcRn (open
diamonds), W311A (open triangles), L322A/L323A
(open circles), W311A/L322A/L323A (open
squares).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chain (Asn-301, Arg-302, Met-303,
and Arg-304). We left these four amino acids in place because they
might act as a stop-transfer signal during translocation of the FcRn
chain through the endoplasmic reticulum membrane. The ability of
this tailless receptor to undergo endocytosis was greatly reduced but
not eliminated. This is consistent with the observation that a chimera
comprising the extracellular and transmembrane regions of Fc
RII-B2
fused to the amino acids Asn-Arg-Met-Arg is internalized much less
efficiently than a chimera that includes the complete FcRn tail
(32).
RII-B2/FcRn chimera lacked the last 20 amino acids of the
cytoplasmic domain. It is possible that the truncation inactivates
Trp-311 by altering the conformation of the polypeptide in this region.
Second, absence of the extracellular and transmembrane domains of FcRn
from the chimera might preclude the formation of dimers in which
cytoplasmic domains interact (46, 47), again altering the contexts of
the tryptophan- and dileucine-based signals. Third, the rat FcRn
cytoplasmic domain might not interact normally with adaptor complexes
in the dog cells in which the chimera was expressed. We avoided these
potential complications by studying full-length rat FcRn in a rat cell line.
3 to
5
with respect to the first residue of most dileucine motifs that
function in endocytosis (51, 52). Leu-322 and Leu-323 of FcRn, together
with Asp-317 and/or Asp-318, are thus parts of a typical
dileucine-based endocytosis signal.
RII-B2/rat FcRn
chimera (32) and human FcRn (53) expressed in Madin-Darby canine kidney cells.
3 domains form part of the interface
between FcRn molecules in dimers seen by x-ray crystallography (20).
The immature glycoform of FcRn might therefore be impaired in
dimerization, with two significant consequences. First, because in a
dimer the cytoplasmic domains are close enough to interact (58), the
unpaired tail of the low molecular weight form of FcRn might cause this
glycoform to be sorted differently from dimers of the high molecular
weight form. Second, because FcRn must dimerize to bind IgG with high
affinity (47, 59), the low molecular weight form might be impaired in
IgG binding. We speculate that these mechanisms allow the immature
glycoform to return to the Golgi unoccupied by IgG. This is consistent
with the detection of FcRn (14) but not internalized IgG (15) in the
Golgi cisternae of intestinal epithelial cells in neonatal rats.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank J. Schwartz for IMCD cells and K. M. McCarthy for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants HD27691 and HD01146.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: Brandeis University MS
029, Waltham, MA 02254-9110. Tel.: 781-736-4952; Fax:
781-736-2405; E-mail: simister@brandeis.edu.
Published, JBC Papers in Press, November 28, DOI 10.1074/jbc.M006684200
2 McCarthy, K. M., Lam, M., Subramanian, L., Shakya, R., Wu, Z., Newton, E. E., and Simister, N. E., J. Cell Sci., in press.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
FcRn, neonatal Fc
receptor;
FcRII-B2, Fc
receptor II-B2;
IMCD, inner medullary
collecting duct;
304t, truncated after Arg-304;
DMEM, Dulbecco's
modified Eagle's medium.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Simister, N. E., and Mostov, K. E. (1989) Nature 337, 184-187[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Israel, E. J.,
Patel, V. K.,
Taylor, S. F.,
Marshak-Rothstein, A.,
and Simister, N. E.
(1995)
J. Immunol.
154,
6246-6251 |
3. | Ghetie, V., Hubbard, J. G., Kim, J. K., Tsen, M. F., Lee, Y., and Ward, E. S. (1996) Eur. J. Immunol. 26, 690-696[Medline] [Order article via Infotrieve] |
4. |
Junghans, R. P.,
and Anderson, C. L.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
5512-5516 |
5. | Israel, E. J., Wilsker, D. F., Hayes, K. C., Schoenfeld, D., and Simister, N. E. (1996) Immunology 89, 573-578[Medline] [Order article via Infotrieve] |
6. | Zijlstra, M., Bix, M., Simister, N. E., Loring, J. M., Raulet, D. H., and Jaenisch, R. (1990) Nature 344, 742-746[CrossRef][Medline] [Order article via Infotrieve] |
7. | Roberts, D. M., Guenthert, M., and Rodewald, R. (1990) J. Cell Biol. 111, 1867-1876[Abstract] |
8. |
Ahouse, J. J.,
Hagerman, C. L.,
Mittal, P.,
Gilbert, D. J.,
Copeland, N. G.,
Jenkins, N. A.,
and Simister, N. E.
(1993)
J. Immunol.
151,
6076-6088 |
9. | Simister, N. E., Story, C. M., Chen, H.-L., and Hunt, J. S. (1996) Eur. J. Immunol. 26, 1527-1531[Medline] [Order article via Infotrieve] |
10. | Kristoffersen, E. K., and Matre, R. (1996) Eur. J. Immunol. 26, 1668-1671[Medline] [Order article via Infotrieve] |
11. | Leach, J. L., Sedmak, D. D., Osborne, J. M., Rahill, B., Lairmore, M. D., and Anderson, C. L. (1996) J. Immunol. 157, 3317-3322[Abstract] |
12. | Simister, N. E. (1998) in The Immunoglobulin Receptors and Their Physiological and Pathological Roles in Immunity (van de Winkel, J. G. J. , and Hogarth, P. M., eds) , pp. 63-71, Kluwer, Dordrecht |
13. | Borvak, J., Richardson, J., Medesan, C., Antohe, F., Radu, C., Simionescu, M., Ghetie, V., and Ward, E. S. (1998) Int. Immunol. 10, 1289-1298[Abstract] |
14. |
Berryman, M.,
and Rodewald, R.
(1995)
J. Cell Sci.
108,
2347-2360 |
15. |
Rodewald, R.
(1973)
J. Cell Biol.
58,
189-211 |
16. | Rodewald, R., and Kraehenbuhl, J.-P. (1984) J. Cell Biol. 99, 159S-164S[Medline] [Order article via Infotrieve] |
17. | Rodewald, R. (1976) J. Cell Biol. 71, 666-670[Abstract] |
18. | Jones, E. A., and Waldman, T. A. (1972) J. Clin. Invest. 51, 2916-2927[Medline] [Order article via Infotrieve] |
19. | Simister, N. E., and Mostov, K. E. (1989) Cold Spring Harbor Symp. Quant. Biol. 54, 571-580[Medline] [Order article via Infotrieve] |
20. | Vaughn, D. E., and Bjorkman, P. J. (1998) Structure 6, 63-73[Medline] [Order article via Infotrieve] |
21. | Benlounes, N., Chedid, R., Thuillier, F., Desjeux, J. F., Rousselet, F., and Heyman, M. (1995) Biol. Neonate 67, 254-263[Medline] [Order article via Infotrieve] |
22. | Leach, L., Eaton, B. M., Firth, J. A., and Contractor, S. F. (1991) Histochem. J. 23, 444-449[Medline] [Order article via Infotrieve] |
23. | Story, C. M., Mikulska, J. E., and Simister, N. E. (1994) J. Exp. Med. 180, 2377-2381[Abstract] |
24. | Meads, T. J., and Wild, A. E. (1994) Placenta 15, 525-539[Medline] [Order article via Infotrieve] |
25. | Trowbridge, I. S., Collawn, J. F., and Hopkins, C. R. (1993) Annu. Rev. Cell Biol. 9, 129-161[CrossRef] |
26. | Sandoval, I. V., and Bakke, O. (1994) Trends Cell Biol. 4, 292-297[CrossRef] |
27. | Johnson, K. F., Chan, W., and Kornfeld, S. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 10010-10014[Abstract] |
28. |
Canfield, W. M.,
Johnson, K. F.,
Ye, R. D.,
Gregory, W.,
and Kornfeld, S.
(1991)
J. Biol. Chem.
266,
5682-5688 |
29. |
Davis, C. G.,
van Driel, I. R.,
Russel, D. W.,
Brown, M. S.,
and Goldstein, J. L.
(1987)
J. Biol. Chem.
262,
4075-4082 |
30. | McGraw, T. E., and Maxfield, F. R. (1990) Cell Regul. 1, 369-377[Medline] [Order article via Infotrieve] |
31. | Mostov, K. E., and Cardone, M. H. (1995) Bioessays 17, 129-138[Medline] [Order article via Infotrieve] |
32. |
Stefaner, I.,
Praetor, A.,
and Hunziker, W.
(1999)
J. Biol. Chem.
274,
8998-9005 |
33. |
McCarthy, K. M.,
Yoong, Y.,
and Simister, N. E.
(2000)
J. Cell Sci.
113,
1277-1285 |
34. | Higuchi, R., Krummel, B., and Saiki, K. (1988) Nucleic Acids Res. 16, 7351-7367[Abstract] |
35. | Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 61-68[CrossRef][Medline] [Order article via Infotrieve] |
36. | Gorman, C. M., Gies, D. R., and McCray, G. (1990) DNA Prot. Eng. Tech. 2, 3-10 |
37. | Wallace, K. H., and Rees, A. R. (1980) Biochem. J. 188, 9-16[Medline] [Order article via Infotrieve] |
38. |
Okamoto, C. T.,
Shia, S. P.,
Bird, C.,
Mostov, K. E.,
and Roth, M. G.
(1992)
J. Biol. Chem.
267,
9925-9932 |
39. | Letourneur, F., and Klausner, R. D. (1992) Cell 69, 1143-1157[Medline] [Order article via Infotrieve] |
40. | Hunziker, W., and Fumey, C. (1994) EMBO J. 13, 2963-2967[Abstract] |
41. | Greenberg, M., DeTulleo, L., Rapoport, I., Skowronski, J., and Kirchhausen, T. (1998) Curr. Biol. 8, 1239-1242[Medline] [Order article via Infotrieve] |
42. | Bresnahan, P. A., Yonemoto, W., Ferrell, S., Williams-Herman, D., Geleziunas, R., and Greene, W. C. (1998) Curr. Biol. 8, 1235-1238[Medline] [Order article via Infotrieve] |
43. | Davis, C. G., Lehrman, M. A., Russell, D. W., Anderson, R. G., Brown, M. S., and Goldstein, J. L. (1986) Cell 45, 15-24[Medline] [Order article via Infotrieve] |
44. | Jing, S. Q., Spencer, T., Miller, K., Hopkins, C., and Trowbridge, I. S. (1990) J. Cell Biol. 110, 283-294[Abstract] |
45. |
Breitfeld, P. P.,
Casanova, J. E.,
McKinnon, W. C.,
and Mostov, K. E.
(1990)
J. Biol. Chem.
265,
13750-13757 |
46. | Burmeister, W. P., Gastinel, L. N., Simister, N. E., Blum, M. L., and Bjorkman, P. J. (1994) Nature 372, 336-343[CrossRef][Medline] [Order article via Infotrieve] |
47. | Raghavan, M., Wang, Y., and Bjorkman, P. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11200-11204[Abstract] |
48. |
Jadot, M.,
Canfield, W. M.,
Gregory, W.,
and Kornfeld, S.
(1992)
J. Biol. Chem.
267,
11069-11077 |
49. | Collawn, J. F., Stangel, M., Kuhn, L. A., Esekogwu, V., Jing, S. Q., Trowbridge, I. S., and Tainer, J. A. (1990) Cell 63, 1061-1072[Medline] [Order article via Infotrieve] |
50. | Spiess, M. (1990) Biochemistry 29, 10009-10018[Medline] [Order article via Infotrieve] |
51. | Dietrich, J., Kastrup, J., Nielsen, B. L., Ødum, N., and Geisler, C. (1997) J. Cell Biol. 138, 272-281 |
52. |
Pitcher, C.,
Honing, S.,
Fingerhut, A.,
Bowers, K.,
and Marsh, M.
(1999)
Mol. Biol. Cell
10,
677-691 |
53. |
Praetor, A.,
Ellinger, I.,
and Hunziker, W.
(1999)
J. Cell Sci.
112,
2291-2299 |
54. | Snider, M. D., and Rogers, O. C. (1985) J. Cell Biol. 100, 826-834[Abstract] |
55. | Duncan, J. R., and Kornfeld, S. (1988) J. Cell Biol. 106, 617-628[Abstract] |
56. |
Bos, C. R.,
Shank, S. L.,
and Snider, M. D.
(1995)
J. Biol. Chem.
270,
665-671 |
57. | Broadwell, R. D., Baker-Cairns, B. J., Friden, P. M., Oliver, C., and Villegas, J. C. (1996) Exp. Neurol. 142, 47-65[CrossRef][Medline] [Order article via Infotrieve] |
58. | Burmeister, W. P., Huber, A. H., and Bjorkman, P. J. (1994) Nature 372, 379-383[CrossRef][Medline] [Order article via Infotrieve] |
59. | Raghavan, M., Chen, M. Y., Gastinel, L. N., and Bjorkman, P. J. (1994) Immunity 1, 303-315[Medline] [Order article via Infotrieve] |
60. | Butor, C., and Davoust, J. (1992) Exp. Cell Res. 203, 115-127[Medline] [Order article via Infotrieve] |