The Journal of Immunology
Brief Residence at the Plasma Membrane of the MHC Class
I-Related Chain B Is Due to Clathrin-Mediated
Cholesterol-Dependent Endocytosis and Shedding1
Sonia Agüera-González,2 Philippe Boutet,2 Hugh T. Reyburn, and Mar Valés-Gómez3
Recognition of MHC class I-related chain (MIC) molecules on the surface of target cells by the activating receptor NKG2D leads
to their lysis by immune effector cells. Up-regulation of NKG2D ligands is broadly related to stress, although the detailed molecular mechanisms that control the presence of these molecules at the plasma membrane are unclear. To investigate the posttranslational mechanisms that control surface expression of the human NKG2D ligand MICB, we studied the subcellular localization and trafficking of this molecule. We found that in several cellular systems, the expression of MICB molecules on the cell
surface is accompanied by an intracellular accumulation of the molecule in the trans-Golgi network and late endosome-related
compartments. Surprisingly, MICB has a much shorter half-life at the plasma membrane than MHC molecules and this depends
on both recycling to internal compartments and shedding to the extracellular medium. Internalization of MICB depends partially
on clathrin, but importantly, the lipid environment of the membrane also plays a crucial role in this process. We suggest that the
brief residence of MICB at the plasma membrane modulates, at least in part, the function of this molecule in the immune
system. The Journal of Immunology, 2009, 182: 4800 – 4808.
M
ajor histocompatibility complex class I-related
chain (MIC)4 A and B are two MHC-like molecules
encoded within the human MHC, but distinct from
classical HLA class I proteins in that they bind neither ␤2microglobulin nor antigenic peptides (1– 4). MIC proteins bind
the activating receptor NKG2D that is constitutively expressed
on all NK cells and CD8⫹ T cell subpopulations and is upregulated after activation on CD4⫹ T cells (5). Engagement of
NKG2D by its ligands leads to the activation of lysis and cytokine secretion by NK cells and T cells either directly or as a
costimulatory factor (for review, see Ref. 6). Thus, the presence
of MIC at the surface of putative target cells must be tightly
regulated so that the immune response is not triggered or hidden
inappropriately (which would lead to autoimmunity in the first
instance and immune evasion in the latter). In fact, with the
exception of gastrointestinal epithelium, the vast majority of
normal cells do not express MIC molecules at the cell surface;
Department of Pathology, University of Cambridge, United Kingdom
Received for publication March 4, 2008. Accepted for publication February 4, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was funded by a Leukemia Research Fund Project Grant (to H.T.R. and
M.V.G.). M.V.G. is a recipient of a New Investigator Grant from the Medical Research Council and an International Joint Project from the Royal Society. S.A. was
supported by fellowships from the Fundación Caja Madrid and Ibercaja. P.B. was
supported in part by The Newton Trust.
2
S.A.-G. and P.B. contributed equally to the work presented.
3
Address correspondence and reprint requests to Dr. Mar Valés-Gómez, Department
of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP,
United Kingdom
4
Abbreviations used in this paper: MICA/B, MHC class I chain-related gene A/B;
MESNA, 2,mercaptoethane sulfonic acid; DRM, detergent-resistant membrane;
HCMV, human CMV; TGN, trans-Golgi network; CI-M6PR, cation-independent
mannose-6-phosphate receptor; ER, endoplasmic reticulum; EEA1, early endosome
A1; LAMP-1, lysosome-activated membrane protein 1; CHX, cycloheximide; BFA,
brefeldin A; CHL, chloroquine; M␤CD, methyl-␤-cyclodextrin.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0800713
instead, the expression of these proteins is up-regulated in a
number of pathological situations, mainly cancer and autoimmunity (for review, see Refs. 7 and 8). For example, MIC expression has been found in tumors of various origins and the
presence of pathogens like Mycobacterium tuberculosis has
been shown to up-regulate its expression (9). Recently, a number of external stimuli such as DNA-damaging agents and proteasome inhibitors have been shown to induce expression of
NKG2D ligands (10, 11). Thus, although the precise mechanisms that regulate MIC expression are still unknown, the presence of MIC at the plasma membrane can be related broadly
with stress and this coincides with the presence of heat shock
elements in the MIC promoters (3). The fact that MIC was
detected by Western blot in a number of nontransformed cell
types, including keratinocytes, endothelial cells, and monocytes, although they were not found at the cell surface of all of
them (2, 12–14), suggests the existence of posttranscriptional
and posttranslational mechanisms regulating MIC expression,
in addition to transcriptional regulation. That NKG2D ligands
can also be shed from the cells as soluble molecules represents
an additional level of complexity in this system (15–17).
MICB is a target for several proteins expressed by pathogens
that are involved in immune evasion. Interestingly, many of
them are selective toward MICB and they do not affect the
related protein MICA (18). There are still many questions about
the large number of NKG2D ligands and whether this diversity
reflects somehow a difference in function (for review, see
Ref. 19).
With the goal of investigating the mechanisms that control
surface expression of MICB molecules at a posttranslational
level, we studied the subcellular localization and trafficking of
MICB. In the present study, we show, in several cell lines, that
the expression of MICB molecules on the cell surface is accompanied by an intracellular accumulation of the molecule in
the trans-Golgi network (TGN) and late endosome-related compartments. We further show that MICB has a short half-life at
the plasma membrane and that this depends on shedding and
The Journal of Immunology
clathrin-dependent endocytosis, although the integrity of cholesterol in the plasma membrane also influences the internalization of MICB protein.
Materials and Methods
Cells, reagents, and Abs
HeLa, U373, and CV1 cells were maintained in DMEM supplemented with
10% FCS and antibiotics (DMEM/10% FCS). 721.221 and HCT116 were
cultured in RPMI 1640 medium with 10% FCS and antibiotics.
721.221, U373, and CV1 transfected with MICB were previously described (20, 21). Anti-MICA/B monoclonal and polyclonal Abs were purchased from R&D Systems, anti-MICA (AMO1) and anti-MICB (BMO2)
were from Immatics, anti-MICA/B 6D4 was from Santa Cruz Biotechnology, and isotype control mouse mAbs were purchased from Sigma-Aldrich. For microscopy, sheep Ab directed against human TGN46 was from
Serotec, mouse mAb specific for GM130 and early endosome A1 (EEA1)
were from BD Biosciences, mouse anti-human CD107a (lysosome-activated membrane (LAMP-1)) was from Southern Biotechnology Associates; mouse KDEL mAb was from Bioquote; rabbit anti-mouse tetramethyl
rhodamine isothiocyanate from DakoCytomation; Alexa Fluor 564 donkey
anti-sheep, Alexa Fluor 488 donkey anti-sheep, Alexa Fluor 488 goat
anti-mouse, and Alexa Fluor 488 and 564 goat anti-rabbit Abs were
from Molecular Probes; and anti-CI-M6PR polyclonal rabbit Ab was a
gift from P. Luzio (Cambridge Institute for Medical Research, Cambridge, U.K.). Anti-caveolin Ab was from BD Transduction Laboratories. Unless otherwise indicated, chemicals were purchased from
Sigma-Aldrich.
Flow cytometry
For flow cytometry, 105 cells were incubated with mouse mAbs and bound
Ab was visualized using either PE- or FITC-labeled F(ab⬘)2 of goat antimouse Ig (DakoCytomation). Samples were analyzed using a FACScan II
flow cytometer (BD Biosciences).
Confocal microscopy
Cells were fixed with 4% p-formaldehyde at room temperature for 10 min,
permeabilized by incubation with 0.1% saponin at room temperature for 10
min, stained with commercial anti-MICB Abs (R&D Systems) or costained
with markers for intracellular compartments, and analyzed by confocal
microscopy as previously described (22).
All fluorescence images were obtained using a confocal microscope
(either a Leica DM IRE2 or IRBE confocal laser-scanning microscope).
Excitation wavelengths and filter sets were as supplied by the manufacturer
(lines at 488 and 568 nm for Alexa Fluor 488 and Alexa Fluor 568, respectively). To reduce Förster resonance energy transfer, the emission
spectrum for the first fluorochrome was made distinct from the excitation
spectrum of the second, i.e., the setting of the spectra was narrowed down
to 500 –560 and 580 – 660 nm for Alexa Fluor 488 and Alexa Fluor 568,
respectively. Images of fixed cells were taken using a 63 ⫻ 1.32 aperture
objective with the confocal pinhole set to 1Airy unit.
Image acquisition. Images were obtained by scanning either a single focal
plane or a series of them across the cell using Leica TCS software. For
colocalization experiments, acquisition of images was conducted by successive scanning of the 488 (green) and the 568 (red) channel, exciting one
fluorochrome at a time by successive scanning of the green and the red
channels independently. To explore the whole intracellular area, series
of sections (total interval z ⫽ 2–4 ␮m) were acquired. Image acquisition
of the subsequent sections was conducted near the resolution limit of
the optical system used, i.e., the optimal for a good analysis of colocalization: 1024 ⫻ 1024 pixels at z intervals of 0.1221 ␮m with a
magnification of 3–4 times. Colors and overlays shown in the figures
were as collected by the microscope software and no further threshold
adjustments were made. Adobe Photoshop software was used for composition of the figures.
Image analysis and quantitation. For visualization of colocalization, images of composed overlays are presented in the manuscript; however, to
analyze the degree of colocalization taking in account the three-dimensional nature of the sample, imaging processing was performed (data are
included in Table I). Huygens Deconvolution Software (Scientific Volume
Imaging) was used for deconvolution and analysis.
ELISA
Detection of soluble MICB was performed using a sandwich ELISA procedure. The anti-MICB mAb from R&D Systems was used at 5 ␮g/ml.
4801
Table I. Colocalization Coefficients
Pearson’s Coefficient
Overlap
0.458
0.347
0.200
0.383
0.099
0.641
0.527
0.299
0.520
0.190
M6PR
KDEL
CD63
EEA1
LAMP-1
After blocking for 1 h with PBS containing 2% BSA, tissue culture supernatant was incubated for 1 h at 37°C. The assay was developed using
biotinylated goat anti-MICB (0.1 ␮g/ml) followed by incubation with
streptavidin-HRP (1/2000; Amersham Biosciences) and a peroxidase substrate system (2,2⬘-azino-di-[3-ethylbenzthiazoline sulfonate] and H2O2 in
a glycine/citric acid buffer; Roche). The absorbance was measured at 410
nm with a reference wavelength of 490. Samples were analyzed in
duplicates.
Internalization experiments
Ab uptake. Ab uptake experiments were performed as previously described (23). Cells were incubated with Ab for 10 min at 4°C and then
transferred to 37°C for different lengths of time. To detect internalized
protein, cells were fixed, permeabilized, stained with secondary Ab, and
analyzed by confocal microscopy.
Cleavable biotinylation. For cleavable biotinylation experiments, the cells
were washed three times with ice-cold PBS. Then incubated 30 min with
0.25 mg/ml sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate
(EZ-Link Sulfo-NHS-SS-Biotin; Pierce) in ice-cold PBS containing 1 mM
MgCl2 and 0.1 mM CaCl2 (PBS⫹). The reaction was quenched by washing
the cells three times with ice-cold serum-free medium containing 0.1%
BSA and then once with ice-cold PBS⫹. Prewarmed (to 37°C) serum-free
medium was added for the desired time period(s) to allow internalization of
the biotinylated surface components, after which they were returned to
4°C. After one rinse with PBS⫹, biotin was stripped by three 20-min
incubations with ice-cold 100 mM sodium 2-mercaptoethane sulfonic acid
(MESNA) in 50 mM Tris-HCl (pH 8.6), 100 mM NaCl, 1 mM EDTA, and
0.2% BSA. The cells were rinsed quickly twice again with PBS⫹, and
residual MESNA was quenched by a 10-min incubation with ice-cold 120
mM iodoacetamide in PBS⫹. After two additional rinses with ice-cold
PBS, the cells were lysed and MICB was immunoprecipitated. The proteins
were resolved in SDS-PAGE and biotinylated proteins detected by Western
blot with either streptavidin-HRP or MICB-specific Ab.
Estimation of MICB internalization by flow cytometry. Internalization of
MICB molecules was quantitated using a variant of a previously described
flow cytometric assay for estimating receptor internalization (24). Briefly,
cells stably transfected with MICB were transiently transfected with plasmids encoding the AP180 C terminus, epsin1 R63L ⫹H73L, and epsinR
(gift from Dr. H McMahon, LMB, Cambridge, U.K.) and EH29 using
JETPEI (PolyPlus Transfection SA). Twenty-four hours later, the cells
were detached by incubation in PBS/0.2% BSA/10 mM EDTA, washed
twice (PBS/BSA), and resuspended in binding buffer (Ham’s F12 medium
with 0.2% BSA, 10 ␮M GM6001, and 1 mM HEPES). Transfected cells
were divided into two aliquots and while one was held at 37oC, the other
was cooled to 4oC. MICA/B-specific mAb (2.5 ␮g/ml) was then added to
each aliquot and the cells were incubated either at 37oC or on ice for 30
min. After this time, the cells were washed into PBS/0.2% BSA/0.1%
sodium azide and placed on ice followed by staining with PE-conjugated
secondary goat anti-mouse Ig. Cells overexpressing the various constructs
were gated on GFP fluorescence and the amount of MICB molecules remaining in the plasma membrane after the surface labeling with specific
mAb was determined for each condition. Duplicate samples (5 ⫻ 104 cells/
sample) were analyzed in each experiment. The reduction in surface receptor fluorescence after incubation at 37oC compared with 4oC represents
internalization of receptors.
Pulse-chase experiments
Cells were washed and resuspended in either methionine-cysteine-free medium alone or with 100 ␮M chloroquine or control. After a 30-min incubation at 37 °C, cells were spun and resuspended at 107 cells/ml with
PRO-MIX L-[35S] In Vitro Cell Labeling Mix (Amersham Biosciences) at
0.3 mCi/ml. Cells were incubated at 37°C for 15 min and then chased using
complete medium with 10% FCS for the indicated times. Each time point
sample was washed in cold PBS and resuspended in lysis buffer (50 mM
4802
CELLULAR TRAFFICKING OF MICB
Tris (pH 7.6), 150 mM NaCl, 5 mM EDTA, 1% (v/v) Nonidet P-40, 1
␮g/ml leupeptin, 1 ␮g/ml pepstatin, and 5 mM iodoacetamide). After a
30-min incubation on ice, samples were precleared by incubation, first with
Pansorbin (Calbiochem), followed by protein A-Sepharose beads (Pharmacia). MICB was immunoprecipitated by incubation on ice with the appropriate Ab and recovered using protein A beads. Samples were washed
three times and resuspended in SDS-PAGE-loading buffer. Proteins were
resolved in SDS-PAGE. Gels were stained and exposed on Biomax MR
film (Kodak).
Immunoprecipitation
Cells were lysed in 50 mM Tris (pH 7.6), 150 mM NaCl, 5 mM EDTA, 1%
Nonidet P-40, 1 ␮g/ml leupeptin, and 1 ␮g/ml pepstatin. After a 30-min
incubation on ice, samples were precleared by incubation with Pansorbin
(Calbiochem). MICB was immunoprecipitated by incubation on ice with a
mixture of the MICB-specific Abs BMO2 and 6D4 and recovered using
protein G beads (Pharmacia). Samples were washed three times and SDSPAGE-loading buffer was added. Proteins were resolved in SDS-PAGE.
Western and dot blots
Cell lysates were run on 12% SDS-PAGE gels and transferred to Immobilon-P (Millipore). The membrane was blocked using PBS containing
0.1% Tween 20 (PBS-T) and 5% nonfat dry milk. Detection of MICB was
performed by incubating the membrane with biotinylated goat polyclonal
anti-MICB Ab, followed by HRP-conjugated secondary reagent. Proteins
were visualized using the ECL system (Amersham Pharmacia). Detection
of ganglioside GM1 was performed by immobilizing 5 ␮l of the above
fractions in nitrocellulose membrane, incubating with cholera toxin-HRP
(Sigma-Aldrich), and detecting with ECL.
Detergent-resistant membrane (DRM) fractionation
Cells were lysed in TNE buffer (20 mM Tris (pH 7.5), 150 mM NaCl, and
5 mM EDTA) containing 1% Brij-58 and protease inhibitors and homogenized using a Dounce homogenizer. Samples were diluted with an equal
volume of 85% sucrose in TNE and then placed at the bottom of a step
sucrose gradient and fractionated by centrifugation for 18 –20 h at
200,000 ⫻ g (25). One-milliliter fractions were collected from top to bottom and made to a final concentration of 0.2% deoxycholate. Proteins were
separated on SDS-PAGE. Western blot was performed as described above
using Abs against MICA/B and caveolin-1.
Efficiency of fractionation was assessed by detection of ganglioside
GM1 using cholera toxin-HRP.
Results
MIC expression on the cell surface is accompanied by an
intracellular accumulation of the molecule in late
endosome/TGN-related compartments
In experiments exploring the mechanism of immune evasion used
by human CMV (HCMV), we previously showed that HCMVinfected cells lost surface expression of MICB and accumulated
MIC intracellularly. Interestingly, however, we observed that uninfected cells also expressed a large amount of intracellular protein
(23). To study this observation further, we analyzed the distribution of these molecules in a number of cell lines: HeLa and the
colon carcinoma HCT116 naturally express MICA and MICB; the
glioma cell line U373 and the monkey cell line CV1 were stably
transfected with MIC molecules (U373/CV1-MICA and -MICB).
The surface expression of MICA and MICB was studied by flow
cytometry (Fig. 1A). In general, MICB expression at the plasma
membrane was lower than that of MICA, but MICB was found
inside HCT116 cells in a larger proportion than MICA (Fig. 1B;
whole intracellular area supplemental Fig. 15). Examination of
both U373 and CV1 transfectants by confocal microscopy also
showed quite a large proportion of MICB intracellularly (Fig. 1C).
Since the tumor cell lines HCT116 and HeLa express both MICA
and MICB, it is difficult to confidently discriminate between these
molecules, particularly since the Abs raised against either MICA
or MICB could have different cross-reactivity depending on the
5
The online version of this article contains supplemental material.
FIGURE 1. A large proportion of MIC is intracellular. A, Flow cytometry analysis of surface MIC molecules. HCT116, HeLa, U373-MICA, and
U373-MICB were stained with anti-MICA (AMO1) and anti-MICB
(BMO2) Abs as indicated or isotype control. B, Confocal microscopy of
intracellular MICA and MICB in HCT116 cells. Cells were stained with
anti-MICA (AMO1) and anti-MICB (BMO2) Abs and processed for confocal microscopy. Images show a single focal plane across the cell (depth
of the plane 0.2849 ␮m) using the ⫻63 objective with a ⫻4 magnification.
The series of images corresponding to the central part of the same cell are
shown in supplemental Fig. 1. Scale bar, 4 ␮m. C, Confocal microscopy of
intracellular MICB in U373- and CV1-MICB transfectants. Cells were
stained with anti-MICA/B Ab and processed for confocal microscopy. Images were obtained by scanning a single focal plane across the cell using
the ⫻63 objective without magnification. Scale bar, 20 ␮m.
alleles present in each cell line. For these reasons, we decided to
focus on the study of transfectants in the following experiments.
To study the intracellular distribution of MICB molecules, cells
were costained with markers for intracellular compartments and
analyzed in confocal microscopy. Visualization of colocalization is
presented as an overlay of the channels corresponding to MICB
and the markers for different compartments (Fig. 2). The perinuclear area where more MICB staining is found was magnified for
intracellular analysis and is also included in Fig. 2. To explore the
whole intracellular area, series of sections were acquired through
the cell (supplemental Fig. 2) and qualitative evaluation and quantification of data for colocalization purposes were performed (see
Materials and Methods). The colocalization coefficients with the
different markers are shown in Table I.
MICB colocalized partially with the TGN markers TGN46 (data
not shown) and cation-independent mannose-6-phosphate receptor
(CI-M6PR), as well as partially with the endoplasmic reticulum
The Journal of Immunology
4803
To confirm the TGN localization of MICB, cellular subfractionation was performed and the fractions were analyzed for the presence of MICB and organelle markers (supplemental Fig. 3). MICB
was detected in fractions enriched for markers of TGN, as well as
in the fractions in which ER and early endosomes markers were
found. The confocal microscopy and cellular subfractionation data
would be consistent with MICB traveling between the TGN and
the late endosomal system.
MICB has a short half-life at the plasma membrane
FIGURE 2. MIC colocalizes with markers for TGN and endosomes.
CV1-MICB transfectants were fixed, permeabilized, and costained for
MICB (green panels) and markers of intracellular compartments (CIM6PR (TGN), KDEL (ER), CD63 (late endosome, MVB), EEA1 (early
endosome), LAMP1 (lysosome, late endosome), and GM130 medial
Golgi)] (red panels, as indicated). Images were obtained by successive
scanning of the green and red fluorochromes (see Materials and Methods).
The first three columns show a single focal plane of a central region in the
z-axis of the series of focal planes acquired (full series shown in supplemental Fig. 2) using the ⫻63 objective. In parallel, images of the same cells
were obtained with a ⫻4 magnification (shown in the last column) to
optimize the optical parameters for colocalization analysis. The experiment
was performed five times and an average of four cells per marker were
analyzed per experiment. Similar results were obtained with the U373MICB transfectants. Scale bars: merge, 20 ␮m; zoom, 5 ␮m.
(ER) marker, KDEL, early endosomes (EEA1), and the late endosome marker CD63. Little colocalization was found with the other
markers used: ER (PDI, ERp72; data not shown), medial Golgi
(GM130), and LAMP-1. The CI-M6PR is involved in the sorting and
transport of newly synthesized lysosomal hydrolases that contain
mannose-6-phosphate moieties on their N-glycan chains, which serve
as sorting signals for TGN-to-endosome transport and in steady state
can be used as a marker for TGN (26). LAMPs are lysosomal integral
membrane glycoproteins and LAMP-1 is often used as a lysosomal
marker, whereas CD63 (LIMP-I, LAMP-3) is a member of the tetraspanin superfamily enriched in intraluminal vesicles in late endosomes/lysosomes that can travel from the TGN to lysosomes (27). It
has been shown recently that LAMP-1 and LAMP-2 are present in
separate TGN-derived vesicles from those containing mannose phosphate receptor and the adaptor protein AP-1 (28).
The presence of MICB molecules in late endosome/TGN-type organelles suggests that the molecule might be traveling within various compartments of the endocytic pathway and that some of the
observed intracellular molecules could be the result of down-modulation from the plasma membrane. To follow the fate of the protein after it reaches the plasma membrane, Ab uptake experiments
were performed and analyzed by confocal microscopy. After a
30-min incubation with Ab, a large proportion of MICB previously
present at the plasma membrane was found intracellularly. The
internalized MICB was then costained with markers for intracellular compartments (supplemental Fig. 4, A and B). At short incubation times, MICB was observed in vesicles that did not colocalize with CI-M6PR, although after longer incubation times, some
colocalization between MICB and CI-M6PR was noted.
The kinetics of MICB endocytosis was also studied in flow cytometry experiments. 721.221 cells transfected with MICB were
stained with PE-labeled MICB-specific Ab and incubated for different periods of time to allow internalization. After the indicated
times, Ab bound at the plasma membrane was removed by acid
wash (29) and the Ab that remained in the internal compartments
was detected by flow cytometry. Surface-bound Ab was efficiently
removed by acid wash without affecting viability of the cells (at
time 0 min, 94% of cells lost all of the staining after the acid wash
and were still viable), and after incubation to allow internalization,
endocytosed MICB staining was still observed (supplemental
Fig. 4c).
To rule out that the observed internalization of the protein
was due to cross-linking with Ab, the phenomenon was studied
in a biochemical assay independent of Ab. Cells were surface
labeled with cleavable biotin and, after incubation at 37°C to
allow internalization of the protein, the cells were treated with
MESNA (a cell-impermeant reducing agent) to remove the biotin moiety from the labeled proteins that remain at the plasma
membrane, after which only intracellular labeled proteins are
detected. As a control for the proper elimination of surfacelabeled proteins, cells were incubated at 4°C and treated with
MESNA. No biotinylated protein was detected, indicating that
no MIC protein had been internalized (Fig. 3A, lane 2). As a
positive control, cells incubated at 4°C but not treated with
MESNA showed the total amount of cell surface biotinylated
MIC (Fig. 3A, lane 1). This experiment shows a band corresponding to internalized protein in cells treated with MESNA
after incubation at 37°C. This result confirms the ability of
MICB to internalize. The various size bands of MICB observed
in these experiments probably represent posttranslational modifications such as glycosylation. However it is also clear from
this experiment that a significant amount of MIC protein is lost
during this incubation at 37oC. This could include both degradation and shedding to the extracellular medium.
Given that MIC proteins are known to be shed from the cell
surface (15, 16), we next studied the dynamics of MIC expression
and shedding in this system. To assess the stability of MICB
molecules at the cell surface, flow cytometry experiments were
4804
CELLULAR TRAFFICKING OF MICB
FIGURE 3. MICB has a short half-life at the plasma membrane. A, Cleavable biotinylation. U373-MICB cells were labeled with disulfide-biotin. Labeled
proteins were allowed to internalize for 2 h at 37°C and, after that period of time, surface biotin was removed by treatment with MESNA. Biotinylated
MICB (left panel) and total MICB (right panel) were assessed by Western blot (WB). The total amount of surface biotinylation can be observed in the lane
corresponding to the control at 4°C untreated with MESNA (lane 1); removal of all surface biotinylation labeling corresponds to the control at 4°C treated
with MESNA (lane 2). A band corresponding to internalized MICB can be observed in the lane corresponding to cells treated with MESNA after incubation
at 37°C. This experiment is representative of four experiments. B, Flow cytometry analysis of surface MICB molecules after treatment of U373-MICB cells
with BFA, BFA and CHX (BFA ⫹ CHX) and CHL as indicated. After incubation with the compound for the indicated time, U373-MICB cells were stained
with anti-MICA/B Ab. Similar results were obtained with the cell line 721.221. Data are representative of three experiments. C, Soluble MICB in the tissue
culture supernatant of U373-MICB cells was detected by ELISA. A sandwich ELISA was performed using anti-MICB mAb from R&D Systems as catching
Ab and biotinylated goat anti-MICB for detection, followed by incubation with streptavidin-HRP. The absorbance was measured at 410 nm with a reference
wavelength of 490. Samples were analyzed in duplicates. The dotted line indicates the background for the assay (medium from untransfected cells). The
inset shows SDS-PAGE and Western blot analysis using anti-MICA/B Ab of tissue culture supernatant from untransfected cells (Med) and U373-MICB
transfectants. The 50-kDa molecular mass marker is indicated. Similar results were obtained with the CV1-MICB transfectants. Experiments were repeated
three times. D, The maturation of MICB does not change in the presence of CHL. Pulse-chase experiment followed by immunoprecipitation of MICB (see
Materials and Methods) from cells either untreated or treated with 100 ␮M CHL (raises the pH of lysosomes). The proteins were resolved in SDS-PAGE.
This experiment was performed twice.
performed using cycloheximide (CHX, to block protein synthesis), brefeldin A (BFA, inhibitor of vesicular trafficking), and a
combination of both (Fig. 3B). Interestingly, treatment with BFA
and CHX led to a significant loss of MICB after 4 h, as did treatment with CHX alone (data not shown). A significant component
of surface MICB must be involved in vesicular trafficking since the
inclusion of BFA alone in the experiment also provoked a marked
decrease in surface staining. Similar data were obtained in experiments with 721.221 cells transfected with MICB (data not
shown). These data show that MICB is different than class I MHC
proteins, which remain stably at the plasma membrane for 18 –24
h (30). An inhibitor of protein degradation, such as the weak base
chloroquine (CHL), that raises the pH of late endosomes and lysosomes, impairing their function, was also included in the experiments. The stability of MICB at the cell surface was not affected
by the presence of this compound. The shedding of MICB to the
tissue culture medium was analyzed by ELISA (Fig. 3C). Significant shedding of MICB was observed after 1 h of incubation and
this soluble MIC accumulated over time (Fig. 3, inset). Treatment
with BFA, CHX, or CHL did not inhibit shedding. The fact that
MICB is present in lysosome-related organelles, but that the levels
of surface and soluble protein do not change when CHL is added
suggest that the protein must be trafficking through these compartments rather than merely being degraded in lysosomes. This was
confirmed by the inability of CHL treatment to alter the loss of
MICB observed in pulse-chase experiments (Fig. 3D). The marked
loss of MICB seen by 3 h of chase confirmed biochemically the
short half-life of MICB molecules suggested by the FACS
experiments.
All together, these data suggest that, at steady state, MICB remains only for a short time at the plasma membrane and that the
amount of MICB at the cell surface is most probably regulated by
a combination of endocytosis and protein loss (by degradation and
shedding).
MICB endocytosis and clathrin-dependent pathways
The mechanism of MICB endocytosis was next investigated. It
is generally accepted that there are two main endocytic pathways based on the dependence or independence of clathrin (for
review, see Refs. 31–35). Both processes can be explored by the
use of mutants or drugs that interfere with specific components
necessary for the correct function of the pathway (reviewed in
Ref. 36).
Because the cytoplasmic tail of MICA/B molecules contain a
dileucine motif that could bind AP2, we first studied clathrindependent endocytosis, a process that requires a variety of
adaptor proteins and the generation of coated pits that bud into
vesicles.
Eps15 is an adaptor protein found in clathrin-coated pits required for the early steps of clathrin-dependent endocytosis.
The EH29 dominant-negative mutant of Eps15 affects the assembly of clathrin-coated pits and strongly inhibits transferrin
The Journal of Immunology
4805
FIGURE 4. MICB endocytosis and clathrin-mediated pathways. A, U373-MICB cells were transfected with the indicated dominant-negative
mutants. EH29 affects the generation of coated pits by inhibiting Eps15; the inactive variant of Eps15 (DIII⌬2) was used as a control. All of the
dominant-negative mutants are expressed as fusions to GFP to allow visualization of transfected cells. Cells were incubated with anti-MIC Ab at
4°C, followed by uptake of the Ab at 37°C for 20 min. After that, cells were fixed, permeabilized, and stained with secondary Ab coupled to Alexa
Fluor 568 (red) and analyzed by confocal microscopy. Similar results were obtained with the CV1-MICB transfectants. Scale bar, 20 ␮m. This
experiment was performed four times. B, Flow cytometric estimation of MICB internalization. U373-MICB cells were transiently transfected with
plasmids to disrupt endocytosis by overexpression of AP180 C terminus, epsin1 R63L ⫹ H73L, epsinR, and EH29. The reduction of MICB at the
plasma membrane produced by incubation of cells at 37°C for 30 min was determined by flow cytometry. Bars represent reduction in surface receptor
fluorescence (relative to control U373-MICB cells held at 4°C). ⴱⴱ, p ⬍ 0.05 in a t test compared with mock. Cells were cultured in the presence
of GM6001 to inhibit metalloprotease-mediated shedding during the 30-min incubation with Ab. Data were obtained in duplicate and are representative of two experiments. C, U373-MICB cells were preincubated for 30 min with 8 ␮g/ml CHL. After the treatment, Ab uptake experiments
were performed as in A. Scale bar, 40 ␮m. This experiment was repeated three times.
receptor internalization (37, 38). The effect of this construct on
MICB endocytosis was studied in Ab uptake experiments, in
which an inactive variant of Eps15 (DIII⌬2) was used as a
control. To identify transfected cells, these variants of Eps15
are tagged with GFP (Fig. 4A). After 20 min of Ab uptake, we
could observe an accumulation of MICB in mock-transfected
cells and cells expressing the inactive mutant, whereas in cells
transfected with EH29 a considerable proportion of MICB was
still at the plasma membrane. In supplemental Figs. 5 and 6,
parallel experiments performing series of sections through the
whole intracellular area are depicted. In these experiments, it is
still possible to observe some intracellular MICB after inhibition of clathrin in the EH29 mutant, although not as much as in
the inactive mutant DIII⌬2. Since clathrin blockade did not
completely abolish MIC endocytosis, these data suggest that
another mechanism of internalization might be involved.
To estimate the amount of MICB that was internalized via
clathrin, flow cytometry experiments were performed. U373MICB cells were transiently transfected with plasmids encoding
the AP180 C terminus (inhibits clathrin-dependent budding
events), epsin1 R63L ⫹ H73L (inhibits AP2 budding events),
epsinR (inhibits AP1 budding events), and EH29 (Fig. 4B). We
observed a marked reduction in internalization of cell surface
MICB in cells transfected with AP180, epsin1 R63L ⫹ H73L,
and EH29.
In parallel, experiments using other methods to interfere with
clathrin-mediated endocytosis were also performed. Hypertonic
medium promotes the disappearance of clathrin-coated vesicles
and plasma membrane-coated pits by preventing the interaction of
clathrin with adaptors required for endocytosis (39). Clathrin-dependent endocytosis can also be inhibited by the use of chlorpromazine (40). Thus, Ab uptake experiments were also performed in
the presence of chlorpromazine (Fig. 4C) and 0.32 M sucrose (supplemental Fig. 7A). The presence of these drugs impaired the uptake of MICB, confirming the data obtained previously.
MICB endocytosis and clathrin-independent pathways
Clathrin-independent endocytosis includes a group of pathways
still not well characterized, but often associated with plasma membrane domains known as lipid rafts/caveolae. These microdomains
are enriched in cholesterol, sphingolipids, and caveolin (for a review, see Refs. 41 and 42). Endocytosis via lipid rafts/caveolae is
4806
CELLULAR TRAFFICKING OF MICB
FIGURE 5. MICB endocytosis and
clathrin-independent pathways. A,
U373-MICB cells were preincubated
for 30 min with 10 mM M␤CD. For
labeling, either fluorescent-cholera
toxin or fluorescent transferrin were
added during the preincubation period
as indicated. Ab uptake experiments
were performed as in Fig. 4A. B, Flow
cytometric estimation of MICB internalization. U373-MICB cells were
treated with either M␤CD or sucrose.
The reduction of MICB at the plasma
membrane produced by incubation of
cells at 37°C for 30 min was determined by flow cytometry. Bars represent reduction in surface receptor
fluorescence (relative to control
U373-MICB cells held at 4°C). Cells
were cultured in the presence of
GM6001 to inhibit metalloproteasemediated shedding during the 30-min
incubation with Ab. ⴱⴱ, p ⬍ 0.05 and
ⴱⴱⴱ, p ⬍ 0.005 in a t test compared
with mock. Data were obtained in duplicate and are representative of two
experiments. Scale bar, 40 ␮m.
frequently defined using agents that disrupt or deplete cholesterol
at the plasma membrane.
To find out whether “caveolin-mediated” endocytosis is involved in MICB down-modulation, we studied the ability of the
protein to recycle from the plasma membrane in the presence of
methyl-␤-cyclodextrin (M␤CD). M␤CD binds to cholesterol
and selectively extracts it from the outer leaflet of the plasma
membrane. We observed that cells preincubated with M␤CD
accumulated MICB at the plasma membrane (Fig. 5A), suggesting a role for cholesterol in the trafficking of MICB. As controls, fluorescently labeled transferrin and cholera toxin were
included in the experiment. As expected, when M␤CD is
present, a large proportion of cholera toxin remains at the
plasma membrane, indicating a lower efficiency in its uptake.
However transferrin, a protein internalized through clathrincoated pits, did get internalized, although with lower efficiency
than in control cells. It has been previously shown that acute
depletion of cholesterol also reduces the rate of internalization
of transferrin (43, 44). In supplementary Fig. 7B, a parallel
experiment performing series of sections through the whole intracellular area is depicted. Flow cytometry analysis confirmed
that treatment with M␤CD reduced the uptake of mAb specific
for MICA/B (Fig. 5B).
These results suggest that MICB is internalized through a clathrin-mediated, cholesterol-dependent pathway. Such a mechanism
has been previously described for the entry of viruses and bacterial
toxins in the cell (45, 46).
MICB associates with DRMs
The finding that the trafficking of MICB could be influenced by the
lipid environment led us to investigate the association of this protein with DRM domains. DRMs are microdomains rich in cholesterol and sphingolipids, especially GM1 ganglioside (41, 42).
DRMs can be separated from other membrane components by
ultracentrifugation in sucrose gradients after solubilizing using detergents such as Triton X-100 or Brij-58 (47). Caveolin-1, a component of caveolae, is usually found in DRMs and can be used as
a marker for the efficiency of fractionation. As shown in Fig. 6, a
proportion of MICB was found in detergent-insoluble fractions
rich in GM1 sphingolipid and caveolin-1. This finding confirms the
location of at least some MICB molecules in domains of the membrane rich in cholesterol.
The Journal of Immunology
FIGURE 6. Association of MICB with DRMs. U373-MICB cells
were lysed in buffer containing 1% Brij-58 and homogenized using a
Dounce homogenizer. Samples were fractionated in a discontinuous
sucrose gradient. Fractions were collected and proteins were separated
on SDS-PAGE. Western blot was performed using Abs against MICA/B and
caveolin-1 (Cav-1). Efficiency of fractionation was assessed by detection of
ganglioside GM1 using cholera toxin-HRP on dot blots. This result is representative of at least five independent experiments and similar results were
obtained with the CV1-MICB transfectants.
Discussion
Given that recognition of NKG2D-L at the cell surface by their
receptor leads to killing of the target cell, a very tight regulation of
the expression of these proteins must exist. There are examples in
the literature of cells that express mRNA for one or more of the
NKG2D-L but do not show any expression of protein at the plasma
membrane (48). A posttranslational mechanism that involves the
degradation of NKG2D-L RNA has been suggested as a manner of
regulating the amount of these proteins in the cell (14). In this
study, we propose that the amount of MICB present at the cell
surface can also be modulated by the particular features of the
trafficking of the protein. Indeed, the main finding of the study is
the short time of residence of MICB at the cell surface, confirmed
by its rapid loss from the plasma membrane upon incubation with
inhibitors of protein synthesis. Several cellular processes seem to
be involved in the loss of MICB from the cell surface, including
internalization toward intracellular compartments and shedding to
the extracellular medium.
We show that MICB is endocytosed and trafficks through TGN
and the endosomal/lysosomal system but most probably recycles
back to the plasma membrane, since inhibitors of vesicular trafficking affect the amount of MICB at the cell surface (Fig. 2B). It
is clear that MICB is not present in lysosomes (membrane-bound
acidic organelles that contain mature acid-dependent hydrolases
and LAMPs but lack mannose-6-phosphate receptors), since it colocalizes with CI-M6PR and the increase of lysosomal pH does not
affect the expression and shedding of MICB. Thus, MICB could be
present in specialized compartments that share features of late endosomes/lysosomes but are functionally and morphologically distinct, such as the MHC class II compartment or other secretory
organelles (49, 50). However, multiple routes converge in the TGN
and it will be important to further characterize the pathway followed by these molecules.
The short half-life of MICB at the plasma membrane of putative
target cells is likely to have a direct effect on their function in
recognition by the immune system. Given that NKG2D-L are indicators of cellular stress, their brief permanence at the cell surface
suggests that continued expression requires continued stress. This
may be a mechanism to minimize immune elimination of cells
during transient situations of cell damage. Moreover, a rapid loss
of MICB protein might be of crucial importance for the recognition of pathogens that affect the cellular machinery of transcription/translation. For example, it has long been known that herpesviruses switch off the synthesis of cellular proteins (51). In this
4807
context, the short half-life of MICB would mean a rapid loss of
surface protein, even after induction of MIC transcription.
There are still outstanding questions about the large number of
ligands recognized by NKG2D and the way that several pathogens
have evolved to affect particular ligands (18, 19). For example,
although only one HCMV product has been found to down-modulate MICA (52), several mechanisms target MICB, including
UL16 (21, 53) and recently discovered virally encoded microRNAs (54). The features of MICB trafficking described here
might favor encounter with the hijacker UL16 protein within
the cytoplasmic compartments or, alternatively, the viral protein could simply enhance a process that already happens in the
uninfected cell.
Another finding from this work is that the mechanism of MICB
endocytosis is dependent on clathrin but also affected by cholesterol. The involvement of lipids in clathrin-mediated pathways has
been previously reported, especially for the uptake of toxins and
viruses toward the cytoplasm (45, 46). Also, internalization of the
apolipoprotein E receptor 2 internalizes through a clathrin-mediated pathway after association with caveolar and noncaveolar lipid
rafts (55). Therefore, recent data support the existence of “hybrid”
endocytosis and trafficking pathways in the cell whose mechanisms and functional significance remain unclear. The possibility
of association of MICA with membrane lipids has been suggested
(56); however, the presence of MIC protein in DRMs was not
demonstrated biochemically. In this study, we could observe a proportion of MICB in DRMs and future work will focus on exploring
the extent and significance of this finding. Further studies are also
needed to fully understand the mechanisms by which MICB associates with lipids and the implications for trafficking and organization in the plasma membrane.
Acknowledgments
We thank N. Miller for assistance with cell sorting; P. Luzio for CI-M6PRspecific Ab; E. Veiga, R. Cassady-Cain, P. Roda-Navarro, and C. Chan for
helpful discussions; J. Trowsdale and A. Kelly for critical reading of this
manuscript; J. Skepper for assistance with quantitation analysis; and
T. Fischer for helpful discussions.
Disclosures
The authors have no financial conflict of interest.
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