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 Agu¨era-Gonza´lez,2 Philippe Boutet,2 Hugh T. Reyburn, and Mar Vale´s-Go´mez3 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 mo- lecular mechanisms that control the presence of these molecules at the plasma membrane are unclear. To investigate the post- translational mechanisms that control surface expression of the human NKG2D ligand MICB, we studied the subcellular local- ization 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. ajor histocompatibility complex class I-related instead, the expression of these proteins is up-regulated in a chain (MIC)4 A and B are two MHC-like molecules number of pathological situations, mainly cancer and autoim- M encoded within the human MHC, but distinct from munity (for review, see Refs. 7 and 8). For example, MIC ex- ␤ classical HLA class I proteins in that they bind neither 2- pression has been found in tumors of various origins and the microglobulin nor antigenic peptides (1–4). MIC proteins bind presence of pathogens like Mycobacterium tuberculosis has the activating receptor NKG2D that is constitutively expressed been shown to up-regulate its expression (9). Recently, a num- ϩ on all NK cells and CD8 T cell subpopulations and is up- ber of external stimuli such as DNA-damaging agents and pro- ϩ regulated after activation on CD4 T cells (5). Engagement of teasome inhibitors have been shown to induce expression of NKG2D by its ligands leads to the activation of lysis and cy- NKG2D ligands (10, 11). Thus, although the precise mecha- tokine secretion by NK cells and T cells either directly or as a nisms that regulate MIC expression are still unknown, the pres- costimulatory factor (for review, see Ref. 6). Thus, the presence ence of MIC at the plasma membrane can be related broadly of MIC at the surface of putative target cells must be tightly with stress and this coincides with the presence of heat shock regulated so that the immune response is not triggered or hidden elements in the MIC promoters (3). The fact that MIC was inappropriately (which would lead to autoimmunity in the first detected by Western blot in a number of nontransformed cell instance and immune evasion in the latter). In fact, with the types, including keratinocytes, endothelial cells, and mono- exception of gastrointestinal epithelium, the vast majority of cytes, although they were not found at the cell surface of all of normal cells do not express MIC molecules at the cell surface; them (2, 12–14), suggests the existence of posttranscriptional and posttranslational mechanisms regulating MIC expression, in addition to transcriptional regulation. That NKG2D ligands Department of Pathology, University of Cambridge, United Kingdom can also be shed from the cells as soluble molecules represents Received for publication March 4, 2008. Accepted for publication February 4, 2009. an additional level of complexity in this system (15–17). The costs of publication of this article were defrayed in part by the payment of page MICB is a target for several proteins expressed by pathogens charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. that are involved in immune evasion. Interestingly, many of 1 This work was funded by a Leukemia Research Fund Project Grant (to H.T.R. and them are selective toward MICB and they do not affect the M.V.G.). M.V.G. is a recipient of a New Investigator Grant from the Medical Re- related protein MICA (18). There are still many questions about search Council and an International Joint Project from the Royal Society. S.A. was the large number of NKG2D ligands and whether this diversity supported by fellowships from the Fundacio´n Caja Madrid and Ibercaja. P.B. was supported in part by The Newton Trust. reflects somehow a difference in function (for review, see 2 S.A.-G. and P.B. contributed equally to the work presented. Ref. 19). With the goal of investigating the mechanisms that control 3 Address correspondence and reprint requests to Dr. Mar Vale´s-Go´mez, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, surface expression of MICB molecules at a posttranslational United Kingdom level, we studied the subcellular localization and trafficking of 4 Abbreviations used in this paper: MICA/B, MHC class I chain-related gene A/B; MICB. In the present study, we show, in several cell lines, that MESNA, 2,mercaptoethane sulfonic acid; DRM, detergent-resistant membrane; HCMV, human CMV; TGN, trans-Golgi network; CI-M6PR, cation-independent the expression of MICB molecules on the cell surface is ac- mannose-6-phosphate receptor; ER, endoplasmic reticulum; EEA1, early endosome companied by an intracellular accumulation of the molecule in A1; LAMP-1, lysosome-activated membrane protein 1; CHX, cycloheximide; BFA, the trans-Golgi network (TGN) and late endosome-related com- brefeldin A; CHL, chloroquine; M␤CD, methyl-␤-cyclodextrin. partments. We further show that MICB has a short half-life at Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 the plasma membrane and that this depends on shedding and www.jimmunol.org/cgi/doi/10.4049/jimmunol.0800713 The Journal of Immunology 4801 clathrin-dependent endocytosis, although the integrity of cho- Table I. Colocalization Coefficients lesterol in the plasma membrane also influences the internal- ization of MICB protein. Pearson’s Coefficient Overlap M6PR 0.458 0.641 Materials and Methods KDEL 0.347 0.527 Cells, reagents, and Abs CD63 0.200 0.299 EEA1 0.383 0.520 HeLa, U373, and CV1 cells were maintained in DMEM supplemented with LAMP-1 0.099 0.190 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 de- scribed (20, 21). Anti-MICA/B monoclonal and polyclonal Abs were pur- chased from R&D Systems, anti-MICA (AMO1) and anti-MICB (BMO2) After blocking for 1 h with PBS containing 2% BSA, tissue culture super- were from Immatics, anti-MICA/B 6D4 was from Santa Cruz Biotechnol- natant was incubated for1hat37°C. The assay was developed using ogy, and isotype control mouse mAbs were purchased from Sigma-Al- biotinylated goat anti-MICB (0.1 ␮g/ml) followed by incubation with drich. For microscopy, sheep Ab directed against human TGN46 was from streptavidin-HRP (1/2000; Amersham Biosciences) and a peroxidase sub- Ј Serotec, mouse mAb specific for GM130 and early endosome A1 (EEA1) strate system (2,2 -azino-di-[3-ethylbenzthiazoline sulfonate] and H2O2 in were from BD Biosciences, mouse anti-human CD107a (lysosome-acti- a glycine/citric acid buffer; Roche). The absorbance was measured at 410 vated membrane (LAMP-1)) was from Southern Biotechnology Associ- nm with a reference wavelength of 490. Samples were analyzed in ates; mouse KDEL mAb was from Bioquote; rabbit anti-mouse tetramethyl duplicates. rhodamine isothiocyanate from DakoCytomation; Alexa Fluor 564 donkey anti-sheep, Alexa Fluor 488 donkey anti-sheep, Alexa Fluor 488 goat Internalization experiments anti-mouse, and Alexa Fluor 488 and 564 goat anti-rabbit Abs were Ab uptake. Ab uptake experiments were performed as previously de- from Molecular Probes; and anti-CI-M6PR polyclonal rabbit Ab was a scribed (23). Cells were incubated with Ab for 10 min at 4°C and then gift from P. Luzio (Cambridge Institute for Medical Research, Cam- transferred to 37°C for different lengths of time. To detect internalized bridge, U.K.). Anti-caveolin Ab was from BD Transduction Laborato- protein, cells were fixed, permeabilized, stained with secondary Ab, and ries. Unless otherwise indicated, chemicals were purchased from analyzed by confocal microscopy. Sigma-Aldrich. Cleavable biotinylation. For cleavable biotinylation experiments, the cells were washed three times with ice-cold PBS. Then incubated 30 min with Flow cytometry 0.25 mg/ml sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate For flow cytometry, 105 cells were incubated with mouse mAbs and bound (EZ-Link Sulfo-NHS-SS-Biotin; Pierce) in ice-cold PBS containing 1 mM Ј MgCl and 0.1 mM CaCl (PBSϩ). The reaction was quenched by washing Ab was visualized using either PE- or FITC-labeled F(ab )2 of goat anti- 2 2 mouse Ig (DakoCytomation). Samples were analyzed using a FACScan II the cells three times with ice-cold serum-free medium containing 0.1% flow cytometer (BD Biosciences). BSA and then once with ice-cold PBSϩ. Prewarmed (to 37°C) serum-free medium was added for the desired time period(s) to allow internalization of Confocal microscopy the biotinylated surface components, after which they were returned to 4°C.