The Mouse CD1d Cytoplasmic Tail Mediates CD1d Trafficking and Antigen Presentation by Adaptor Protein 3-Dependent and -Independent Mechanism
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免疫学杂志 2005年第6期
Abstract
The short cytoplasmic tail of mouse CD1d (mCD1d) is required for its endosomal localization, for the presentation of some glycolipid Ags, and for the development of V14i NKT cells. This tail has a four-amino acid Tyr-containing motif, Tyr-Gln-Asp-Ile (YQDI), similar to those sequences known to be important for the interaction with adaptor protein complexes (AP) that mediate the endosomal localization of many different proteins. In fact, mCD1d has been shown previously to interact with the AP-3 adaptor complex. In the present study, we mutated each amino acid in the YQDI motif to determine the importance of the entire motif sequence in influencing mCD1d trafficking, its interaction with adaptors, and its intracellular localization. The results indicate that the Y, D, and I amino acids are significant functionally because mutations at each of these positions altered the intracellular distribution of mCD1d and reduced its ability to present glycosphingolipids to NKT cells. However, the three amino acids are not all acting in the same way because they differ with regard to how they influence the intracellular distribution of CD1d, its rate of internalization, and its ability to interact with the μ subunit of AP-3. Our results emphasize that multiple steps, including interactions with the adaptors AP-2 and AP-3, are required for normal trafficking of mCD1d and that these different steps are mediated by only a few cytoplasmic amino acids.
Introduction
The CD1 family of proteins is a group of nonclassical, class I-like Ag-presenting molecules (1). Whereas MHC-encoded class I and class II molecules present peptides, CD1 molecules present various lipid Ags (2). Group I CD1 molecules, which include human CD1a, CD1b, and CD1c, present mycobacteria-derived and brain-derived glycolipids (2, 3, 4). Group II CD1 molecules, e.g., CD1d, present glycolipids to V14i NKT cells in mice and their homologues in other species, and they regulate their development in mice (5).
V14i NKT cells are a distinct sublineage of T lymphocytes, which may be involved in immune regulation and host defense (6). V14i NKT cells are autoreactive to mouse CD1d (mCD1d)5 (7), and this response is enhanced greatly by a synthetic phytosphingolipid, -D-galactosyl ceramide (GalCer) (8, 9). Current studies of the cellular requirements for lipid-Ag presentation by CD1 molecules provide evidence of both endosomal and nonendosomal pathways for glycolipid-Ag presentation to T cells (10). Whereas the presentation of GalCer by mCD1d does not require internalization because even plate-bound recombinant mCD1d protein can present GalCer to V14i NKT cells (11), analogues of GalCer that have additional sugars such as the 2' and/or 3' carbon of the galactose have to be internalized and processed to generate the monosaccharide GalCer before they can be recognized by TCR. The processing of these analogues presumably occurs in the lysosomes (11).
Analyses of tail deletion mutations demonstrates that the cytoplasmic tail of mCD1d is critical for its localization to low-pH endosomal compartments, Ag presentation, and the development of V14i NKT cells (12, 13, 14, 15). The cytoplasmic tail of mCD1d contains a Tyr-based endosomal-targeting motif, YXX (X = any amino acid and = hydrophobic amino acid). This sequence is likely to govern the endosomal localization of mCD1d, based on its similarity to those in a number of other proteins, including human CD1b, CD1c, and CD1d.
The Tyr motif is predicted to bind one or more of adaptor protein complexes (AP), AP-1, AP-2, AP-3, and AP-4, which are involved in targeting integral membrane proteins, to intracellular compartments (16, 17, 18, 19). AP-1 and AP-2 are components of clathrin coats associated with the trans-Golgi network (TGN)/endosomes and the plasma membrane, respectively (20). AP-1 is important for the trafficking of proteins from the TGN to endosomes, and AP-2 is involved in cargo recruitment in endocytosis (21). AP-3 has been shown to be part of both clathrin and nonclathrin coats localized to endosomes, and it is important in the localization of membrane proteins to lysosome-related vesicles (22). AP-4 is associated with the TGN, transport vesicles, and endosomes, and it might be associated with a clathrin coat (23, 24, 25). In epithelial cells, both AP-4 and a μ-specific isoform of AP-1, called μ1B, is thought to be involved in targeting of proteins bearing tyrosine motifs (26, 27). In such cells, human CD1d is in fact sorted basolaterally, but it is not known which adaptor complex is involved (28). Each AP molecule is composed of four subunits, two large chains, for AP-1 to AP-4, respectively, and 1 to 4, one medium (μ1 to μ4), and one small chain (1 to 4) (18). The Tyr motif normally binds to μ subunits of AP (29, 30, 31, 32). We and others (33, 34, 35) have shown recently that the Tyr endosomal motif of human CD1b and mCD1d indeed interacts with the μ subunit of AP-3.
Other than for the tyrosine, there is relatively little information directly implicating particular amino acids in the amino acid sequence motif of the cytoplasmic tail in mCD1d traffic and Ag presentation. In the present study, we demonstrate that three amino acids in the YXX motif are important for mCD1d function, and the diverse effects of these mutations demonstrate AP-3-dependent and -independent steps in mCD1d trafficking.
Materials and Methods
Reagents and cell lines
Glycosphingolipid Ags, GalCer and Gal(12)GalCer, have been described previously (9, 11) and were a gift from the Kirin Pharmaceutical Research Corporation (Gunma, Japan). V14i NKT cell hybridomas 1.2, 1.4, and 3C3 and non-V14i NKT cell hybridoma 24 were described previously (9, 12).
Expression of mCD1d and Ag presentation assays
The cloning and generation of A20 B lymphoma cells expressing wild-type mCD1d, Tyr332Ala mutant (Y332A), and tail-deleted mutant were described previously (12). The Glu333Ala, Asp334Ala, and Ile335Ala constructs were made by oligonucleotide-directed mutagenesis. Final PCR products were cloned into pHAprNeo, and A20 transfectants were generated according to procedures described previously (12). Ag presentation assays, using A20 cells transfected with wild-type or mutant mCD1d molecules as APC and V14i and non-V14i NKT cell hybridomas, have been described previously (9, 11, 34).
Immunofluorescent labeling and confocal microscopy
A20 transfectants were fixed, permeabilized, and blocked before the addition of the Abs, as described previously (9, 11). mCD1d molecules were labeled with biotinylated mAb 1B1, followed by Cy3-conjugated streptavidin (Jackson ImmunoResearch Laboratories). For the colocalization experiments, cells were stained with either FITC-conjugated rat anti-mouse transferrin receptor (TfR) or anti-mouse Lamp-2 mAbs (BD Pharmingen). The fluorescently labeled cells were analyzed with a Bio-Rad Micro Radiance Confocal 1024 laser scanning confocal microscope. Quantitation of colocalization was done using LaserPix program (Bio-Rad).
Flow cytometry
Cells were washed and blocked in staining buffer (PBS, 10% FCS, and 0.02% NaN3) containing anti-FcR Ab 2.4G2 for 15 min at 4°C. Cells were then stained with PE-conjugated 1B1 mAb to CD1d (BD Pharmingen). After washing, cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences).
Internalization assay
The rate of internalization of wild-type and mutant mCD1d molecules was measured using a modified flow cytometry-based internalization assay (36). A20 transfectants expressing wild-type and mutant mCD1d (30 x 106 cells/sample) were washed and blocked in staining buffer containing anti-FcR Ab 2.4G2 (BD Pharmingen) for 30 min on ice. Cells were then stained with PE-1B1 and FITC-anti-TfR or FITC-anti-H-2Kd (BD Pharmingen) at 30 μg/sample in 300 μl of staining buffer on ice for 30 min. After two washes with ice-cold PBS to remove unbound Abs, cells were divided into various sets, warmed to 37°C, and chilled at different time points to terminate internalization at 37°C. Uninternalized Abs were removed by a 45-s treatment with acid buffer (PBS (pH 2), 0.03 M sucrose, and 10% FCS). After washing in RPMI 1640 medium, 10% FCS, and 100 mM HEPES, cells were analyzed on a FACSCalibur cytometer. The quantitation of the rate of internalization at specific time points was done using the following formula: percentage of internalization = (median fluorescence intensity of cells with acid treatment/median fluorescence intensity of cells without acid treatment) x 100.
Yeast two-hybrid assays
The constructs Gal4AD-μ1A, Gal4AD-μ2, Gal4AD-μ3A, and Gal4AD-μ4 in the pACTII (LEU2) plasmid and Gal4BD-TGN38 in the pGBT9 (TRP1) plasmid have been described previously (29, 30). The MATCHMAKER GAL4 Two-Hybrid System 3 (BD Clontech) was used for the generation of GAL4BD-mCD1d, containing the wild-type mCD1d cytoplasmic tail, and the four mutants GAL4BD-YA, GAL4BD-QA, GAL4BD-DA, and GAL4BD-IA, followed by yeast transformation. GAL4BD-mCD1d was generated as described previously (34). GAL4BD-YA, GAL4BD-QA, GAL4BD-DA, and GAL4BD-IA were generated by ligation of pGBKT7, the GAL4BD vector, with synthetic double-strand DNA encoding the entire 10-amino acid cytoplasmic domain of mCD1d containing various point mutations. The constructs were confirmed by sequencing. The Saccharomyces cerevisiae strain AH109 was transformed according to the manufacturer’s instructions with various combinations of Gal4AD-μ1A, Gal4AD-μ2, Gal4AD-μ3A, or Gal4AD-μ4 with GAL4BD-mCD1d, GAL4BD-YA, GAL4BD-QA, GAL4BD-DA, GAL4BD-IA, GAL4BD-TGN38, or the pGBKT7 vector and selected in medium lacking leucine and tryptophan. For plate colony growth assays, AH109 transformants were streaked on plates lacking adenine, histidine, leucine, and tryptophan and allowed to grow at 30°C for 3 days. Quantitative growth of AH109 transformants was measured by inoculating colonies of various transformants in liquid medium lacking adenine, histidine, leucine, and tryptophan and allowing them to grow at 30°C. The OD at 600 nm was measured at various times.
Cloning, expression, and purification of soluble adaptor μ-chains
The full-length cDNA clones of mouse μ1A and rat μ3A were kindly provided by P. Schu (University of G?ttingen, G?ttingen, Germany) and M. S. Robinson (Medical Research Council, Cambridge, U.K.), respectively. The pET28a vector (Novagen) containing N-terminally 6x His-tagged rat μ2 (residues 158–435) was a gift from V. Haucke (Freie Universit?t Berlin, Berlin, Germany). These plasmids were transfected into the Escherichia coli strain BL21, and protein production was induced by addition of isopropyl -D-thiogalactoside for 3 h at 30°C, followed by purification of the μ-chain proteins from 1 liter of bacterial culture. The purification was performed according to a standard protocol (Qiagen) using Ni-NTA agarose as an affinity matrix. The purity of the μ-chain proteins stored in buffer A (10 mM HEPES-KOH (pH 7.4), 500 mM NaCl, and 10 mM 2-ME) was controlled by SDS-PAGE. Before BIAcore experiments, the proteins were centrifuged for 30 min at 100,000 x g to remove possible protein aggregates.
Detection of AP μ-chain binding to sorting signals by surface plasmon resonance (SPR)
The binding of recombinant μ-chains of the adaptor complexes, μ1 to μ3, to cytoplasmic tail sorting signals was recorded in real time using a SPR-based biosensor (BIAcore 3000; BIAcore AB). The 6x His-tagged truncated μ-chain proteins were used at concentrations ranging from 500 nM to 2.5 μM. Synthetic peptides corresponding to the sorting signals of CD1d (-CIWRRRSAYQDIR), TGN38 (-CKVTRRPKASDYQRL), and the TfR (-CGEPLSYTRFSLARQVDG), as well as their mutants in which the critical tyrosine was substituted for alanine, were immobilized on a CM5 sensor surface using the thiol coupling method in the manufacturer’s instructions. All peptides were immobilized according to their molecular weights at equal densities of 800-1100 resonance units. After immobilization, the sensor surface was washed with short pulse injections of 50 mM sodium hydroxide to remove any nonspecifically attached material. The μ-chains were injected in buffer A (with 5 mM DTT instead of 2-ME) at a flow rate of 10 μl/min for 1 min, followed by washing with buffer for 5 min. Subsequently, any μ-chain protein still bound to the sensor surface was removed by a 1-min pulse injection of 50 mM sodium hydroxide. The μ-chains were passed simultaneously over surfaces containing a sorting signal and the respective tyrosine mutant peptides. The binding curve of the latter one was subtracted from the curve obtained for the wild-type sorting signal before the kinetic rate constants were calculated, as described elsewhere (37).
Results
Expression of mutated mCD1d molecules
To assess how the individual amino acids in the Tyr motif might affect the subcellular localization and Ag presentation ability of the mCD1d molecules, each amino acid in the YQDI motif was substituted with alanine, as schematically presented in Fig. 1A. Flow cytometry analysis showed that the sorted A20 transfectants expressing mutant molecules have a similar or higher surface expression of mCD1d compared with transfectants expressing wild-type mCD1d (Fig. 1B).
FIGURE 1. Expression of mCD1d cytoplasmic tail point mutants. A, Point mutations of the four-amino acid endosomal-targeting motif (indicated in bold) localized at the cytoplasmic terminus. B, Surface expression of mCD1d point mutants. Transfectants with the indicated point mutations were stained with PE-conjugated rat anti-mCD1d mAb, and the expression level of surface mCD1d was determined by flow cytometry. Isotype control stainings are shown with shaded-fill under the curve.
Ag presentation by mutant mCD1d molecules
The Ag presentation ability of the various mCD1d point mutants was tested using V14i NKT cell hybridomas responding to GalCer or Gal(12)GalCer. The presentation of GalCer does not absolutely require internalization, and all of the mCD1d cytoplasmic tail mutants presented GalCer (Fig. 2A). Three mutants, Y332A, D334A, and I335A, had a dramatically reduced ability to present Gal(12)GalCer, which requires mCD1d localization to late endosomes or lysosomes (34). Although the Q333A mutant had lower surface expression than the Y332A, D334A, and I335A mutants, the ability of Q333A molecules to present Gal(12)GalCer was comparable to wild-type mCD1d. Therefore, substitutions of three of four amino acids within the Tyr-based motif affect the ability of mCD1d to present glycolipid Ags to V14i NKT cells, especially those requiring internalization.
FIGURE 2. Ag presentation by mCD1d point mutants. A, A20 transfectants expressing wild-type or point mutant mCD1d molecules were pulsed with the indicated amounts of Ags, either GalCer or Gal(12)GalCer, washed, and cultured with NKT cell hybridoma 3C3. Cell-free supernatants were tested by ELISA for IL-2 production. The error bars indicate the SD of triplicate measurements. The responses of the 1.2 and 1.4 V14i NKT cell hybridomas were similar (data not shown). The data are representative of at least three independent experiments. B, A20 transfectants expressing wild-type or the Y332A, D334A, and I335A mutants were cultured with the mCD1d-autoreactive hybridoma 24, which does not express a V14iTCR. Cell-free supernatants were tested by ELISA for IL-2 production. The error bars indicate the SD of triplicate measurements. Shown here are data from one of four independent experiments.
The ability of the tail-mutated mCD1d molecules to present GalCer suggests that the extracellular domain has not been compromised. To provide an additional control for this, we tested the autoreactivity of a CD1d-dependent hybridoma, 24 (9), which does not have the V14i rearrangement and which is independent presumably of the CD1d tail motif (38). The mutants retained approximately the same ability to stimulate hybridoma 24 (Fig. 2B). The I335A mutant caused a 2-fold less stimulation of the autoreactive hybridoma 24 (Fig. 2B), and it presented GalCer less effectively than the other mCD1d molecules (Fig. 2A). This is almost certainly a reflection of the lower mCD1d surface level in cells expressing this mutant (Fig. 1). However, most important is that the ability of I335A to present Gal(12)GalCer was decreased drastically in comparison with its ability to present GalCer, as with the Y332A and D334A mutants. This drastic decrease is indicative of the importance of I335 and, by the same token, Y332 and D334 in regulating the intracellular localization of mCD1d.
Subcelullar localization of the cytoplasmic tail mutants
We examined the subcellular localization of wild-type and mutant mCD1d molecules using confocal microscopic analysis of fixed and permeabilized A20 cells (Fig. 3A). Quantitation of the degree of colocalization is presented in Fig. 3B. Wild-type CD1d molecules are distributed throughout the endocytic pathway, colocalizing with the late endosomal/lysosomal marker, Lamp-2, but also with the early/recycling endosomal marker TfR. Nearly 100% of vesicles containing Lamp-2 also contained detectable mCD1d, and the same also was true for vesicles containing the TfR (Fig. 3B). For the different mutations, there was a good correlation between mutants that affect Ag presentation and those that affect the intracellular distribution of mCD1d. The Q333A mutant displayed an intracellular distribution similar to wild-type mCD1d. By contrast, the Y332A, D334A, and I335A mutant molecules have decreased localization in endosomal vesicles. Among these three, the Y332A mutant has the highest degree of colocalization with the TfR, with more than half of the TfR-containing vesicles having some mCD1d. However, the disruption of trafficking to late endosomes is most severe in this mutant with a few Lamp-2-positive vesicles containing mCD1d. The DA and IA mutants behave similarly, with affects on mCD1d colocalization to both early TfR-containing and late Lamp-2-positive endosomes. These data suggest that the Tyr might affect mCD1d intracellular localization through a partially different mechanism than the Asp and Ile.
FIGURE 3. Steady-state distribution of wild-type and mutant mCD1d molecules in A20 cells as analyzed by immunofluorescence and confocal microscopy. Colocalization of wild-type and mutant mCD1d molecules (red) with markers (green) for early/recycling endosomes (A, TfR) and lysosomes (B, Lamp-2) was revealed as yellow in the overlay images. The colocalization was quantified using the Laserpix program (Bio-Rad) and shown in C. n denotes the numbers of cells used for each quantitation. Scale bar, 50 μm.
Internalization of mutant mCD1d molecules
Previous studies of cells from mice expressing mCD1d with a truncated cytoplasmic tail indicated that mCD1d molecules depend on the cytoplasmic tail, presumably the Tyr motif, for internalization (15). The internalization of human CD1d in MDCK cells also depends on the Tyr motif (28). To further investigate the mechanism whereby the other amino acids within the sorting motif of the mCD1d cytoplasmic tail regulate mCD1d trafficking and Ag presentation, we examined the internalization rates of the different mutant molecules using a flow cytometry-based internalization assay (Fig. 4). As demonstrated for the human TfR (39), internalization of the mouse TfR was very efficient. Within 30 min, 60% of the surface TfR was internalized (Fig. 4A). Wild-type mCD1d internalized more slowly than TfR, with <40% of mCD1d internalized by 60 min. However, this rate of internalization is still considerably faster than that of MHC class I molecules with only 5% internalized by 60 min. Among all the mutant mCD1d molecules, the Y332A mutation affected the internalization rate to the greatest degree, comparable to the tail-deleted mCD1d molecules (Fig. 4B). The I335A mutation also resulted in a decreased rate of internalization, although to a lesser degree. However, even these mCD1d mutants internalized at a higher rate than MHC class I molecules. The internalization rates of the Q333A and D334A mutant molecules were not different significantly from wild-type mCD1d. Therefore, Tyr and, to a lesser extent, Ile appear to be important in governing the internalization of the mCD1d molecules, presumably through interaction with the machinery responsible for endocytosis.
FIGURE 4. Internalization rate of wild-type and mutant mCD1d molecules. A20 transfectants expressing wild-type or point mutant mCD1d molecules were stained with FITC-anti-TfR, PE-anti-mCD1d, and/or FITC-anti-H-2Kd. The surface-bound Abs were allowed to internalize at 37°C. After acid treatment to remove the remaining surface-bound Ab, cells were permeabilized and analyzed by flow cytometry. A, Comparison of internalization rates for TfR, mCD1d, and H-2Kd in A20 transfectants expressing wild-type mCD1d molecules. The data are representative of two independent experiments. B, Comparison of internalization rates of wild-type and mutant mCD1d molecules in A20 transfectants. The data are average of four to six independent experiments. To allow for comparison between different experiments, the rates of internalization for the mutant molecules were normalized to wild-type mCD1d.
The ability of mCD1d Tyr motif to interact with the μ subunits of AP
Adaptor proteins (AP-1 to AP-4) of distinct subunit composition are currently understood to direct the trafficking of many proteins to multiple vesicular destinations. We have previously shown that the Tyr is important for the interaction between the mCD1d cytoplasmic tail and the AP-3 complex, presumably through the μ3A subunit (34). To further understand whether the Tyr motif interacts with other AP and how individual amino acids within the Tyr motif contribute to these interactions, we used a yeast two-hybrid system in which the μ subunits from AP-1, AP-2, or AP-3 were expressed as fusions with the activation domain of GAL4. Additionally, point mutants of the mCD1d cytoplasmic tail were expressed as fusions with the binding domain. The mCD1d cytoplasmic tail did not interact with the μ subunits of AP-1 and AP-2 in yeast solid plate and liquid culture growth assays, whereas these assays confirmed the previously reported interaction of the cytoplasmic tail with the μ3A subunit by a -galactosidase assay (Fig. 5, A and B; Ref. 34). We then used SPR to measure the interaction of synthetic peptides corresponding to the cytoplasmic tail of wild-type and Y332A mutant mCD1d to purified soluble μ1 to μ3 subunits (Table I). Data from SPR agreed with those from the yeast two-hybrid experiments, in that the cytoplasmic tail of mCD1d interacted with the μ3A subunit but do not with μ1. The mCD1d tail also interacted with the μ2 subunit in SPR, although this interaction is weaker compared with the μ3A. Optimal μ2 binding to CD1d requires a functional tyrosine motif (YQDIR) because the Y332A mutant peptide binds only weakly to μ2 (Fig. 5C).
FIGURE 5. Interaction of wild-type (WT) and mutant mCD1d cytoplasmic tails with the μ subunits of AP. A, Growth of AH109 yeast transformants coexpressing various μ subunits and WT mCD1d. Transformants were streaked on medium plates lacking adenine, histidine, leucine, and tryptophan. B, Quantitation of the growth rate of AH109 yeast transformants coexpressing various μ subunits and WT mCD1d. Transformants were selected in medium lacking histidine, leucine, and tryptophan at 30°C, and absorbance at wavelength 600 nm was measured with a spectrophotometer. The data are representative of three independent experiments. C, SPR sensorgrams of the binding of peptides corresponding to WT and Y332A mutant (YA) mCD1d tails to μ2. Synthetic peptides corresponding to the cytoplasmic tails of WT and the YA mCD1d were immobilized on a CM5 sensor surface as described in Materials and Methods. A surface without peptide served as an additional control (curve 4 in YA). Purified soluble μ2 was passed over the surfaces at the indicated concentrations followed by washing with running buffer. The rate constants for the binding μ2 to WT CD1d were calculated after subtraction of the binding to the YA. D, Quantitation of the growth rate of AH109 yeast transformants coexpressing μ3A along with constructs encoding WT or mutant mCD1d cytoplasmic tails. Transformants were selected in the medium, as described above, and absorbance at 600 nm was measured. The data are representatives of three independent experiments. E, SPR sensorgrams of the binding of μ3 to peptides corresponding to WT and mutant mCD1d cytoplasmic tails. Purified soluble μ3 was passed at the concentration of 15 μM over a CM5 surface derivatized with the indicated mCD1d peptides.
Table I. Kinetic rate constants for adaptor μ-chain binding to tail peptidesa
As shown in Fig. 5D, yeast transformants containing μ3A along with wild-type CD1d cytoplasmic tail, QA, or DA mutants grew in medium lacking histidine, leucine, and tryptophan at comparable rates, whereas those containing the μ3A subunit along with YA or IA did not grow at all. This observation was confirmed by SPR experiments in which we tested μ3A binding with peptides corresponding to the wild-type and mutant mCD1d cytoplasmic tails. We used micromolar concentrations of μ3A (4–20 μM) to ensure the detection of any weak interaction with the mutant mCD1d peptides. As shown in Fig. 5E, the binding of the Q333A and D334A mutants to μ3A was comparable to that of wild-type mCD1d, although they both exhibited a <2-fold increase in the dissociation rate. In contrast, the binding of Y332A and I335A was reduced to levels observed for an unrelated control peptide or a blank surface (data not shown). Therefore, the Y and I amino acids are critical for the interaction of mCD1d with the μ3A subunit, whereas the D amino acid mostly affects CD1d localization and Ag presentation through some other mechanism.
Discussion
Ag presentation by mCD1d and the development of V14i NKT cells depend on the 10-amino acid cytoplasmic tail of mCD1d. In human CD1d, a similarly short cytoplasmic sequence contains a tyrosine internalization motif together within an overlapping leucine-based signal and mediates basolateral sorting (40), which is consistent with the hypothesis that the multiple steps required for proper CD1d localization are mediated by these short sequences. Although they both contain a YXX motif, human CD1d and mCD1d differ for their cytoplasmic sequences and in their trafficking, however, as illustrated by the inability of human CD1d to interact with AP-3μ, in a yeast two-hybrid analysis (33).
mCD1d molecules potentially could take two routes to the endocytic pathway, either directly from the Golgi complex or via internalization from the cell surface. The currently accepted hypothesis for mCD1d trafficking is that the Tyr-based endosomal localization motif within the cytoplasmic tail regulates the recycling of mCD1d that occurs relatively early after its biosynthesis (38). There are no data so far demonstrating that a population of newly synthesized mCD1d molecules traffic directly from the TGN to endosomes, although this has been reported for other proteins that contain tyrosine signals that bind AP-3 (41). The cytoplasmic tail also regulates the subsequent trafficking of mCD1d to late endosomes/lysosomes through its interaction with μ3A of the AP-3 complex (34, 35). The data reported here represent an effort to understand additionally the molecular basis of the regulation of mCD1d localization. We conducted an analysis of the roles of individual amino acids within the YXX motif on mCD1d internalization, intracellular localization, and Ag presentation. The data demonstrate that three of the four amino acids in this motif are important. Furthermore, because the affects of Ala substitution of the individual amino acids are different, they illustrate AP-3-dependent mechanisms influenced by Tyr and Ile and AP-3-independent mechanisms revealed by Ala substitution for Asp.
Consistent with previous reports indicating the importance of the cytoplasmic tail Tyr in human CD1d internalization (28), our data show that the Y332A mutation reduced the rate of internalization to the greatest degree. When the four point mutants were compared, the effect of the Y332A mutation and was similar to a complete cytoplasmic tail truncation. Despite this, steady-state measurements indicated that the Y332A mutation affected colocalization with the TfR to a lesser extent than either the D334A and I335A mutations. However, the steady-state distribution of mCD1d in early/recycling endosomes is determined by several factors, including the internalization rate from the cell surface, the recycling rate, the rate of entry to late endosomal/lysosomal compartments from recycling compartments, and possibly the rate of exit from the TGN directly to endosomes. Because tail-deleted mCD1d has been reported to recycle normally (38) and Tyr is most critical for the interaction of mCD1d with μ3A and its localization to Lamp-2-positive vesicles, a possible reason for the accumulation of Y332A mutant mCD1d in early/recycling endosomes, compared with the I335A and D334A mutants, could be its impaired transport to late endosomes/lysosomes.
The molecular basis for mCD1d internalization is yet to be understood. The results from a direct binding study using SPR suggested that the internalization of mCD1d is most likely mediated by the AP-2 complex through the interaction of the Tyr motif of mCD1d with μ2. The interaction is 2-fold weaker than the interaction with the μ3A subunit of AP-3, and it could not be detected in the yeast two-hybrid assay. Therefore, negative results from the two-hybrid assay using CD1d cytoplasmic tails must be interpreted cautiously. We then have confirmed the interaction of the mCD1d tail with AP-2 by SPR-binding experiments using the purified AP-2 complex as opposed to the recombinant, purified μ subunit (O. Bakke and S. H?ning, data not shown). The selective strength of the interaction of the mCD1d cytoplasmic tail with μ3A compared with μ2 may be due to favored interaction to μ3A by the Arg at position Y-3 and the Ile at position Y + 3 of the motif (31). The absence of this Y + 3 Ile in human CD1d may in part account for the inability of the human homologue to interact with AP-3A when analyzed by a yeast two-hybrid experiment. Furthermore, the physiological significance of mCD1d internalization and recycling has not been established. Internalized mCD1d may acquire certain Ags in early endosomes, and although a normal internalization rate may be required for presentation of some of the Ags that stimulate V14i NKT cells, analysis of the DA mutant suggests this is not sufficient for a correct intracellular localization and Ag presentation capability. This is consistent with our earlier observation that chimeric mCD1d molecules that localize to early but not late endosomes are deficient in the presentation of glycolipids that require internalization and processing (34).
Ala substitution of the Ile at the Y + 3 position in the YXX motif had global effects on Ag presentation, intracellular localization, internalization, and interaction with the AP-3μA-chain. Consistent with this, it was shown previously that Ala substitution of the Val at the Y + 3 position of the cytoplasmic tail of human CD1d reduced internalization (28). However, the effects of the I335A and Y332A mutations were not equivalent with the I335A mutant having a reduced affect on the internalization rate and an increased ability to localize to Lamp-2-positive vesicles.
The effects of Ala substitution for the Asp in the Y + 2 position of the mCD1d cytoplasmic tail were striking. These mutant mCD1d molecules internalized at a normal rate, but they have a drastically decreased ability to present a glycolipid Ag that requires internalization and processing, and they displayed an altered steady-state intracellular distribution in a manner similar to the I335A mutant. Despite this, Asp is not critical for the interaction with the AP-3μA subunit. However, the Asp at position Y + 2 could be a good candidate for involvement in alternative processes, such as a role in mediating a direct route from the Golgi to late endosomes/lysosomes. This direct trafficking to endosomes remains an attractive if speculative possibility, based on the composition of residues at positions Y + 1 and Y + 2 of the Tyr motif (19, 42) of mCD1d and the six-amino acid distance from the membrane to the Tyr (19, 43).
In conclusion, analysis of the Tyr motif demonstrates the complexity of mCD1d trafficking and its effects on glycolipid Ag presentation and the multifaceted roles of the amino acids in the short cytoplasmic tail.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Dr. Jeffery A. Lawton for critical reading of this manuscript and Dr. Juan S. Bonifacino (Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD) for providing Gal4AD-μ1/2/3 and -TGN yeast two-hybrid constructs.
Footnotes
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 supported by National Institutes of Health Grant RO1 AI40617 (to M.K.), a grant from the Human Frontiers of Science Program (to M.K.), and grants from the University of Oslo and the Research Council of Norway (to O.B.). A.P.L. is the recipient of a National Research Service Award from National Institutes of Health (Grant AI52552).
2 Current address: Tampa Bay Research Institute, St. Petersburg, FL 33712.
3 Current address: Department of Molecular Microbiology and Immunology and Graduate Program in Pathobiology, Division of Biology and Medicine, Brown University, Providence, RI 02912.
4 Address correspondence and reprint requests to Dr. Mitchell Kronenberg, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address: mitch{at}liai.org
5 Abbreviations used in this paper: mCD1d, mouse CD1d; GalCer, -D-galctosyl ceramide; AP, adaptor protein complex; TGN, trans-Golgi network; Gal(12)GalCer, -2'-galactosyl-GalCer; TfR, transferrin receptor; SPR, surface plasmon resonance.
Received for publication August 6, 2004. Accepted for publication December 1, 2004.
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The short cytoplasmic tail of mouse CD1d (mCD1d) is required for its endosomal localization, for the presentation of some glycolipid Ags, and for the development of V14i NKT cells. This tail has a four-amino acid Tyr-containing motif, Tyr-Gln-Asp-Ile (YQDI), similar to those sequences known to be important for the interaction with adaptor protein complexes (AP) that mediate the endosomal localization of many different proteins. In fact, mCD1d has been shown previously to interact with the AP-3 adaptor complex. In the present study, we mutated each amino acid in the YQDI motif to determine the importance of the entire motif sequence in influencing mCD1d trafficking, its interaction with adaptors, and its intracellular localization. The results indicate that the Y, D, and I amino acids are significant functionally because mutations at each of these positions altered the intracellular distribution of mCD1d and reduced its ability to present glycosphingolipids to NKT cells. However, the three amino acids are not all acting in the same way because they differ with regard to how they influence the intracellular distribution of CD1d, its rate of internalization, and its ability to interact with the μ subunit of AP-3. Our results emphasize that multiple steps, including interactions with the adaptors AP-2 and AP-3, are required for normal trafficking of mCD1d and that these different steps are mediated by only a few cytoplasmic amino acids.
Introduction
The CD1 family of proteins is a group of nonclassical, class I-like Ag-presenting molecules (1). Whereas MHC-encoded class I and class II molecules present peptides, CD1 molecules present various lipid Ags (2). Group I CD1 molecules, which include human CD1a, CD1b, and CD1c, present mycobacteria-derived and brain-derived glycolipids (2, 3, 4). Group II CD1 molecules, e.g., CD1d, present glycolipids to V14i NKT cells in mice and their homologues in other species, and they regulate their development in mice (5).
V14i NKT cells are a distinct sublineage of T lymphocytes, which may be involved in immune regulation and host defense (6). V14i NKT cells are autoreactive to mouse CD1d (mCD1d)5 (7), and this response is enhanced greatly by a synthetic phytosphingolipid, -D-galactosyl ceramide (GalCer) (8, 9). Current studies of the cellular requirements for lipid-Ag presentation by CD1 molecules provide evidence of both endosomal and nonendosomal pathways for glycolipid-Ag presentation to T cells (10). Whereas the presentation of GalCer by mCD1d does not require internalization because even plate-bound recombinant mCD1d protein can present GalCer to V14i NKT cells (11), analogues of GalCer that have additional sugars such as the 2' and/or 3' carbon of the galactose have to be internalized and processed to generate the monosaccharide GalCer before they can be recognized by TCR. The processing of these analogues presumably occurs in the lysosomes (11).
Analyses of tail deletion mutations demonstrates that the cytoplasmic tail of mCD1d is critical for its localization to low-pH endosomal compartments, Ag presentation, and the development of V14i NKT cells (12, 13, 14, 15). The cytoplasmic tail of mCD1d contains a Tyr-based endosomal-targeting motif, YXX (X = any amino acid and = hydrophobic amino acid). This sequence is likely to govern the endosomal localization of mCD1d, based on its similarity to those in a number of other proteins, including human CD1b, CD1c, and CD1d.
The Tyr motif is predicted to bind one or more of adaptor protein complexes (AP), AP-1, AP-2, AP-3, and AP-4, which are involved in targeting integral membrane proteins, to intracellular compartments (16, 17, 18, 19). AP-1 and AP-2 are components of clathrin coats associated with the trans-Golgi network (TGN)/endosomes and the plasma membrane, respectively (20). AP-1 is important for the trafficking of proteins from the TGN to endosomes, and AP-2 is involved in cargo recruitment in endocytosis (21). AP-3 has been shown to be part of both clathrin and nonclathrin coats localized to endosomes, and it is important in the localization of membrane proteins to lysosome-related vesicles (22). AP-4 is associated with the TGN, transport vesicles, and endosomes, and it might be associated with a clathrin coat (23, 24, 25). In epithelial cells, both AP-4 and a μ-specific isoform of AP-1, called μ1B, is thought to be involved in targeting of proteins bearing tyrosine motifs (26, 27). In such cells, human CD1d is in fact sorted basolaterally, but it is not known which adaptor complex is involved (28). Each AP molecule is composed of four subunits, two large chains, for AP-1 to AP-4, respectively, and 1 to 4, one medium (μ1 to μ4), and one small chain (1 to 4) (18). The Tyr motif normally binds to μ subunits of AP (29, 30, 31, 32). We and others (33, 34, 35) have shown recently that the Tyr endosomal motif of human CD1b and mCD1d indeed interacts with the μ subunit of AP-3.
Other than for the tyrosine, there is relatively little information directly implicating particular amino acids in the amino acid sequence motif of the cytoplasmic tail in mCD1d traffic and Ag presentation. In the present study, we demonstrate that three amino acids in the YXX motif are important for mCD1d function, and the diverse effects of these mutations demonstrate AP-3-dependent and -independent steps in mCD1d trafficking.
Materials and Methods
Reagents and cell lines
Glycosphingolipid Ags, GalCer and Gal(12)GalCer, have been described previously (9, 11) and were a gift from the Kirin Pharmaceutical Research Corporation (Gunma, Japan). V14i NKT cell hybridomas 1.2, 1.4, and 3C3 and non-V14i NKT cell hybridoma 24 were described previously (9, 12).
Expression of mCD1d and Ag presentation assays
The cloning and generation of A20 B lymphoma cells expressing wild-type mCD1d, Tyr332Ala mutant (Y332A), and tail-deleted mutant were described previously (12). The Glu333Ala, Asp334Ala, and Ile335Ala constructs were made by oligonucleotide-directed mutagenesis. Final PCR products were cloned into pHAprNeo, and A20 transfectants were generated according to procedures described previously (12). Ag presentation assays, using A20 cells transfected with wild-type or mutant mCD1d molecules as APC and V14i and non-V14i NKT cell hybridomas, have been described previously (9, 11, 34).
Immunofluorescent labeling and confocal microscopy
A20 transfectants were fixed, permeabilized, and blocked before the addition of the Abs, as described previously (9, 11). mCD1d molecules were labeled with biotinylated mAb 1B1, followed by Cy3-conjugated streptavidin (Jackson ImmunoResearch Laboratories). For the colocalization experiments, cells were stained with either FITC-conjugated rat anti-mouse transferrin receptor (TfR) or anti-mouse Lamp-2 mAbs (BD Pharmingen). The fluorescently labeled cells were analyzed with a Bio-Rad Micro Radiance Confocal 1024 laser scanning confocal microscope. Quantitation of colocalization was done using LaserPix program (Bio-Rad).
Flow cytometry
Cells were washed and blocked in staining buffer (PBS, 10% FCS, and 0.02% NaN3) containing anti-FcR Ab 2.4G2 for 15 min at 4°C. Cells were then stained with PE-conjugated 1B1 mAb to CD1d (BD Pharmingen). After washing, cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences).
Internalization assay
The rate of internalization of wild-type and mutant mCD1d molecules was measured using a modified flow cytometry-based internalization assay (36). A20 transfectants expressing wild-type and mutant mCD1d (30 x 106 cells/sample) were washed and blocked in staining buffer containing anti-FcR Ab 2.4G2 (BD Pharmingen) for 30 min on ice. Cells were then stained with PE-1B1 and FITC-anti-TfR or FITC-anti-H-2Kd (BD Pharmingen) at 30 μg/sample in 300 μl of staining buffer on ice for 30 min. After two washes with ice-cold PBS to remove unbound Abs, cells were divided into various sets, warmed to 37°C, and chilled at different time points to terminate internalization at 37°C. Uninternalized Abs were removed by a 45-s treatment with acid buffer (PBS (pH 2), 0.03 M sucrose, and 10% FCS). After washing in RPMI 1640 medium, 10% FCS, and 100 mM HEPES, cells were analyzed on a FACSCalibur cytometer. The quantitation of the rate of internalization at specific time points was done using the following formula: percentage of internalization = (median fluorescence intensity of cells with acid treatment/median fluorescence intensity of cells without acid treatment) x 100.
Yeast two-hybrid assays
The constructs Gal4AD-μ1A, Gal4AD-μ2, Gal4AD-μ3A, and Gal4AD-μ4 in the pACTII (LEU2) plasmid and Gal4BD-TGN38 in the pGBT9 (TRP1) plasmid have been described previously (29, 30). The MATCHMAKER GAL4 Two-Hybrid System 3 (BD Clontech) was used for the generation of GAL4BD-mCD1d, containing the wild-type mCD1d cytoplasmic tail, and the four mutants GAL4BD-YA, GAL4BD-QA, GAL4BD-DA, and GAL4BD-IA, followed by yeast transformation. GAL4BD-mCD1d was generated as described previously (34). GAL4BD-YA, GAL4BD-QA, GAL4BD-DA, and GAL4BD-IA were generated by ligation of pGBKT7, the GAL4BD vector, with synthetic double-strand DNA encoding the entire 10-amino acid cytoplasmic domain of mCD1d containing various point mutations. The constructs were confirmed by sequencing. The Saccharomyces cerevisiae strain AH109 was transformed according to the manufacturer’s instructions with various combinations of Gal4AD-μ1A, Gal4AD-μ2, Gal4AD-μ3A, or Gal4AD-μ4 with GAL4BD-mCD1d, GAL4BD-YA, GAL4BD-QA, GAL4BD-DA, GAL4BD-IA, GAL4BD-TGN38, or the pGBKT7 vector and selected in medium lacking leucine and tryptophan. For plate colony growth assays, AH109 transformants were streaked on plates lacking adenine, histidine, leucine, and tryptophan and allowed to grow at 30°C for 3 days. Quantitative growth of AH109 transformants was measured by inoculating colonies of various transformants in liquid medium lacking adenine, histidine, leucine, and tryptophan and allowing them to grow at 30°C. The OD at 600 nm was measured at various times.
Cloning, expression, and purification of soluble adaptor μ-chains
The full-length cDNA clones of mouse μ1A and rat μ3A were kindly provided by P. Schu (University of G?ttingen, G?ttingen, Germany) and M. S. Robinson (Medical Research Council, Cambridge, U.K.), respectively. The pET28a vector (Novagen) containing N-terminally 6x His-tagged rat μ2 (residues 158–435) was a gift from V. Haucke (Freie Universit?t Berlin, Berlin, Germany). These plasmids were transfected into the Escherichia coli strain BL21, and protein production was induced by addition of isopropyl -D-thiogalactoside for 3 h at 30°C, followed by purification of the μ-chain proteins from 1 liter of bacterial culture. The purification was performed according to a standard protocol (Qiagen) using Ni-NTA agarose as an affinity matrix. The purity of the μ-chain proteins stored in buffer A (10 mM HEPES-KOH (pH 7.4), 500 mM NaCl, and 10 mM 2-ME) was controlled by SDS-PAGE. Before BIAcore experiments, the proteins were centrifuged for 30 min at 100,000 x g to remove possible protein aggregates.
Detection of AP μ-chain binding to sorting signals by surface plasmon resonance (SPR)
The binding of recombinant μ-chains of the adaptor complexes, μ1 to μ3, to cytoplasmic tail sorting signals was recorded in real time using a SPR-based biosensor (BIAcore 3000; BIAcore AB). The 6x His-tagged truncated μ-chain proteins were used at concentrations ranging from 500 nM to 2.5 μM. Synthetic peptides corresponding to the sorting signals of CD1d (-CIWRRRSAYQDIR), TGN38 (-CKVTRRPKASDYQRL), and the TfR (-CGEPLSYTRFSLARQVDG), as well as their mutants in which the critical tyrosine was substituted for alanine, were immobilized on a CM5 sensor surface using the thiol coupling method in the manufacturer’s instructions. All peptides were immobilized according to their molecular weights at equal densities of 800-1100 resonance units. After immobilization, the sensor surface was washed with short pulse injections of 50 mM sodium hydroxide to remove any nonspecifically attached material. The μ-chains were injected in buffer A (with 5 mM DTT instead of 2-ME) at a flow rate of 10 μl/min for 1 min, followed by washing with buffer for 5 min. Subsequently, any μ-chain protein still bound to the sensor surface was removed by a 1-min pulse injection of 50 mM sodium hydroxide. The μ-chains were passed simultaneously over surfaces containing a sorting signal and the respective tyrosine mutant peptides. The binding curve of the latter one was subtracted from the curve obtained for the wild-type sorting signal before the kinetic rate constants were calculated, as described elsewhere (37).
Results
Expression of mutated mCD1d molecules
To assess how the individual amino acids in the Tyr motif might affect the subcellular localization and Ag presentation ability of the mCD1d molecules, each amino acid in the YQDI motif was substituted with alanine, as schematically presented in Fig. 1A. Flow cytometry analysis showed that the sorted A20 transfectants expressing mutant molecules have a similar or higher surface expression of mCD1d compared with transfectants expressing wild-type mCD1d (Fig. 1B).
FIGURE 1. Expression of mCD1d cytoplasmic tail point mutants. A, Point mutations of the four-amino acid endosomal-targeting motif (indicated in bold) localized at the cytoplasmic terminus. B, Surface expression of mCD1d point mutants. Transfectants with the indicated point mutations were stained with PE-conjugated rat anti-mCD1d mAb, and the expression level of surface mCD1d was determined by flow cytometry. Isotype control stainings are shown with shaded-fill under the curve.
Ag presentation by mutant mCD1d molecules
The Ag presentation ability of the various mCD1d point mutants was tested using V14i NKT cell hybridomas responding to GalCer or Gal(12)GalCer. The presentation of GalCer does not absolutely require internalization, and all of the mCD1d cytoplasmic tail mutants presented GalCer (Fig. 2A). Three mutants, Y332A, D334A, and I335A, had a dramatically reduced ability to present Gal(12)GalCer, which requires mCD1d localization to late endosomes or lysosomes (34). Although the Q333A mutant had lower surface expression than the Y332A, D334A, and I335A mutants, the ability of Q333A molecules to present Gal(12)GalCer was comparable to wild-type mCD1d. Therefore, substitutions of three of four amino acids within the Tyr-based motif affect the ability of mCD1d to present glycolipid Ags to V14i NKT cells, especially those requiring internalization.
FIGURE 2. Ag presentation by mCD1d point mutants. A, A20 transfectants expressing wild-type or point mutant mCD1d molecules were pulsed with the indicated amounts of Ags, either GalCer or Gal(12)GalCer, washed, and cultured with NKT cell hybridoma 3C3. Cell-free supernatants were tested by ELISA for IL-2 production. The error bars indicate the SD of triplicate measurements. The responses of the 1.2 and 1.4 V14i NKT cell hybridomas were similar (data not shown). The data are representative of at least three independent experiments. B, A20 transfectants expressing wild-type or the Y332A, D334A, and I335A mutants were cultured with the mCD1d-autoreactive hybridoma 24, which does not express a V14iTCR. Cell-free supernatants were tested by ELISA for IL-2 production. The error bars indicate the SD of triplicate measurements. Shown here are data from one of four independent experiments.
The ability of the tail-mutated mCD1d molecules to present GalCer suggests that the extracellular domain has not been compromised. To provide an additional control for this, we tested the autoreactivity of a CD1d-dependent hybridoma, 24 (9), which does not have the V14i rearrangement and which is independent presumably of the CD1d tail motif (38). The mutants retained approximately the same ability to stimulate hybridoma 24 (Fig. 2B). The I335A mutant caused a 2-fold less stimulation of the autoreactive hybridoma 24 (Fig. 2B), and it presented GalCer less effectively than the other mCD1d molecules (Fig. 2A). This is almost certainly a reflection of the lower mCD1d surface level in cells expressing this mutant (Fig. 1). However, most important is that the ability of I335A to present Gal(12)GalCer was decreased drastically in comparison with its ability to present GalCer, as with the Y332A and D334A mutants. This drastic decrease is indicative of the importance of I335 and, by the same token, Y332 and D334 in regulating the intracellular localization of mCD1d.
Subcelullar localization of the cytoplasmic tail mutants
We examined the subcellular localization of wild-type and mutant mCD1d molecules using confocal microscopic analysis of fixed and permeabilized A20 cells (Fig. 3A). Quantitation of the degree of colocalization is presented in Fig. 3B. Wild-type CD1d molecules are distributed throughout the endocytic pathway, colocalizing with the late endosomal/lysosomal marker, Lamp-2, but also with the early/recycling endosomal marker TfR. Nearly 100% of vesicles containing Lamp-2 also contained detectable mCD1d, and the same also was true for vesicles containing the TfR (Fig. 3B). For the different mutations, there was a good correlation between mutants that affect Ag presentation and those that affect the intracellular distribution of mCD1d. The Q333A mutant displayed an intracellular distribution similar to wild-type mCD1d. By contrast, the Y332A, D334A, and I335A mutant molecules have decreased localization in endosomal vesicles. Among these three, the Y332A mutant has the highest degree of colocalization with the TfR, with more than half of the TfR-containing vesicles having some mCD1d. However, the disruption of trafficking to late endosomes is most severe in this mutant with a few Lamp-2-positive vesicles containing mCD1d. The DA and IA mutants behave similarly, with affects on mCD1d colocalization to both early TfR-containing and late Lamp-2-positive endosomes. These data suggest that the Tyr might affect mCD1d intracellular localization through a partially different mechanism than the Asp and Ile.
FIGURE 3. Steady-state distribution of wild-type and mutant mCD1d molecules in A20 cells as analyzed by immunofluorescence and confocal microscopy. Colocalization of wild-type and mutant mCD1d molecules (red) with markers (green) for early/recycling endosomes (A, TfR) and lysosomes (B, Lamp-2) was revealed as yellow in the overlay images. The colocalization was quantified using the Laserpix program (Bio-Rad) and shown in C. n denotes the numbers of cells used for each quantitation. Scale bar, 50 μm.
Internalization of mutant mCD1d molecules
Previous studies of cells from mice expressing mCD1d with a truncated cytoplasmic tail indicated that mCD1d molecules depend on the cytoplasmic tail, presumably the Tyr motif, for internalization (15). The internalization of human CD1d in MDCK cells also depends on the Tyr motif (28). To further investigate the mechanism whereby the other amino acids within the sorting motif of the mCD1d cytoplasmic tail regulate mCD1d trafficking and Ag presentation, we examined the internalization rates of the different mutant molecules using a flow cytometry-based internalization assay (Fig. 4). As demonstrated for the human TfR (39), internalization of the mouse TfR was very efficient. Within 30 min, 60% of the surface TfR was internalized (Fig. 4A). Wild-type mCD1d internalized more slowly than TfR, with <40% of mCD1d internalized by 60 min. However, this rate of internalization is still considerably faster than that of MHC class I molecules with only 5% internalized by 60 min. Among all the mutant mCD1d molecules, the Y332A mutation affected the internalization rate to the greatest degree, comparable to the tail-deleted mCD1d molecules (Fig. 4B). The I335A mutation also resulted in a decreased rate of internalization, although to a lesser degree. However, even these mCD1d mutants internalized at a higher rate than MHC class I molecules. The internalization rates of the Q333A and D334A mutant molecules were not different significantly from wild-type mCD1d. Therefore, Tyr and, to a lesser extent, Ile appear to be important in governing the internalization of the mCD1d molecules, presumably through interaction with the machinery responsible for endocytosis.
FIGURE 4. Internalization rate of wild-type and mutant mCD1d molecules. A20 transfectants expressing wild-type or point mutant mCD1d molecules were stained with FITC-anti-TfR, PE-anti-mCD1d, and/or FITC-anti-H-2Kd. The surface-bound Abs were allowed to internalize at 37°C. After acid treatment to remove the remaining surface-bound Ab, cells were permeabilized and analyzed by flow cytometry. A, Comparison of internalization rates for TfR, mCD1d, and H-2Kd in A20 transfectants expressing wild-type mCD1d molecules. The data are representative of two independent experiments. B, Comparison of internalization rates of wild-type and mutant mCD1d molecules in A20 transfectants. The data are average of four to six independent experiments. To allow for comparison between different experiments, the rates of internalization for the mutant molecules were normalized to wild-type mCD1d.
The ability of mCD1d Tyr motif to interact with the μ subunits of AP
Adaptor proteins (AP-1 to AP-4) of distinct subunit composition are currently understood to direct the trafficking of many proteins to multiple vesicular destinations. We have previously shown that the Tyr is important for the interaction between the mCD1d cytoplasmic tail and the AP-3 complex, presumably through the μ3A subunit (34). To further understand whether the Tyr motif interacts with other AP and how individual amino acids within the Tyr motif contribute to these interactions, we used a yeast two-hybrid system in which the μ subunits from AP-1, AP-2, or AP-3 were expressed as fusions with the activation domain of GAL4. Additionally, point mutants of the mCD1d cytoplasmic tail were expressed as fusions with the binding domain. The mCD1d cytoplasmic tail did not interact with the μ subunits of AP-1 and AP-2 in yeast solid plate and liquid culture growth assays, whereas these assays confirmed the previously reported interaction of the cytoplasmic tail with the μ3A subunit by a -galactosidase assay (Fig. 5, A and B; Ref. 34). We then used SPR to measure the interaction of synthetic peptides corresponding to the cytoplasmic tail of wild-type and Y332A mutant mCD1d to purified soluble μ1 to μ3 subunits (Table I). Data from SPR agreed with those from the yeast two-hybrid experiments, in that the cytoplasmic tail of mCD1d interacted with the μ3A subunit but do not with μ1. The mCD1d tail also interacted with the μ2 subunit in SPR, although this interaction is weaker compared with the μ3A. Optimal μ2 binding to CD1d requires a functional tyrosine motif (YQDIR) because the Y332A mutant peptide binds only weakly to μ2 (Fig. 5C).
FIGURE 5. Interaction of wild-type (WT) and mutant mCD1d cytoplasmic tails with the μ subunits of AP. A, Growth of AH109 yeast transformants coexpressing various μ subunits and WT mCD1d. Transformants were streaked on medium plates lacking adenine, histidine, leucine, and tryptophan. B, Quantitation of the growth rate of AH109 yeast transformants coexpressing various μ subunits and WT mCD1d. Transformants were selected in medium lacking histidine, leucine, and tryptophan at 30°C, and absorbance at wavelength 600 nm was measured with a spectrophotometer. The data are representative of three independent experiments. C, SPR sensorgrams of the binding of peptides corresponding to WT and Y332A mutant (YA) mCD1d tails to μ2. Synthetic peptides corresponding to the cytoplasmic tails of WT and the YA mCD1d were immobilized on a CM5 sensor surface as described in Materials and Methods. A surface without peptide served as an additional control (curve 4 in YA). Purified soluble μ2 was passed over the surfaces at the indicated concentrations followed by washing with running buffer. The rate constants for the binding μ2 to WT CD1d were calculated after subtraction of the binding to the YA. D, Quantitation of the growth rate of AH109 yeast transformants coexpressing μ3A along with constructs encoding WT or mutant mCD1d cytoplasmic tails. Transformants were selected in the medium, as described above, and absorbance at 600 nm was measured. The data are representatives of three independent experiments. E, SPR sensorgrams of the binding of μ3 to peptides corresponding to WT and mutant mCD1d cytoplasmic tails. Purified soluble μ3 was passed at the concentration of 15 μM over a CM5 surface derivatized with the indicated mCD1d peptides.
Table I. Kinetic rate constants for adaptor μ-chain binding to tail peptidesa
As shown in Fig. 5D, yeast transformants containing μ3A along with wild-type CD1d cytoplasmic tail, QA, or DA mutants grew in medium lacking histidine, leucine, and tryptophan at comparable rates, whereas those containing the μ3A subunit along with YA or IA did not grow at all. This observation was confirmed by SPR experiments in which we tested μ3A binding with peptides corresponding to the wild-type and mutant mCD1d cytoplasmic tails. We used micromolar concentrations of μ3A (4–20 μM) to ensure the detection of any weak interaction with the mutant mCD1d peptides. As shown in Fig. 5E, the binding of the Q333A and D334A mutants to μ3A was comparable to that of wild-type mCD1d, although they both exhibited a <2-fold increase in the dissociation rate. In contrast, the binding of Y332A and I335A was reduced to levels observed for an unrelated control peptide or a blank surface (data not shown). Therefore, the Y and I amino acids are critical for the interaction of mCD1d with the μ3A subunit, whereas the D amino acid mostly affects CD1d localization and Ag presentation through some other mechanism.
Discussion
Ag presentation by mCD1d and the development of V14i NKT cells depend on the 10-amino acid cytoplasmic tail of mCD1d. In human CD1d, a similarly short cytoplasmic sequence contains a tyrosine internalization motif together within an overlapping leucine-based signal and mediates basolateral sorting (40), which is consistent with the hypothesis that the multiple steps required for proper CD1d localization are mediated by these short sequences. Although they both contain a YXX motif, human CD1d and mCD1d differ for their cytoplasmic sequences and in their trafficking, however, as illustrated by the inability of human CD1d to interact with AP-3μ, in a yeast two-hybrid analysis (33).
mCD1d molecules potentially could take two routes to the endocytic pathway, either directly from the Golgi complex or via internalization from the cell surface. The currently accepted hypothesis for mCD1d trafficking is that the Tyr-based endosomal localization motif within the cytoplasmic tail regulates the recycling of mCD1d that occurs relatively early after its biosynthesis (38). There are no data so far demonstrating that a population of newly synthesized mCD1d molecules traffic directly from the TGN to endosomes, although this has been reported for other proteins that contain tyrosine signals that bind AP-3 (41). The cytoplasmic tail also regulates the subsequent trafficking of mCD1d to late endosomes/lysosomes through its interaction with μ3A of the AP-3 complex (34, 35). The data reported here represent an effort to understand additionally the molecular basis of the regulation of mCD1d localization. We conducted an analysis of the roles of individual amino acids within the YXX motif on mCD1d internalization, intracellular localization, and Ag presentation. The data demonstrate that three of the four amino acids in this motif are important. Furthermore, because the affects of Ala substitution of the individual amino acids are different, they illustrate AP-3-dependent mechanisms influenced by Tyr and Ile and AP-3-independent mechanisms revealed by Ala substitution for Asp.
Consistent with previous reports indicating the importance of the cytoplasmic tail Tyr in human CD1d internalization (28), our data show that the Y332A mutation reduced the rate of internalization to the greatest degree. When the four point mutants were compared, the effect of the Y332A mutation and was similar to a complete cytoplasmic tail truncation. Despite this, steady-state measurements indicated that the Y332A mutation affected colocalization with the TfR to a lesser extent than either the D334A and I335A mutations. However, the steady-state distribution of mCD1d in early/recycling endosomes is determined by several factors, including the internalization rate from the cell surface, the recycling rate, the rate of entry to late endosomal/lysosomal compartments from recycling compartments, and possibly the rate of exit from the TGN directly to endosomes. Because tail-deleted mCD1d has been reported to recycle normally (38) and Tyr is most critical for the interaction of mCD1d with μ3A and its localization to Lamp-2-positive vesicles, a possible reason for the accumulation of Y332A mutant mCD1d in early/recycling endosomes, compared with the I335A and D334A mutants, could be its impaired transport to late endosomes/lysosomes.
The molecular basis for mCD1d internalization is yet to be understood. The results from a direct binding study using SPR suggested that the internalization of mCD1d is most likely mediated by the AP-2 complex through the interaction of the Tyr motif of mCD1d with μ2. The interaction is 2-fold weaker than the interaction with the μ3A subunit of AP-3, and it could not be detected in the yeast two-hybrid assay. Therefore, negative results from the two-hybrid assay using CD1d cytoplasmic tails must be interpreted cautiously. We then have confirmed the interaction of the mCD1d tail with AP-2 by SPR-binding experiments using the purified AP-2 complex as opposed to the recombinant, purified μ subunit (O. Bakke and S. H?ning, data not shown). The selective strength of the interaction of the mCD1d cytoplasmic tail with μ3A compared with μ2 may be due to favored interaction to μ3A by the Arg at position Y-3 and the Ile at position Y + 3 of the motif (31). The absence of this Y + 3 Ile in human CD1d may in part account for the inability of the human homologue to interact with AP-3A when analyzed by a yeast two-hybrid experiment. Furthermore, the physiological significance of mCD1d internalization and recycling has not been established. Internalized mCD1d may acquire certain Ags in early endosomes, and although a normal internalization rate may be required for presentation of some of the Ags that stimulate V14i NKT cells, analysis of the DA mutant suggests this is not sufficient for a correct intracellular localization and Ag presentation capability. This is consistent with our earlier observation that chimeric mCD1d molecules that localize to early but not late endosomes are deficient in the presentation of glycolipids that require internalization and processing (34).
Ala substitution of the Ile at the Y + 3 position in the YXX motif had global effects on Ag presentation, intracellular localization, internalization, and interaction with the AP-3μA-chain. Consistent with this, it was shown previously that Ala substitution of the Val at the Y + 3 position of the cytoplasmic tail of human CD1d reduced internalization (28). However, the effects of the I335A and Y332A mutations were not equivalent with the I335A mutant having a reduced affect on the internalization rate and an increased ability to localize to Lamp-2-positive vesicles.
The effects of Ala substitution for the Asp in the Y + 2 position of the mCD1d cytoplasmic tail were striking. These mutant mCD1d molecules internalized at a normal rate, but they have a drastically decreased ability to present a glycolipid Ag that requires internalization and processing, and they displayed an altered steady-state intracellular distribution in a manner similar to the I335A mutant. Despite this, Asp is not critical for the interaction with the AP-3μA subunit. However, the Asp at position Y + 2 could be a good candidate for involvement in alternative processes, such as a role in mediating a direct route from the Golgi to late endosomes/lysosomes. This direct trafficking to endosomes remains an attractive if speculative possibility, based on the composition of residues at positions Y + 1 and Y + 2 of the Tyr motif (19, 42) of mCD1d and the six-amino acid distance from the membrane to the Tyr (19, 43).
In conclusion, analysis of the Tyr motif demonstrates the complexity of mCD1d trafficking and its effects on glycolipid Ag presentation and the multifaceted roles of the amino acids in the short cytoplasmic tail.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Dr. Jeffery A. Lawton for critical reading of this manuscript and Dr. Juan S. Bonifacino (Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD) for providing Gal4AD-μ1/2/3 and -TGN yeast two-hybrid constructs.
Footnotes
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 supported by National Institutes of Health Grant RO1 AI40617 (to M.K.), a grant from the Human Frontiers of Science Program (to M.K.), and grants from the University of Oslo and the Research Council of Norway (to O.B.). A.P.L. is the recipient of a National Research Service Award from National Institutes of Health (Grant AI52552).
2 Current address: Tampa Bay Research Institute, St. Petersburg, FL 33712.
3 Current address: Department of Molecular Microbiology and Immunology and Graduate Program in Pathobiology, Division of Biology and Medicine, Brown University, Providence, RI 02912.
4 Address correspondence and reprint requests to Dr. Mitchell Kronenberg, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address: mitch{at}liai.org
5 Abbreviations used in this paper: mCD1d, mouse CD1d; GalCer, -D-galctosyl ceramide; AP, adaptor protein complex; TGN, trans-Golgi network; Gal(12)GalCer, -2'-galactosyl-GalCer; TfR, transferrin receptor; SPR, surface plasmon resonance.
Received for publication August 6, 2004. Accepted for publication December 1, 2004.
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