Plasticity and Rigidity in Adaptor Protein-2-Mediated Internalization of the TCR:CD3 Complex
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免疫学杂志 2005年第7期
Abstract
Many cell surface proteins are internalized via dileucine- or tyrosine-based motifs within their cytoplasmic domains by the heterotetrameric adaptor protein complex, AP-2. In this study we have examined how AP-2 mediates internalization of large cell surface receptors, such as the eight-chain TCR:CD3 complex. Although most receptors have a single signal that drives internalization, the TCR complex has two (D/E)xxxL(L/I) motifs and 20 Yxx? motifs. Using 293T cells, we show that AP-2 is completely dependent on both signals to mediate TCR internalization, because deletion of either completely blocks this process. Significant plasticity and redundancy were observed in the use of the Yxx? motifs, with a clear hierarchy in their use (CD3 > CD3 CD3 >> CD3). Remarkably, a single, membrane-distal Yxx? motif in CD3 could mediate 75% of receptor internalization, whereas its removal only reduced internalization by 20%. In contrast, significant rigidity was observed in use of the (D/E)xxxL(L/I) motif in CD3. This was due to an absolute requirement for the position of this signal in the context of the TCR complex and for a highly conserved lysine residue, K128, which is not present in CD3. These contrasting requirements suggest a general principle by which AP-2 may mediate the internalization of large, multichain complexes.
Introduction
Many cell surface receptors are constitutively internalized via clathrin-coated pits and subsequently recycled back to the cell surface (1, 2). This process is mediated by the heterotetrameric clathrin-associated adaptor protein complex AP-2, which recognizes short amino acid sequences within the cytoplasmic tails of these receptors. These primarily consist of either tyrosine-based signals, Yxx? (x, any amino acid; ?, a hydrophobic amino acid) or dileucine-based signals, (D/E)xxxL(L/I) (3). The Yxx? signal is recognized by the medium subunit (μ2) of the AP-2 complex and mediates the rapid internalization of a wide variety of molecules, such as the transferrin receptor (CD71) and CTLA-4 (CD152) (4, 5, 6, 7). The (D/E)xxxL(L/I) signal, which has been shown to mediate the internalization of CD4 and CD3, is known to interact with AP-2, but does not compete with tyrosine-based signals (8, 9, 10, 11, 12, 13). Although the exact binding site remains to be resolved, it has been suggested that (D/E)xxxL(L/I) signals bind to the μ2-chain and/or the trunk domain of the 2 subunit (14, 15, 16, 17, 18, 19).
The TCR is expressed as a large, multisubunit complex composed of at least eight polypeptide chains (TCR and CD3, -, and -) (20, 21, 22). In resting cells, the TCR complex is constitutively internalized and recycled via motifs in the cytoplasmic domains of the CD3 molecules (23, 24, 25). Although the physiological importance of this process is unclear, it may play a role in receptor down-modulation, serial ligation, and/or control of surface expression (24, 26). Several studies have shown that AP-2 can bind to the TCR complex (8, 27), and that the CD3 dileucine-based signal can mediate internalization (8, 28, 29). However, it is not clear whether all signals that mediate TCR internalization have been defined, because it has been suggested that tyrosine-based signals in the CD3 chains may contribute (24).
The cytoplasmic tails of the CD3 chains contain ITAMs ((D/E)X2YX2(L/I)X7YX2(L/I)) that are phosphorylated upon TCR ligation and mediate downstream signal transduction and T cell activation (30, 31, 32). Within those ITAMs there are at least 20 Yxx? motifs that could act as signals for AP-2 binding, based on the currently accepted model of the TCR:CD3 complex (22, 33). Given that a single Yxx? motif in CTLA-4 mediates its rapid internalization, largely preventing stable cell surface expression, it is paradoxical that the TCR complex remains stably expressed on the cell surface.
Because most studies have analyzed the interaction between AP-2 and single-chain transmembrane proteins or chimeras containing isolated cytoplasmic domains, it is unclear how AP-2 mediates the internalization of large, multichain complexes that contain many potential internalization motifs. For instance, is there cooperative or selective use of multiple or individual signals? Tackling this problem has been hampered by the lack of mutant cell lines for each or all of the CD3 chains. We recently showed that cell surface TCR:CD3 complex expression could be obtained by transient transfection of 293T cells (24). Using this system, we have shown that the TCR is internalized and recycled in a manner comparable to that in normal T cells, and that this process is mediated by signals in the CD3 chains. This provided a useful system to decipher the mechanism of TCR internalization, because many of the signals and proteins involved in this process are evolutionarily conserved (3). In this study we have addressed the following questions. 1) Which CD3 Yxx? motifs can bind to AP-2μ? 2) Which tyrosine- and dileucine-based motifs can mediate TCR internalization, and what is their relative contribution to this process? 3) Is a single motif adequate for TCR internalization, as suggested by experiments using single-chain CD3 chimeric molecules, or is there cooperative use of multiple motifs?
Materials and Methods
Plasmids
Mutant and wild-type TCR:CD3 chains were generated by recombinant PCR and cloned in the murine stem cell virus-based vector, MSCV.IRES.GFP, as previously described (34, 35, 36). Details of primers and cloning strategy can be obtained from A. Szymczak (andrea.szymczak@stjude.org).
Yeast two-hybrid
CD3:AP-2μ interaction was assessed using a galactosidase-4 (GAL4)3-based yeast two-hybrid system (provided by S. Elledge, Baylor College of Medicine, Houston, TX). The entire cytoplasmic domains of wild-type or mutant CD3 chains were generated by PCR and cloned into pAS2, which confers leucine auxotrophy. AP-2μ in pACT, which confers tryptophan auxotrophy, was provided by J. Bonifacino (National Institutes of Health, Bethesda, MD). The day before transformation, 400 ml of YPAD was inoculated with 40 μl of an overnight Y153 yeast stock (MATa GAL4 GAL180 His3 Trp1–901 Ura3–52 Leu2–3,-112 + URA3::GALlacZ, Lys2::GALHIS3) and grown at 30°C to 1 x 107 cells/ml (OD600, 0.4). Cells were washed in HPLC grade H2O and resuspended in 10 ml of 100 mM lithium acetate in Tris-EDTA (TE) (pH 7.5). Competent yeast were transformed with 1 μg of pACT and 1 μg of pAS2 vectors with 100 μg of denatured salmon sperm DNA (Sigma-Aldrich), 30% polyethylene glycol 4000, and 100 mM lithium acetate/TE. Yeast were shaken for 30 min at 30°C and heat-shocked for 15 min at 42°C. Yeast were spun, resuspended in TE, and plated out on simple defined dropout plates, SD-Trp-Leu and SD-Trp-Leu-His + 3AT (Bio 101). All yeast grew on the former. Interaction was determined by growth on the latter (histidine auxotrophy) and -GAL activity, as determined by a lacZ assay. Liquid cultures were set up by inoculation of SD-Trp-Leu broth and were grown at 30°C overnight to 1 x 107 cells/ml in SD-Trp-Leu broth. Four hundred microliters of culture was placed onto nitrocellulose filters using dot-blot apparatus (Bio-Rad), snap-frozen in liquid N2, placed on 3M filter paper saturated in Z buffer (60 mM Na2HPO4·7H2O, 40 mM NaH2PO4·H2O, 10 mM KCl, 1 mM MgSO4·7H2O, and 50 mM 2-ME, pH 7.0) containing 1 mg/ml X-GAL (Sigma-Aldrich), and incubated overnight at 30°C.
Transient transfection of 293T and TCR internalization assay
Transfections and internalization assays were performed as described with some modification (24). 293T cells (human embryonic kidney cells; provided by D. Baltimore, Caltech, and E. Vanin, St. Jude Children’s Research Hospital, Memphis, TN) were incubated in 10-cm plates at 2 x 106/plate overnight at 37°C. DMEM (470 μl) was mixed with 30 μl of FuGene 6 transfection reagent (Roche) for 5 min at room temperature. Each group of transfectants received 2A peptide-linked 3A9.TCR -chains as well as the CD3 chains indicated (36). DNA (2 μg of each chain) was mixed with FuGene/DMEM and incubated for 15 min at room temperature. The mixture was incubated with the cells for 2 days. On day 3, cells were stained with anti-V8.1/2-PE (F23.1) and anti-CD3-allophycocyanin (BD Pharmingen). GFP+/TCR+/CD3+ cells were sorted and cultured in 96-well, flat-bottom plates at 5 x 104/well overnight. Brefeldin A (BFA; Epicentre Technologies) was added at 10 μg/ml for the times indicated. Cells were stained with anti-TCR--CyChrome (H57-597) and analyzed for TCR modulation (FACSCalibur; BD Biosciences). The percent internalization was determined from the median fluorescence values using untreated controls as reference.
Results
AP-2μ specifically interacts with tyrosine residues in CD3 and to a lesser extent CD3
Yeast two-hybrid assays have been successfully used to determine the relative abilities of various Yxx? motifs to bind to the AP-2μ-chain (14). Although AP-2 has been shown to bind to the TCR:CD3 complex (8, 27), it is unclear which Yxx? motifs in CD3 can be bound by AP-2μ (Fig. 1A). Initially, we used a GAL4-based, yeast two-hybrid assay to determine which CD3 cytoplasmic tails can bind directly to AP-2μ. The CTLA-4 cytoplasmic tail, which is known to interact with AP-2μ (4, 27), mediated significant growth on minimal medium lacking histidine and production of -GAL in yeast transformed with the AP-2μ vector, but not an empty vector control (Fig. 1B). In contrast, no interaction was observed with the CD28 cytoplasmic tail, as expected.
FIGURE 1. AP-2μ-mediated internalization of the TCR:CD3 complex is mediated by tyrosine residues in the cytoplasmic tails of the CD3 chains. A, Amino acid sequence of the CD3 cytoplasmic domains. The ITAMs are boxed, and the tyrosine residues (Y) are in bold. The number above the tyrosine residues denotes the tyrosines of the Yxx? motifs detailed in B and Figs. 2 and 3. TM, transmembrane domain; CY, cytoplasmic domain. B, Interaction between AP-2μ and the CD3 cytoplasmic domains was determined in a yeast two-hybrid assay. The entire cytoplasmic domains of CD28, CTLA-4, and wild-type or mutant CD3 were cloned into pAS2, which contains the GAL4 DNA-binding domain. AP-2μ was cloned into pACT, which contains the GAL4 activation domain. Interaction was determined by the production of -GAL using a standard lacZ assay (right) and growth on minimal medium plates that lack histidine (SD-Leu-Trp-His + 3AT; see Materials and Methods; left). All yeast grew comparably on SD-Leu-Trp plates. Y-F, Yxx? Tyr residues mutated to Phe. C and D, 293T cells were transiently transfected with plasmids carrying the 3A9 TCR chains and the wild-type (WT) CD3 chains or CD3 chains with all Yxx? Tyr mutated to Phe. After 2 days, TCR+/CD3+/GFP+ cells were sorted and treated with BFA (10 μg/ml) for the times indicated (C) or for 30 min (D), and TCR internalization was determined. C, A representative experiment is shown. D, The mean ± SE from 14 experiments are shown as a percentage of the CD3.WT transfectants.
Using this assay, AP-2μ was found to specifically interact with the cytoplasmic tail of CD3 and, to a lesser extent, with that of CD3, but not of CD3 or CD3. Importantly, mutation of the two tyrosine residues in CD3 and CD3 to phenylalanine (Y-F) substantially reduced this interaction. A small amount of growth was observed with the CD3.Y1/2-F mutant. This growth does not appear to be due to any interaction with the dileucine motif in CD3, because mutation of these residues did not affect the growth or production of -GAL in this assay (data not shown). Although this could be due to background growth or another as yet undefined motif, it is clear that the CD3-AP-2μ interaction is substantially reduced by these substitutions. Mutation of either Y1 or Y2 completely abrogated the interaction with AP-2μ, suggesting that both are required for the weak association seen with wild-type CD3. In contrast, reduced, but significant, interaction with AP-2μ was observed with single tyrosine mutations in the CD3 cytoplasmic tail, suggesting that either CD3 Yxx? motif alone can mediate interaction with AP-2.
Internalization of TCR is mediated by tyrosine-based signals in CD3 chains
BFA blocks anterograde transport from the endoplasmic reticulum to the Golgi complex and causes tubulation and fusion of early endosomes with the trans-Golgi network, essentially blocking recycling of receptors back to the cell surface (37, 38). We have previously exploited this property to study TCR internalization in resting and activated T cells (24). We also showed that TCR:CD3 complexes ectopically expressed on 293T cells were internalized with kinetics comparable to those of normal T cells. This is consistent with the idea that TCR internalization is mediated by a general biological process that uses basic cell machinery (e.g., AP-2, dynamin, clathrin, etc.) that is present in all cells.
Using this system, we first introduced a series of tyrosine to phenylalanine (Y-F) substitutions in the Yxx? motifs of all four CD3 chains to determine whether these tyrosine-based motifs mediated TCR complex internalization. Wild-type and mutant TCR:CD3 complexes were expressed on 293T cells by transient transfection with plasmids expressing all six TCR:CD3 chains, and the extent of TCR internalization was evaluated using BFA. All constructs generated gave TCR surface expression levels comparable to those of wild type, and any small differences were normalized in calculating the percentage of TCR internalization. As we have previously shown, the effect of BFA is not complete, and TCR levels reach a steady state equilibrium after 20–30 min (24, 37, 38) (Fig. 1C). This appears to be remarkably consistent, as shown by the predominantly small error seen in all experiments in this study. As the data clearly show, mutation of all Yxx? tyrosine residues in the CD3 chains (CD3.Y-F) completely abrogated TCR internalization, suggesting that one or more of these residues mediated this process (Fig. 1, C and D). These data confirmed previous suggestions that TCR internalization is mediated by motifs within the CD3 cytoplasmic domains (8, 24, 39). However, we were surprised that mutation of the tyrosine-based motifs was sufficient to completely block internalization, because previous studies with TAC:CD3 chimeras had suggested that the dileucine- and tyrosine-based motifs act independently to mediate endocytosis (11). Thus, even though these CD3.Y-F mutant complexes have an intact CD3 dileucine motif, they cannot be internalized, suggesting that experiments with chimeric molecules may not recapitulate the requirements for intact, multichain complexes.
TCR internalization is mediated by a broad hierarchy of tyrosine-based motifs
Additional analysis of the individual CD3 chains was performed in two ways. First, we expressed TCR complexes with Y-F mutations within the ITAMs of a single CD3 chain (e.g., CD3 mutant, CD3 wild type; Fig. 2A, left). Second, we expressed TCR complexes in which ITAM tyrosine residues of a single CD3 chain remained intact, whereas all remaining Yxx? tyrosine residues in the CD3 complex were mutated (e.g., CD3 wild type and CD3 mutant; Fig. 2A, right). The majority of the constructs mediated TCR surface expression levels comparable to those of wild type (Fig. 3).
FIGURE 2. Multiple CD3 Yxx? signals mediate TCR internalization. 293T cells were transfected and sorted as described in Fig. 1. Cells were treated with BFA for 30 min, and TCR internalization was determined. A, Schematic of the two types of transfectants generated where one of the CD3 chains is mutated (left) or one of the CD3 chains is wild type (right). B–F, The mean ± SE from three to 15 experiments are shown as a percentage of the CD3 wild-type (WT) transfectants. Y-F, Yxx? Tyr residues mutated to Phe. TCR expression levels relative to wild type are depicted in Fig. 3.
FIGURE 3. Surface expression of TCR:CD3 complexes possessing Yxx? mutations. A–D, 293T cells were transfected, sorted, and analyzed as described in Fig. 1. The mean ± SE from three to 15 experiments are shown as a percentage of TCR surface expression of CD3.WT transfectants. WT, wild type.
For TCR containing single CD3 chain mutations, internalization appeared to be unaffected when tyrosine residues in CD3, CD3, or CD3 were mutated (Fig. 2B). However, an 50% reduction in TCR internalization was seen after mutation of the CD3 Yxx? tyrosine residues. This suggests the CD3 Yxx? signals are strongly recognized by the AP-2 internalization machinery, consistent with data from the yeast two-hybrid experiments. However, TCR internalization was not completely abrogated as seen when all CD3 Yxx? tyrosine residues are mutated, indicating some cooperativity between CD3 and the other CD3 chains. To address this issue, TCR complexes were expressed that contained Y-F mutations of all Yxx? tyrosines except for one CD3 chain (Fig. 2A, right). Cells expressing TCR complexes in which only the CD3 tyrosine residues were intact showed internalization levels 60% of wild-type transfectants (Fig. 2C). TCR complexes containing intact tyrosine residues only in CD3 or CD3 mediated 30–40% internalization compared with wild type. Because this represents two CD3 Yxx? signals vs 12 CD3 Yxx? signals, it is conceivable that CD3 contributes more to TCR internalization. In contrast, internalization was completely blocked in cells where only CD3 remained the wild-type chain. These data demonstrate the importance of CD3 tyrosine residues in mediating internalization, a process that appears to involve cooperativity among CD3- and CD3- and/or CD3-chains, but not CD3.
Next we assessed the relative contributions of the individual Yxx? signals in the CD3, CD3, and CD3 cytoplasmic domains. Although mutation of the CD3 ITAM tyrosine residues in the presence of an otherwise intact TCR:CD3 complex had little effect on TCR internalization, the CD3 Yxx? signals alone could still mediate 40% TCR internalization (Fig. 2, B and C). Analysis of TCR complexes that contained only one intact CD3 Yxx? signal (and thus only two per complex) mediated only weak internalization (Fig. 2F). Although there were no substantial differences among the six CD3 Yxx? signals, it did appear that CD3.Y3, -Y4, and -Y6 may have been marginally more efficient. As shown previously, significant TCR internalization was observed when only the CD3 Yxx? signals were intact, even though they were largely dispensable in the presence of wild-type CD3 (Fig. 2, B and C). This level of TCR internalization was lost if only CD3.Y2 was intact and was substantially reduced if only CD3.Y1 was intact (Fig. 2E). Clearly, these tyrosines are dispensable, because mutation of either Yxx? signal in the presence of wild-type CD3 had no effect on TCR internalization (Fig. 2D). Somewhat similar observations were made with CD3.Y1, where modest internalization was observed when it was the only intact Yxx? signal (Fig. 2E). In contrast, the membrane-distal Yxx? signal in CD3 (Y2) was clearly the most important contributor to TCR internalization. Indeed, this single tyrosine-based signal could mediate 75% of wild-type TCR internalization (Fig. 2E). Remarkably, its removal only reduced internalization by 20% (Fig. 2D).
These results suggest that there is a hierarchy in the ability of different Yxx? signals to mediate TCR internalization: Y2 >> Y1 Y1 = Y3 = Y4 = Y6 Y1 = Y5 Y2 = Y2 = Y1/2. Although the tyrosine-based signals in CD3, especially Y2, were the most potent mediators of TCR internalization, there was clear redundancy in Yxx? signal use. Taken together, these data suggest that there is significant plasticity in the requirements for AP-2-mediated internalization of the TCR via the tyrosine-based motifs.
The dileucine-based signal in CD3, but not CD3, mediates TCR internalization
It is well established that the dileucine-based motif in CD3 can mediate TCR internalization. The majority of studies have focused on protein kinase C-mediated internalization of the TCR in which the aspartic acid (D127), the two leucines (L131/132), and phosphorylation of a serine residue proximal to the motif (S126) were found to be essential (29, 39). However, less is known about the requirements for constitutive internalization of the TCR in resting cells. Recently, it was found that mutation of the human CD3 dileucine residues completely abolished TCR internalization in an S126 phosphorylation-independent manner (28). It is not known whether the CD3 dileucine-based signal participates in constitutive TCR internalization, although there are some data from experiments with Tac:CD3 chimeras that it is less effective at mediating lysosomal targeting than its CD3 counterpart (11). In these studies we wanted to assess the roles of the CD3 and CD3 dileucine-based signals, in the context of the whole TCR:CD3 complex, in mediating TCR internalization.
Cells were transfected with wild-type or dileucine mutant (LL-AA) CD3- and CD3-chains in the presence of the remaining wild-type TCR:CD3 chains (Fig. 4A). Mutation of the CD3 dileucine residues significantly reduced TCR internalization (Fig. 4, B and C). In contrast, TCR internalization was not affected by mutation of the CD3 dileucine residues. Interestingly, coexpression of the CD3.LL-AA and CD3.Y1/2-F mutants did not further reduce TCR internalization, suggesting that the defects incurred by these mutations were not additive, but cooperative (Fig. 4B). Collectively, these data raised two interesting issues. First, as mutation of the CD3 dileucine residues did not abrogate internalization, were there other proximal residues that contributed to its function? Second, why is the CD3 (D/E)xxxL(L/I) motif unable to mediate TCR internalization?
FIGURE 4. The dileucine residues in CD3, but not CD3, are required for optimal TCR internalization. A, Cytoplasmic domain amino acid sequence of the CD3 and CD3 constructs analyzed. TM, transmembrane domain; CY, cytoplasmic domain. ITAM is boxed. B and C, 293T cells were transfected and sorted as described in Fig. 1. Cells were treated with BFA for 30 min (B) or for the times indicated (C), and TCR internalization was determined. B, The mean ± SE from seven experiments are shown as a percentage of the CD3.WT transfectants. C, A representative experiment is shown. LL-AA, Leu residues (L131/132 in CD3 and L142/143 in CD3) mutated to Ala. TCR expression levels relative to wild type (WT) are depicted in Fig. 6. D, Sequence alignment of the dileucine region of CD3 and CD3. TM, transmembrane domain; CY, cytoplasmic domain; h, hydrophobic residues. Residues that differ between CD3 and CD3 are in bold; the dileucine-based signal used by CD3 is boxed; nonconserved residues are indicated by a dot; acidic residues are indicated by a dash.
Key parameters for TCR internalization by the CD3 dileucine-based signal: position and K128
It was particularly surprising that the CD3 dileucine-based motif did not appear to mediate TCR internalization even though the dileucine and negatively charged residues in the (D/E)xxxL(L/I) motif were present (CD3, DKQTLL; CD3, EVQALL). This inferred that there were other parameters that determined the ability of the CD3 dileucine-based motif to mediate TCR internalization. Alignment of the CD3 and CD3 sequences revealed 11 amino acid residues between the transmembrane domain and the ITAM motif that differ between the two chains (Fig. 4D). Initially, we generated mutants in which only these nonconserved residues were swapped between the two proteins (Fig. 5A, CD3-LL- and CD3-LL-). This was preferred over exchanging the entire cytoplasmic tails to avoid altering the contribution of the tyrosine-based signals. TCR containing CD3-LL- instead of CD3.WT, which essentially contained two CD3 dileucine-based motifs (one in the wild-type CD3-chain and one placed in the context of CD3), were completely refractory to internalization (Fig. 5B). This was surprising, because mutation of the dileucine residues had only reduced TCR internalization by 60% (Fig. 4, B and C).
FIGURE 5. Internalization of the TCR via the CD3 dileucine signal is highly dependent on surrounding amino acid residues. A, Cytoplasmic domain amino acid sequence of the CD3 and CD3 constructs analyzed. TM, transmembrane domain; CY, cytoplasmic domain. ITAM is boxed. B–E, 293T cells were transfected and sorted as described in Fig. 1. Cells were treated with BFA for 30 min, and TCR internalization was determined. The mean ± SE from three to seven experiments are shown as a percentage of the CD3.WT transfectants. TCR expression levels relative to wild type (WT) are depicted in Fig. 6.
Do TCR containing two CD3 dileucine-based motifs, where CD3.WT has been replaced with CD3-LL-, exhibit enhanced internalization? Interestingly, the data infer the opposite, with a 30% reduction in TCR internalization observed in the presence of CD3-LL- (Fig. 5B, third bar). TCR internalization was prevented in this complex when the wild-type CD3 was replaced with the CD3.LL-AA mutant, suggesting that the internalization seen with CD3.WT/CD3-LL- was mediated by the dileucine signal in CD3, rather than the CD3 dileucine motif inserted into CD3 (data not shown). Likewise, when both regional mutants were expressed instead of the wild-type CD3- and CD3-chains (Fig. 5B, fourth bar), essentially transposing the CD3 dileucine-based signal from CD3 to CD3, minimal TCR internalization was observed.
Collectively, these data suggest the following. First, additional residues surrounding the two leucines in CD3, which are not present in CD3, are necessary for TCR internalization. Second, the position of the active dileucine-based signal relative to the rest of the TCR:CD3 complex is important. Third, placement of this signal in the wrong location appears to reduce internalization, suggesting that it may act as a dominant negative by encouraging AP-2 to engage the TCR in a less productive orientation.
Additional mutants were generated to determine which CD3-specific residue(s) was responsible for the efficacy of the CD3 dileucine-based signal (Fig. 5A). Substitution of CD3 residues S126/K128/T130 with the equivalent residues in CD3 (A137/V139/A141) gave essentially the same results as those observed above (Fig. 5C). Single-point mutations of these CD3 residues suggested that T130 was not required (Fig. 5D). Furthermore, mutation of the S126 residue, which is phosphorylated after phorbol ester treatment, has little effect on TCR internalization, consistent with recent findings using CD3-deficient Jurkat T cells (28). Remarkably, mutation of a single lysine residue (K128) completely prevented TCR internalization (Fig. 5D). Conservative substitution of the lysine with arginine reduced internalization by 30%, inferring preference for lysine and an absolute requirement for a positively charged amino acid. This was a surprising finding, because this residue had not previously been shown to be required for AP-2 binding and TCR internalization. Previous studies had shown a strong requirement for a negatively charged residue (D or E) four amino acids N-terminal to the dileucine residues (8). This was confirmed by the complete abrogation of TCR internalization by mutation of D127 (Fig. 5D). It is unclear whether D127 and/or K128 are directly or indirectly involved in AP-2 binding. However, it is clear that the lysine residue is an essential part of the CD3 dileucine-based internalization signal. Furthermore, it is clearly essential, because the K128 residue is not present in CD3, whereas the negatively charged residue (D/E) at 127 is present in both CD3 and CD3.
Interestingly, all but one of the constructs (CD3.T130A) described in this study and above gave TCR surface expression levels comparable to or above wild-type TCR levels and were inversely proportional to their effects on internalization (i.e., if TCR internalization was reduced, expression was increased; Fig. 6). For reasons that are not clear, this correlation was only observed with dileucine-based signal mutants.
FIGURE 6. Surface expression of TCR:CD3 complexes possessing DKxxxLL mutations. A–D, 293T cells were transfected, sorted, and analyzed as described in Figs. 4 and 5. The mean ± SE from three to seven experiments are shown as a percentage of TCR surface expression of CD3.WT transfectants. WT, wild type.
Lastly, we questioned whether this lysine residue was responsible for the inability of CD3-LL- to replace CD3.WT in mediating TCR internalization. Although mutation of K128 in CD3 completely prevented TCR internalization, the lack of this lysine residue in CD3 is not sufficient, as demonstrated with TCR expressing CD3-LLV128K- (Fig. 5E). This suggests that additional residues and/or structural constraints are required. Taken together, these data suggest that there is significant rigidity in the requirements for AP-2-mediated internalization of the TCR via the dileucine-based motif.
Discussion
The TCR:CD3 complex has 20 Yxx? motifs and two (D/E)xxxL(L/I) motifs. In this study we have defined which motifs constitute signals that are used by AP-2 to mediate TCR internalization. In so doing, we have also examined the general principle that AP-2 may use to mediate the internalization of large, multichain transmembrane complexes. Contrary to previous expectations (11), our studies have clearly shown that AP-2 requires both tyrosine- and dileucine-based signals to mediate TCR internalization and revealed both plasticity and rigidity in the engagement of these signals. It should be noted that these experiments were performed in 293T cells, and it remains to be determined whether the same requirements for TCR internalization will apply to lymphoid cells. However, we have previously shown that the TCR is internalized and recycled in a manner comparable to that in normal T cells (24), and others have shown that TCR internalization is mediated by a general biological process that uses basic cell machinery (i.e., AP-2, clathrin, etc.) that is present in all cells (14).
There appears to be significant plasticity in the use of Yxx? motifs by AP-2, which is consistent with the broad specificity of AP-2μ (3). The Yxx? sequences within CD3, CD3, and, in particular, CD3, appear to mediate TCR internalization. Generally, there was a good correlation between data obtained from the yeast two-hybrid AP-2μ binding assay and the 293T TCR internalization assay. In both instances, the CD3 Yxx? signals were clearly the most efficient. The only difference was our observation that CD3 tyrosines alone could mediate some TCR internalization in 293T cells, but we could not detect any binding to AP-2μ. This could be due to an affinity that is below the threshold of this assay. Our use of the entire CD3 cytoplasmic domains, rather than tandemerized repeats of the Yxx? motifs, may also yield different results (14). Alternatively, this difference could simply be due to the duplication of CD3 in the TCR complex.
Our studies suggest that there is a definable hierarchy in the ability of different Yxx? signals to mediate TCR internalization: Y2 >> Y1 Y1 Y1–6 Y2 = Y1/2. The proposed binding motif for AP-2μ suggests a strong preference for arginine (R) or proline (P) at Y + 2 and a preference for leucine (L) rather than isoleucine (I) at Y + 3 (Fig. 7) (40). This is consistent with Y2, Y1, and Y1 being particularly favored and may explain why Y1 is not used.
FIGURE 7. Alignment of the CD3 Yxx? signals and their relationship to the AP-2μ motif (40 ).
Inherent in this plasticity was marked redundancy in the use of different Yxx? signals. Remarkably, 75% of wild-type TCR internalization was mediated by a single tyrosine residue in CD3 of the 20 in the TCR:CD3 complex. However, mutation of this residue only slightly reduces TCR internalization. This infers that AP-2 only requires a single, optimal Yxx? signal to efficiently mediate TCR internalization, but can use several Yxx? signals in the TCR:CD3 complex.
In stark contrast, use of the dileucine-based motif exhibited remarkable rigidity. Even though both CD3 and CD3 have (D/E)xxxL(L/I) motifs, TCR internalization is mediated by the former, but not the latter. AP-2 has been shown to bind to a variety of sequences, including some that do not entirely conform to this motif (3). Thus, it is particularly surprising that for TCR internalization via this motif, AP-2 should exhibit such rigidity. The majority of data concerning this motif has come from its requirement for protein kinase C-mediated TCR internalization (8, 29, 39, 41, 42). In this instance, phosphorylation of the unique CD3 S126 defines this specificity. However, for constitutive TCR internalization, we and others have clearly shown that this residue is not required (28). Our study clearly showed that the aspartic acid (D127) and the two leucine residues (L131/L132) in CD3 were required for TCR internalization, consistent with previous studies (8, 43).
So what is the basis for use of the (D/E)xxxL(L/I) motif in CD3, but not in CD3? Our studies have shown that this specificity is manifest at two levels. First, the position of this motif within the complex appears to be critical, because the transposition of the CD3 (D/E)xxxL(L/I) motif into CD3, in the presence of an inactive CD3 mutant, abrogated TCR internalization. Second, there is an absolute dependency on the highly conserved, CD3-specific lysine at LL-3 (K128). We have shown that a single mutation of either K128 or D127 completely abrogates TCR internalization. However, the latter can be efficiently substituted for by glutamic acid, which is the residue in the analogous position in CD3 (8). These data infer that AP-2 either directly binds to K128 or binds to D127, but only in the presence of K128. Despite the importance of both signal position and the residue K128, these factors alone are insufficient to mediate TCR internalization via this dileucine-based signal. Transposition of the CD3 (D/E)xxxL(L/I) motif into CD3 and replacement of the LL-3 residue from valine to lysine did not restore TCR internalization, inferring that additional CD3-specific residues are required. These attributes confer significant rigidity on the use of the CD3 (D/E)xxxL(L/I) signal for TCR internalization.
Most of the transmembrane proteins studied to date use a single tyrosine- or dileucine-based signal to mediate internalization (3). Indeed, previous studies with TAC/IL-2R:CD3 chimeras had suggested that the dileucine- and tyrosine-based motifs act independently to mediate endocytosis (11). However, our data clearly show that both signals are required for AP-2-mediated internalization of the intact, cell surface TCR:CD3 complex. Why is this necessary, and how might the contrasting features of these signals, detailed above, assist in this process? One possibility is that the large size of the TCR complex necessitates the use of two distinct signals. Indeed, it has been shown that their binding sites do not overlap, inferring that AP-2 could bind both simultaneously (13). It seems unlikely that the requirement for both signals is due to their weak affinity per se, because comparable AP-2μ interaction was observed with the cytoplasmic tails of CD3 and CTLA-4, which is known to mediate rapid internalization. Furthermore, we have seen no additive effect of combining the CD3.LL-AA and CD3.Y-F mutants. It is also possible that with such a large complex, there is some steric hindrance in the ability of AP-2 to effectively associate with these signals. Thus, it is possible that binding to one signal induces a conformational change that facilitates interaction with the other.
If there is rigidity in AP-2 interaction with the CD3 (D/E)xxxL(L/I) signal, how might plasticity in interaction with Yxx? signals be accommodated? The length and flexibility of the large subunit hinge coupled with conformational changes that occur after AP-2 phosphorylation imply that μ2 can orientate in any direction with respect to the rest of the AP-2 complex through its flexible linker, perhaps facilitating binding to different Yxx? signals (44, 45, 46). Our data suggested that the position of the CD3 (D/E)xxxL(L/I) signal in relation to the rest of the TCR:CD3 complex is critical. We were intrigued to find that transposition of the CD3 (D/E)xxxL(L/I) motif into CD3 resulted in a 30% reduction in TCR internalization in the presence of wild-type CD3. These data infer that this mutant may act as a dominant negative, facilitating AP-2 association, but preventing TCR internalization. This suggests that the orientation of AP-2 docking with the TCR:CD3 complex may be important. Whether this relates to the position of preferred Yxx? signals (e.g., in CD3 rather than CD3) or some other factor remains to be determined. We also cannot rule out the possibility that other, as yet undefined, motifs in the TCR:CD3 complex may be contributing to this process.
In conclusion, we have shown that AP-2-mediated internalization of the TCR:CD3 complex requires both Yxx? and (D/E)xxxL(L/I) signals, a requirement not normally afforded to simple transmembrane receptors. Of particular note was our finding that a highly conserved lysine residue in CD3 was essential for TCR internalization. Although this residue was crucial for TCR internalization, it was not found in all dileucine signals (80% of dileucine signals had D/E at LL-4, whereas 45% have K/R at LL-3) (3), suggesting that its use may be restricted to certain types of receptors. The identification of these signals will allow us to manipulate TCR internalization and determine the physiological importance of this process in T cell function, serial ligation, and TCR down-modulation (24, 26). Furthermore, there appears to be both plasticity and rigidity in the use of the Yxx? and (D/E)xxxL(L/I) signals, respectively. This may highlight the difficulties associated with moving large multichain complexes in excess of 200,000 Da. In this instance, the AP-2 complex would need to effectively mediate internalization and thus would require multiple, but distinct, motifs while retaining the flexibility to internalize a variety of large cell surface complexes. It remains to be seen whether these observations form a general principle by which AP-2 and perhaps other adaptor proteins mediate the transport of large, multichain transmembrane complexes.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We are very grateful to David Baltimore, Elio Vanin, Juan Bonifacino, and Steve Elledge for the gifts of reagents. We also thank Kate Vignali and Haiyan Liu for technical assistance, Richard Bram for assistance and advice with the establishment of the yeast two-hybrid system, Richard Cross and Jennifer Hoffrage for assistance with flow cytometry, the staff of Hartwell Center for oligo synthesis and DNA sequencing, and members of the Vignali laboratory for constructive criticisms and comments.
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 Grants AI39480 and AI52199, Cancer Center Support CORE Grant CA21765, and the American Lebanese Syrian Associated Charities.
2 Address correspondence and reprint requests to Dr. Dario A. A. Vignali, Department of Immunology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105-2794. E-mail address: dario.vignali@stjude.org
3 Abbreviations used in this paper: GAL, galactosidase; BFA, brefeldin A.
Received for publication August 23, 2004. Accepted for publication January 11, 2005.
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Many cell surface proteins are internalized via dileucine- or tyrosine-based motifs within their cytoplasmic domains by the heterotetrameric adaptor protein complex, AP-2. In this study we have examined how AP-2 mediates internalization of large cell surface receptors, such as the eight-chain TCR:CD3 complex. Although most receptors have a single signal that drives internalization, the TCR complex has two (D/E)xxxL(L/I) motifs and 20 Yxx? motifs. Using 293T cells, we show that AP-2 is completely dependent on both signals to mediate TCR internalization, because deletion of either completely blocks this process. Significant plasticity and redundancy were observed in the use of the Yxx? motifs, with a clear hierarchy in their use (CD3 > CD3 CD3 >> CD3). Remarkably, a single, membrane-distal Yxx? motif in CD3 could mediate 75% of receptor internalization, whereas its removal only reduced internalization by 20%. In contrast, significant rigidity was observed in use of the (D/E)xxxL(L/I) motif in CD3. This was due to an absolute requirement for the position of this signal in the context of the TCR complex and for a highly conserved lysine residue, K128, which is not present in CD3. These contrasting requirements suggest a general principle by which AP-2 may mediate the internalization of large, multichain complexes.
Introduction
Many cell surface receptors are constitutively internalized via clathrin-coated pits and subsequently recycled back to the cell surface (1, 2). This process is mediated by the heterotetrameric clathrin-associated adaptor protein complex AP-2, which recognizes short amino acid sequences within the cytoplasmic tails of these receptors. These primarily consist of either tyrosine-based signals, Yxx? (x, any amino acid; ?, a hydrophobic amino acid) or dileucine-based signals, (D/E)xxxL(L/I) (3). The Yxx? signal is recognized by the medium subunit (μ2) of the AP-2 complex and mediates the rapid internalization of a wide variety of molecules, such as the transferrin receptor (CD71) and CTLA-4 (CD152) (4, 5, 6, 7). The (D/E)xxxL(L/I) signal, which has been shown to mediate the internalization of CD4 and CD3, is known to interact with AP-2, but does not compete with tyrosine-based signals (8, 9, 10, 11, 12, 13). Although the exact binding site remains to be resolved, it has been suggested that (D/E)xxxL(L/I) signals bind to the μ2-chain and/or the trunk domain of the 2 subunit (14, 15, 16, 17, 18, 19).
The TCR is expressed as a large, multisubunit complex composed of at least eight polypeptide chains (TCR and CD3, -, and -) (20, 21, 22). In resting cells, the TCR complex is constitutively internalized and recycled via motifs in the cytoplasmic domains of the CD3 molecules (23, 24, 25). Although the physiological importance of this process is unclear, it may play a role in receptor down-modulation, serial ligation, and/or control of surface expression (24, 26). Several studies have shown that AP-2 can bind to the TCR complex (8, 27), and that the CD3 dileucine-based signal can mediate internalization (8, 28, 29). However, it is not clear whether all signals that mediate TCR internalization have been defined, because it has been suggested that tyrosine-based signals in the CD3 chains may contribute (24).
The cytoplasmic tails of the CD3 chains contain ITAMs ((D/E)X2YX2(L/I)X7YX2(L/I)) that are phosphorylated upon TCR ligation and mediate downstream signal transduction and T cell activation (30, 31, 32). Within those ITAMs there are at least 20 Yxx? motifs that could act as signals for AP-2 binding, based on the currently accepted model of the TCR:CD3 complex (22, 33). Given that a single Yxx? motif in CTLA-4 mediates its rapid internalization, largely preventing stable cell surface expression, it is paradoxical that the TCR complex remains stably expressed on the cell surface.
Because most studies have analyzed the interaction between AP-2 and single-chain transmembrane proteins or chimeras containing isolated cytoplasmic domains, it is unclear how AP-2 mediates the internalization of large, multichain complexes that contain many potential internalization motifs. For instance, is there cooperative or selective use of multiple or individual signals? Tackling this problem has been hampered by the lack of mutant cell lines for each or all of the CD3 chains. We recently showed that cell surface TCR:CD3 complex expression could be obtained by transient transfection of 293T cells (24). Using this system, we have shown that the TCR is internalized and recycled in a manner comparable to that in normal T cells, and that this process is mediated by signals in the CD3 chains. This provided a useful system to decipher the mechanism of TCR internalization, because many of the signals and proteins involved in this process are evolutionarily conserved (3). In this study we have addressed the following questions. 1) Which CD3 Yxx? motifs can bind to AP-2μ? 2) Which tyrosine- and dileucine-based motifs can mediate TCR internalization, and what is their relative contribution to this process? 3) Is a single motif adequate for TCR internalization, as suggested by experiments using single-chain CD3 chimeric molecules, or is there cooperative use of multiple motifs?
Materials and Methods
Plasmids
Mutant and wild-type TCR:CD3 chains were generated by recombinant PCR and cloned in the murine stem cell virus-based vector, MSCV.IRES.GFP, as previously described (34, 35, 36). Details of primers and cloning strategy can be obtained from A. Szymczak (andrea.szymczak@stjude.org).
Yeast two-hybrid
CD3:AP-2μ interaction was assessed using a galactosidase-4 (GAL4)3-based yeast two-hybrid system (provided by S. Elledge, Baylor College of Medicine, Houston, TX). The entire cytoplasmic domains of wild-type or mutant CD3 chains were generated by PCR and cloned into pAS2, which confers leucine auxotrophy. AP-2μ in pACT, which confers tryptophan auxotrophy, was provided by J. Bonifacino (National Institutes of Health, Bethesda, MD). The day before transformation, 400 ml of YPAD was inoculated with 40 μl of an overnight Y153 yeast stock (MATa GAL4 GAL180 His3 Trp1–901 Ura3–52 Leu2–3,-112 + URA3::GALlacZ, Lys2::GALHIS3) and grown at 30°C to 1 x 107 cells/ml (OD600, 0.4). Cells were washed in HPLC grade H2O and resuspended in 10 ml of 100 mM lithium acetate in Tris-EDTA (TE) (pH 7.5). Competent yeast were transformed with 1 μg of pACT and 1 μg of pAS2 vectors with 100 μg of denatured salmon sperm DNA (Sigma-Aldrich), 30% polyethylene glycol 4000, and 100 mM lithium acetate/TE. Yeast were shaken for 30 min at 30°C and heat-shocked for 15 min at 42°C. Yeast were spun, resuspended in TE, and plated out on simple defined dropout plates, SD-Trp-Leu and SD-Trp-Leu-His + 3AT (Bio 101). All yeast grew on the former. Interaction was determined by growth on the latter (histidine auxotrophy) and -GAL activity, as determined by a lacZ assay. Liquid cultures were set up by inoculation of SD-Trp-Leu broth and were grown at 30°C overnight to 1 x 107 cells/ml in SD-Trp-Leu broth. Four hundred microliters of culture was placed onto nitrocellulose filters using dot-blot apparatus (Bio-Rad), snap-frozen in liquid N2, placed on 3M filter paper saturated in Z buffer (60 mM Na2HPO4·7H2O, 40 mM NaH2PO4·H2O, 10 mM KCl, 1 mM MgSO4·7H2O, and 50 mM 2-ME, pH 7.0) containing 1 mg/ml X-GAL (Sigma-Aldrich), and incubated overnight at 30°C.
Transient transfection of 293T and TCR internalization assay
Transfections and internalization assays were performed as described with some modification (24). 293T cells (human embryonic kidney cells; provided by D. Baltimore, Caltech, and E. Vanin, St. Jude Children’s Research Hospital, Memphis, TN) were incubated in 10-cm plates at 2 x 106/plate overnight at 37°C. DMEM (470 μl) was mixed with 30 μl of FuGene 6 transfection reagent (Roche) for 5 min at room temperature. Each group of transfectants received 2A peptide-linked 3A9.TCR -chains as well as the CD3 chains indicated (36). DNA (2 μg of each chain) was mixed with FuGene/DMEM and incubated for 15 min at room temperature. The mixture was incubated with the cells for 2 days. On day 3, cells were stained with anti-V8.1/2-PE (F23.1) and anti-CD3-allophycocyanin (BD Pharmingen). GFP+/TCR+/CD3+ cells were sorted and cultured in 96-well, flat-bottom plates at 5 x 104/well overnight. Brefeldin A (BFA; Epicentre Technologies) was added at 10 μg/ml for the times indicated. Cells were stained with anti-TCR--CyChrome (H57-597) and analyzed for TCR modulation (FACSCalibur; BD Biosciences). The percent internalization was determined from the median fluorescence values using untreated controls as reference.
Results
AP-2μ specifically interacts with tyrosine residues in CD3 and to a lesser extent CD3
Yeast two-hybrid assays have been successfully used to determine the relative abilities of various Yxx? motifs to bind to the AP-2μ-chain (14). Although AP-2 has been shown to bind to the TCR:CD3 complex (8, 27), it is unclear which Yxx? motifs in CD3 can be bound by AP-2μ (Fig. 1A). Initially, we used a GAL4-based, yeast two-hybrid assay to determine which CD3 cytoplasmic tails can bind directly to AP-2μ. The CTLA-4 cytoplasmic tail, which is known to interact with AP-2μ (4, 27), mediated significant growth on minimal medium lacking histidine and production of -GAL in yeast transformed with the AP-2μ vector, but not an empty vector control (Fig. 1B). In contrast, no interaction was observed with the CD28 cytoplasmic tail, as expected.
FIGURE 1. AP-2μ-mediated internalization of the TCR:CD3 complex is mediated by tyrosine residues in the cytoplasmic tails of the CD3 chains. A, Amino acid sequence of the CD3 cytoplasmic domains. The ITAMs are boxed, and the tyrosine residues (Y) are in bold. The number above the tyrosine residues denotes the tyrosines of the Yxx? motifs detailed in B and Figs. 2 and 3. TM, transmembrane domain; CY, cytoplasmic domain. B, Interaction between AP-2μ and the CD3 cytoplasmic domains was determined in a yeast two-hybrid assay. The entire cytoplasmic domains of CD28, CTLA-4, and wild-type or mutant CD3 were cloned into pAS2, which contains the GAL4 DNA-binding domain. AP-2μ was cloned into pACT, which contains the GAL4 activation domain. Interaction was determined by the production of -GAL using a standard lacZ assay (right) and growth on minimal medium plates that lack histidine (SD-Leu-Trp-His + 3AT; see Materials and Methods; left). All yeast grew comparably on SD-Leu-Trp plates. Y-F, Yxx? Tyr residues mutated to Phe. C and D, 293T cells were transiently transfected with plasmids carrying the 3A9 TCR chains and the wild-type (WT) CD3 chains or CD3 chains with all Yxx? Tyr mutated to Phe. After 2 days, TCR+/CD3+/GFP+ cells were sorted and treated with BFA (10 μg/ml) for the times indicated (C) or for 30 min (D), and TCR internalization was determined. C, A representative experiment is shown. D, The mean ± SE from 14 experiments are shown as a percentage of the CD3.WT transfectants.
Using this assay, AP-2μ was found to specifically interact with the cytoplasmic tail of CD3 and, to a lesser extent, with that of CD3, but not of CD3 or CD3. Importantly, mutation of the two tyrosine residues in CD3 and CD3 to phenylalanine (Y-F) substantially reduced this interaction. A small amount of growth was observed with the CD3.Y1/2-F mutant. This growth does not appear to be due to any interaction with the dileucine motif in CD3, because mutation of these residues did not affect the growth or production of -GAL in this assay (data not shown). Although this could be due to background growth or another as yet undefined motif, it is clear that the CD3-AP-2μ interaction is substantially reduced by these substitutions. Mutation of either Y1 or Y2 completely abrogated the interaction with AP-2μ, suggesting that both are required for the weak association seen with wild-type CD3. In contrast, reduced, but significant, interaction with AP-2μ was observed with single tyrosine mutations in the CD3 cytoplasmic tail, suggesting that either CD3 Yxx? motif alone can mediate interaction with AP-2.
Internalization of TCR is mediated by tyrosine-based signals in CD3 chains
BFA blocks anterograde transport from the endoplasmic reticulum to the Golgi complex and causes tubulation and fusion of early endosomes with the trans-Golgi network, essentially blocking recycling of receptors back to the cell surface (37, 38). We have previously exploited this property to study TCR internalization in resting and activated T cells (24). We also showed that TCR:CD3 complexes ectopically expressed on 293T cells were internalized with kinetics comparable to those of normal T cells. This is consistent with the idea that TCR internalization is mediated by a general biological process that uses basic cell machinery (e.g., AP-2, dynamin, clathrin, etc.) that is present in all cells.
Using this system, we first introduced a series of tyrosine to phenylalanine (Y-F) substitutions in the Yxx? motifs of all four CD3 chains to determine whether these tyrosine-based motifs mediated TCR complex internalization. Wild-type and mutant TCR:CD3 complexes were expressed on 293T cells by transient transfection with plasmids expressing all six TCR:CD3 chains, and the extent of TCR internalization was evaluated using BFA. All constructs generated gave TCR surface expression levels comparable to those of wild type, and any small differences were normalized in calculating the percentage of TCR internalization. As we have previously shown, the effect of BFA is not complete, and TCR levels reach a steady state equilibrium after 20–30 min (24, 37, 38) (Fig. 1C). This appears to be remarkably consistent, as shown by the predominantly small error seen in all experiments in this study. As the data clearly show, mutation of all Yxx? tyrosine residues in the CD3 chains (CD3.Y-F) completely abrogated TCR internalization, suggesting that one or more of these residues mediated this process (Fig. 1, C and D). These data confirmed previous suggestions that TCR internalization is mediated by motifs within the CD3 cytoplasmic domains (8, 24, 39). However, we were surprised that mutation of the tyrosine-based motifs was sufficient to completely block internalization, because previous studies with TAC:CD3 chimeras had suggested that the dileucine- and tyrosine-based motifs act independently to mediate endocytosis (11). Thus, even though these CD3.Y-F mutant complexes have an intact CD3 dileucine motif, they cannot be internalized, suggesting that experiments with chimeric molecules may not recapitulate the requirements for intact, multichain complexes.
TCR internalization is mediated by a broad hierarchy of tyrosine-based motifs
Additional analysis of the individual CD3 chains was performed in two ways. First, we expressed TCR complexes with Y-F mutations within the ITAMs of a single CD3 chain (e.g., CD3 mutant, CD3 wild type; Fig. 2A, left). Second, we expressed TCR complexes in which ITAM tyrosine residues of a single CD3 chain remained intact, whereas all remaining Yxx? tyrosine residues in the CD3 complex were mutated (e.g., CD3 wild type and CD3 mutant; Fig. 2A, right). The majority of the constructs mediated TCR surface expression levels comparable to those of wild type (Fig. 3).
FIGURE 2. Multiple CD3 Yxx? signals mediate TCR internalization. 293T cells were transfected and sorted as described in Fig. 1. Cells were treated with BFA for 30 min, and TCR internalization was determined. A, Schematic of the two types of transfectants generated where one of the CD3 chains is mutated (left) or one of the CD3 chains is wild type (right). B–F, The mean ± SE from three to 15 experiments are shown as a percentage of the CD3 wild-type (WT) transfectants. Y-F, Yxx? Tyr residues mutated to Phe. TCR expression levels relative to wild type are depicted in Fig. 3.
FIGURE 3. Surface expression of TCR:CD3 complexes possessing Yxx? mutations. A–D, 293T cells were transfected, sorted, and analyzed as described in Fig. 1. The mean ± SE from three to 15 experiments are shown as a percentage of TCR surface expression of CD3.WT transfectants. WT, wild type.
For TCR containing single CD3 chain mutations, internalization appeared to be unaffected when tyrosine residues in CD3, CD3, or CD3 were mutated (Fig. 2B). However, an 50% reduction in TCR internalization was seen after mutation of the CD3 Yxx? tyrosine residues. This suggests the CD3 Yxx? signals are strongly recognized by the AP-2 internalization machinery, consistent with data from the yeast two-hybrid experiments. However, TCR internalization was not completely abrogated as seen when all CD3 Yxx? tyrosine residues are mutated, indicating some cooperativity between CD3 and the other CD3 chains. To address this issue, TCR complexes were expressed that contained Y-F mutations of all Yxx? tyrosines except for one CD3 chain (Fig. 2A, right). Cells expressing TCR complexes in which only the CD3 tyrosine residues were intact showed internalization levels 60% of wild-type transfectants (Fig. 2C). TCR complexes containing intact tyrosine residues only in CD3 or CD3 mediated 30–40% internalization compared with wild type. Because this represents two CD3 Yxx? signals vs 12 CD3 Yxx? signals, it is conceivable that CD3 contributes more to TCR internalization. In contrast, internalization was completely blocked in cells where only CD3 remained the wild-type chain. These data demonstrate the importance of CD3 tyrosine residues in mediating internalization, a process that appears to involve cooperativity among CD3- and CD3- and/or CD3-chains, but not CD3.
Next we assessed the relative contributions of the individual Yxx? signals in the CD3, CD3, and CD3 cytoplasmic domains. Although mutation of the CD3 ITAM tyrosine residues in the presence of an otherwise intact TCR:CD3 complex had little effect on TCR internalization, the CD3 Yxx? signals alone could still mediate 40% TCR internalization (Fig. 2, B and C). Analysis of TCR complexes that contained only one intact CD3 Yxx? signal (and thus only two per complex) mediated only weak internalization (Fig. 2F). Although there were no substantial differences among the six CD3 Yxx? signals, it did appear that CD3.Y3, -Y4, and -Y6 may have been marginally more efficient. As shown previously, significant TCR internalization was observed when only the CD3 Yxx? signals were intact, even though they were largely dispensable in the presence of wild-type CD3 (Fig. 2, B and C). This level of TCR internalization was lost if only CD3.Y2 was intact and was substantially reduced if only CD3.Y1 was intact (Fig. 2E). Clearly, these tyrosines are dispensable, because mutation of either Yxx? signal in the presence of wild-type CD3 had no effect on TCR internalization (Fig. 2D). Somewhat similar observations were made with CD3.Y1, where modest internalization was observed when it was the only intact Yxx? signal (Fig. 2E). In contrast, the membrane-distal Yxx? signal in CD3 (Y2) was clearly the most important contributor to TCR internalization. Indeed, this single tyrosine-based signal could mediate 75% of wild-type TCR internalization (Fig. 2E). Remarkably, its removal only reduced internalization by 20% (Fig. 2D).
These results suggest that there is a hierarchy in the ability of different Yxx? signals to mediate TCR internalization: Y2 >> Y1 Y1 = Y3 = Y4 = Y6 Y1 = Y5 Y2 = Y2 = Y1/2. Although the tyrosine-based signals in CD3, especially Y2, were the most potent mediators of TCR internalization, there was clear redundancy in Yxx? signal use. Taken together, these data suggest that there is significant plasticity in the requirements for AP-2-mediated internalization of the TCR via the tyrosine-based motifs.
The dileucine-based signal in CD3, but not CD3, mediates TCR internalization
It is well established that the dileucine-based motif in CD3 can mediate TCR internalization. The majority of studies have focused on protein kinase C-mediated internalization of the TCR in which the aspartic acid (D127), the two leucines (L131/132), and phosphorylation of a serine residue proximal to the motif (S126) were found to be essential (29, 39). However, less is known about the requirements for constitutive internalization of the TCR in resting cells. Recently, it was found that mutation of the human CD3 dileucine residues completely abolished TCR internalization in an S126 phosphorylation-independent manner (28). It is not known whether the CD3 dileucine-based signal participates in constitutive TCR internalization, although there are some data from experiments with Tac:CD3 chimeras that it is less effective at mediating lysosomal targeting than its CD3 counterpart (11). In these studies we wanted to assess the roles of the CD3 and CD3 dileucine-based signals, in the context of the whole TCR:CD3 complex, in mediating TCR internalization.
Cells were transfected with wild-type or dileucine mutant (LL-AA) CD3- and CD3-chains in the presence of the remaining wild-type TCR:CD3 chains (Fig. 4A). Mutation of the CD3 dileucine residues significantly reduced TCR internalization (Fig. 4, B and C). In contrast, TCR internalization was not affected by mutation of the CD3 dileucine residues. Interestingly, coexpression of the CD3.LL-AA and CD3.Y1/2-F mutants did not further reduce TCR internalization, suggesting that the defects incurred by these mutations were not additive, but cooperative (Fig. 4B). Collectively, these data raised two interesting issues. First, as mutation of the CD3 dileucine residues did not abrogate internalization, were there other proximal residues that contributed to its function? Second, why is the CD3 (D/E)xxxL(L/I) motif unable to mediate TCR internalization?
FIGURE 4. The dileucine residues in CD3, but not CD3, are required for optimal TCR internalization. A, Cytoplasmic domain amino acid sequence of the CD3 and CD3 constructs analyzed. TM, transmembrane domain; CY, cytoplasmic domain. ITAM is boxed. B and C, 293T cells were transfected and sorted as described in Fig. 1. Cells were treated with BFA for 30 min (B) or for the times indicated (C), and TCR internalization was determined. B, The mean ± SE from seven experiments are shown as a percentage of the CD3.WT transfectants. C, A representative experiment is shown. LL-AA, Leu residues (L131/132 in CD3 and L142/143 in CD3) mutated to Ala. TCR expression levels relative to wild type (WT) are depicted in Fig. 6. D, Sequence alignment of the dileucine region of CD3 and CD3. TM, transmembrane domain; CY, cytoplasmic domain; h, hydrophobic residues. Residues that differ between CD3 and CD3 are in bold; the dileucine-based signal used by CD3 is boxed; nonconserved residues are indicated by a dot; acidic residues are indicated by a dash.
Key parameters for TCR internalization by the CD3 dileucine-based signal: position and K128
It was particularly surprising that the CD3 dileucine-based motif did not appear to mediate TCR internalization even though the dileucine and negatively charged residues in the (D/E)xxxL(L/I) motif were present (CD3, DKQTLL; CD3, EVQALL). This inferred that there were other parameters that determined the ability of the CD3 dileucine-based motif to mediate TCR internalization. Alignment of the CD3 and CD3 sequences revealed 11 amino acid residues between the transmembrane domain and the ITAM motif that differ between the two chains (Fig. 4D). Initially, we generated mutants in which only these nonconserved residues were swapped between the two proteins (Fig. 5A, CD3-LL- and CD3-LL-). This was preferred over exchanging the entire cytoplasmic tails to avoid altering the contribution of the tyrosine-based signals. TCR containing CD3-LL- instead of CD3.WT, which essentially contained two CD3 dileucine-based motifs (one in the wild-type CD3-chain and one placed in the context of CD3), were completely refractory to internalization (Fig. 5B). This was surprising, because mutation of the dileucine residues had only reduced TCR internalization by 60% (Fig. 4, B and C).
FIGURE 5. Internalization of the TCR via the CD3 dileucine signal is highly dependent on surrounding amino acid residues. A, Cytoplasmic domain amino acid sequence of the CD3 and CD3 constructs analyzed. TM, transmembrane domain; CY, cytoplasmic domain. ITAM is boxed. B–E, 293T cells were transfected and sorted as described in Fig. 1. Cells were treated with BFA for 30 min, and TCR internalization was determined. The mean ± SE from three to seven experiments are shown as a percentage of the CD3.WT transfectants. TCR expression levels relative to wild type (WT) are depicted in Fig. 6.
Do TCR containing two CD3 dileucine-based motifs, where CD3.WT has been replaced with CD3-LL-, exhibit enhanced internalization? Interestingly, the data infer the opposite, with a 30% reduction in TCR internalization observed in the presence of CD3-LL- (Fig. 5B, third bar). TCR internalization was prevented in this complex when the wild-type CD3 was replaced with the CD3.LL-AA mutant, suggesting that the internalization seen with CD3.WT/CD3-LL- was mediated by the dileucine signal in CD3, rather than the CD3 dileucine motif inserted into CD3 (data not shown). Likewise, when both regional mutants were expressed instead of the wild-type CD3- and CD3-chains (Fig. 5B, fourth bar), essentially transposing the CD3 dileucine-based signal from CD3 to CD3, minimal TCR internalization was observed.
Collectively, these data suggest the following. First, additional residues surrounding the two leucines in CD3, which are not present in CD3, are necessary for TCR internalization. Second, the position of the active dileucine-based signal relative to the rest of the TCR:CD3 complex is important. Third, placement of this signal in the wrong location appears to reduce internalization, suggesting that it may act as a dominant negative by encouraging AP-2 to engage the TCR in a less productive orientation.
Additional mutants were generated to determine which CD3-specific residue(s) was responsible for the efficacy of the CD3 dileucine-based signal (Fig. 5A). Substitution of CD3 residues S126/K128/T130 with the equivalent residues in CD3 (A137/V139/A141) gave essentially the same results as those observed above (Fig. 5C). Single-point mutations of these CD3 residues suggested that T130 was not required (Fig. 5D). Furthermore, mutation of the S126 residue, which is phosphorylated after phorbol ester treatment, has little effect on TCR internalization, consistent with recent findings using CD3-deficient Jurkat T cells (28). Remarkably, mutation of a single lysine residue (K128) completely prevented TCR internalization (Fig. 5D). Conservative substitution of the lysine with arginine reduced internalization by 30%, inferring preference for lysine and an absolute requirement for a positively charged amino acid. This was a surprising finding, because this residue had not previously been shown to be required for AP-2 binding and TCR internalization. Previous studies had shown a strong requirement for a negatively charged residue (D or E) four amino acids N-terminal to the dileucine residues (8). This was confirmed by the complete abrogation of TCR internalization by mutation of D127 (Fig. 5D). It is unclear whether D127 and/or K128 are directly or indirectly involved in AP-2 binding. However, it is clear that the lysine residue is an essential part of the CD3 dileucine-based internalization signal. Furthermore, it is clearly essential, because the K128 residue is not present in CD3, whereas the negatively charged residue (D/E) at 127 is present in both CD3 and CD3.
Interestingly, all but one of the constructs (CD3.T130A) described in this study and above gave TCR surface expression levels comparable to or above wild-type TCR levels and were inversely proportional to their effects on internalization (i.e., if TCR internalization was reduced, expression was increased; Fig. 6). For reasons that are not clear, this correlation was only observed with dileucine-based signal mutants.
FIGURE 6. Surface expression of TCR:CD3 complexes possessing DKxxxLL mutations. A–D, 293T cells were transfected, sorted, and analyzed as described in Figs. 4 and 5. The mean ± SE from three to seven experiments are shown as a percentage of TCR surface expression of CD3.WT transfectants. WT, wild type.
Lastly, we questioned whether this lysine residue was responsible for the inability of CD3-LL- to replace CD3.WT in mediating TCR internalization. Although mutation of K128 in CD3 completely prevented TCR internalization, the lack of this lysine residue in CD3 is not sufficient, as demonstrated with TCR expressing CD3-LLV128K- (Fig. 5E). This suggests that additional residues and/or structural constraints are required. Taken together, these data suggest that there is significant rigidity in the requirements for AP-2-mediated internalization of the TCR via the dileucine-based motif.
Discussion
The TCR:CD3 complex has 20 Yxx? motifs and two (D/E)xxxL(L/I) motifs. In this study we have defined which motifs constitute signals that are used by AP-2 to mediate TCR internalization. In so doing, we have also examined the general principle that AP-2 may use to mediate the internalization of large, multichain transmembrane complexes. Contrary to previous expectations (11), our studies have clearly shown that AP-2 requires both tyrosine- and dileucine-based signals to mediate TCR internalization and revealed both plasticity and rigidity in the engagement of these signals. It should be noted that these experiments were performed in 293T cells, and it remains to be determined whether the same requirements for TCR internalization will apply to lymphoid cells. However, we have previously shown that the TCR is internalized and recycled in a manner comparable to that in normal T cells (24), and others have shown that TCR internalization is mediated by a general biological process that uses basic cell machinery (i.e., AP-2, clathrin, etc.) that is present in all cells (14).
There appears to be significant plasticity in the use of Yxx? motifs by AP-2, which is consistent with the broad specificity of AP-2μ (3). The Yxx? sequences within CD3, CD3, and, in particular, CD3, appear to mediate TCR internalization. Generally, there was a good correlation between data obtained from the yeast two-hybrid AP-2μ binding assay and the 293T TCR internalization assay. In both instances, the CD3 Yxx? signals were clearly the most efficient. The only difference was our observation that CD3 tyrosines alone could mediate some TCR internalization in 293T cells, but we could not detect any binding to AP-2μ. This could be due to an affinity that is below the threshold of this assay. Our use of the entire CD3 cytoplasmic domains, rather than tandemerized repeats of the Yxx? motifs, may also yield different results (14). Alternatively, this difference could simply be due to the duplication of CD3 in the TCR complex.
Our studies suggest that there is a definable hierarchy in the ability of different Yxx? signals to mediate TCR internalization: Y2 >> Y1 Y1 Y1–6 Y2 = Y1/2. The proposed binding motif for AP-2μ suggests a strong preference for arginine (R) or proline (P) at Y + 2 and a preference for leucine (L) rather than isoleucine (I) at Y + 3 (Fig. 7) (40). This is consistent with Y2, Y1, and Y1 being particularly favored and may explain why Y1 is not used.
FIGURE 7. Alignment of the CD3 Yxx? signals and their relationship to the AP-2μ motif (40 ).
Inherent in this plasticity was marked redundancy in the use of different Yxx? signals. Remarkably, 75% of wild-type TCR internalization was mediated by a single tyrosine residue in CD3 of the 20 in the TCR:CD3 complex. However, mutation of this residue only slightly reduces TCR internalization. This infers that AP-2 only requires a single, optimal Yxx? signal to efficiently mediate TCR internalization, but can use several Yxx? signals in the TCR:CD3 complex.
In stark contrast, use of the dileucine-based motif exhibited remarkable rigidity. Even though both CD3 and CD3 have (D/E)xxxL(L/I) motifs, TCR internalization is mediated by the former, but not the latter. AP-2 has been shown to bind to a variety of sequences, including some that do not entirely conform to this motif (3). Thus, it is particularly surprising that for TCR internalization via this motif, AP-2 should exhibit such rigidity. The majority of data concerning this motif has come from its requirement for protein kinase C-mediated TCR internalization (8, 29, 39, 41, 42). In this instance, phosphorylation of the unique CD3 S126 defines this specificity. However, for constitutive TCR internalization, we and others have clearly shown that this residue is not required (28). Our study clearly showed that the aspartic acid (D127) and the two leucine residues (L131/L132) in CD3 were required for TCR internalization, consistent with previous studies (8, 43).
So what is the basis for use of the (D/E)xxxL(L/I) motif in CD3, but not in CD3? Our studies have shown that this specificity is manifest at two levels. First, the position of this motif within the complex appears to be critical, because the transposition of the CD3 (D/E)xxxL(L/I) motif into CD3, in the presence of an inactive CD3 mutant, abrogated TCR internalization. Second, there is an absolute dependency on the highly conserved, CD3-specific lysine at LL-3 (K128). We have shown that a single mutation of either K128 or D127 completely abrogates TCR internalization. However, the latter can be efficiently substituted for by glutamic acid, which is the residue in the analogous position in CD3 (8). These data infer that AP-2 either directly binds to K128 or binds to D127, but only in the presence of K128. Despite the importance of both signal position and the residue K128, these factors alone are insufficient to mediate TCR internalization via this dileucine-based signal. Transposition of the CD3 (D/E)xxxL(L/I) motif into CD3 and replacement of the LL-3 residue from valine to lysine did not restore TCR internalization, inferring that additional CD3-specific residues are required. These attributes confer significant rigidity on the use of the CD3 (D/E)xxxL(L/I) signal for TCR internalization.
Most of the transmembrane proteins studied to date use a single tyrosine- or dileucine-based signal to mediate internalization (3). Indeed, previous studies with TAC/IL-2R:CD3 chimeras had suggested that the dileucine- and tyrosine-based motifs act independently to mediate endocytosis (11). However, our data clearly show that both signals are required for AP-2-mediated internalization of the intact, cell surface TCR:CD3 complex. Why is this necessary, and how might the contrasting features of these signals, detailed above, assist in this process? One possibility is that the large size of the TCR complex necessitates the use of two distinct signals. Indeed, it has been shown that their binding sites do not overlap, inferring that AP-2 could bind both simultaneously (13). It seems unlikely that the requirement for both signals is due to their weak affinity per se, because comparable AP-2μ interaction was observed with the cytoplasmic tails of CD3 and CTLA-4, which is known to mediate rapid internalization. Furthermore, we have seen no additive effect of combining the CD3.LL-AA and CD3.Y-F mutants. It is also possible that with such a large complex, there is some steric hindrance in the ability of AP-2 to effectively associate with these signals. Thus, it is possible that binding to one signal induces a conformational change that facilitates interaction with the other.
If there is rigidity in AP-2 interaction with the CD3 (D/E)xxxL(L/I) signal, how might plasticity in interaction with Yxx? signals be accommodated? The length and flexibility of the large subunit hinge coupled with conformational changes that occur after AP-2 phosphorylation imply that μ2 can orientate in any direction with respect to the rest of the AP-2 complex through its flexible linker, perhaps facilitating binding to different Yxx? signals (44, 45, 46). Our data suggested that the position of the CD3 (D/E)xxxL(L/I) signal in relation to the rest of the TCR:CD3 complex is critical. We were intrigued to find that transposition of the CD3 (D/E)xxxL(L/I) motif into CD3 resulted in a 30% reduction in TCR internalization in the presence of wild-type CD3. These data infer that this mutant may act as a dominant negative, facilitating AP-2 association, but preventing TCR internalization. This suggests that the orientation of AP-2 docking with the TCR:CD3 complex may be important. Whether this relates to the position of preferred Yxx? signals (e.g., in CD3 rather than CD3) or some other factor remains to be determined. We also cannot rule out the possibility that other, as yet undefined, motifs in the TCR:CD3 complex may be contributing to this process.
In conclusion, we have shown that AP-2-mediated internalization of the TCR:CD3 complex requires both Yxx? and (D/E)xxxL(L/I) signals, a requirement not normally afforded to simple transmembrane receptors. Of particular note was our finding that a highly conserved lysine residue in CD3 was essential for TCR internalization. Although this residue was crucial for TCR internalization, it was not found in all dileucine signals (80% of dileucine signals had D/E at LL-4, whereas 45% have K/R at LL-3) (3), suggesting that its use may be restricted to certain types of receptors. The identification of these signals will allow us to manipulate TCR internalization and determine the physiological importance of this process in T cell function, serial ligation, and TCR down-modulation (24, 26). Furthermore, there appears to be both plasticity and rigidity in the use of the Yxx? and (D/E)xxxL(L/I) signals, respectively. This may highlight the difficulties associated with moving large multichain complexes in excess of 200,000 Da. In this instance, the AP-2 complex would need to effectively mediate internalization and thus would require multiple, but distinct, motifs while retaining the flexibility to internalize a variety of large cell surface complexes. It remains to be seen whether these observations form a general principle by which AP-2 and perhaps other adaptor proteins mediate the transport of large, multichain transmembrane complexes.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We are very grateful to David Baltimore, Elio Vanin, Juan Bonifacino, and Steve Elledge for the gifts of reagents. We also thank Kate Vignali and Haiyan Liu for technical assistance, Richard Bram for assistance and advice with the establishment of the yeast two-hybrid system, Richard Cross and Jennifer Hoffrage for assistance with flow cytometry, the staff of Hartwell Center for oligo synthesis and DNA sequencing, and members of the Vignali laboratory for constructive criticisms and comments.
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 Grants AI39480 and AI52199, Cancer Center Support CORE Grant CA21765, and the American Lebanese Syrian Associated Charities.
2 Address correspondence and reprint requests to Dr. Dario A. A. Vignali, Department of Immunology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105-2794. E-mail address: dario.vignali@stjude.org
3 Abbreviations used in this paper: GAL, galactosidase; BFA, brefeldin A.
Received for publication August 23, 2004. Accepted for publication January 11, 2005.
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