The CD3 gamma leucine-based receptor-sorting motif is required for efficient ligand-mediated TCR down-regulation

TCR down-regulation plays an important role in modulating T cell responses both during T cell development and in mature T cells. At least two distinct pathways exist for down-regulation of the TCR. One pathway is activated following TCR ligation and is dependent on tyrosine phosphorylation. The other pathway is dependent on protein kinase C (PKC)-mediated activation of the CD3 gamma di-leucine-based receptor-sorting motif. Previous studies have failed to demonstrate a connection between ligand- and PKC-induced TCR down-regulation. Thus, although an apparent paradox, the dogma has been that ligand- and PKC-induced TCR down-regulations are not interrelated. By analyses of a newly developed CD3 gamma-negative T cell variant, freshly isolated and PHA-activated PBMC, and a mouse T cell line, we challenged this dogma and demonstrate in this work that PKC activation and the CD3 gamma di-leucine-based motif are indeed required for efficient ligand-induced TCR down-regulation.     

TCR dynamics in human mature T lymphocytes lacking CD3 gamma

The contribution of CD3gamma to the surface expression, internalization, and intracellular trafficking of the TCR/CD3 complex (TCR) has not been completely defined. However, CD3gamma is believed to be crucial for constitutive as well as for phorbol ester-induced internalization. We have explored TCR dynamics in resting and stimulated mature T lymphocytes derived from two unrelated human congenital CD3gamma-deficient (gamma(-)) individuals. In contrast to gamma(-) mutants of the human T cell line Jurkat, which were selected for their lack of membrane TCR and are therefore constitutively surface TCR negative, these natural gamma(-) T cells constitutively expressed surface TCR, mainly through biosynthesis of new chains other than CD3gamma. However, surface (but not intracellular) TCR expression in these cells was less than wild-type cells, and normal surface expression was clearly CD3gamma-dependent, as it was restored by retroviral transduction of CD3gamma. The reduced surface TCR expression was likely caused by an impaired assembly or membrane transport step during recycling, whereas constitutive internalization and degradation were apparently normal. Ab binding to the mutant TCR, but not phorbol ester treatment, caused its down-modulation from the cell surface, albeit at a slower rate than in normal controls. Kinetic confocal analysis indicated that early ligand-induced endocytosis was impaired. After its complete down-modulation, TCR re-expression was also delayed. The results suggest that CD3gamma contributes to, but is not absolutely required for, the regulation of TCR trafficking in resting and Ag-stimulated mature T lymphocytes. The results also indicate that TCR internalization is regulated differently in each case.     

Selective defect in antigen-induced TCR internalization at the immune synapse of CD8 T cells bearing the ZAP-70(Y292F) mutation

Cbl proteins have been implicated in ligand-induced TCR/CD3 down-modulation, but underlying mechanisms are unclear. We analyzed the effect of mutation of a cbl-binding site on ZAP-70 (ZAP-Y292F) on dynamics, internalization, and degradation of the TCR/CD3 complex in response to distinct stimuli. Naive CD8 T cells expressing the P14 transgenic TCR from ZAP-Y292F mice were selectively affected in TCR/CD3 down-modulation in response to antigenic stimulation, whereas neither anti-CD3 Ab-, and PMA-induced TCR down-modulation, nor constitutive receptor endocytosis/cycling were impaired. We further established that the defect in TCR/CD3 down-modulation in response to Ag was paralleled by an impaired TCR/CD3 internalization and CD3zeta degradation. Analysis of T/APC conjugates revealed that delayed redistribution of TCR at the T/APC contact zone was paralleled by a delay in TCR internalization in the synaptic zone in ZAP-Y292F compared with ZAP-wild-type T cells. Cbl recruitment to the synapse was also retarded in ZAP-Y292F T cells, although F-actin and LFA-1 redistribution was similar for both cell types. This study identifies a step involving ZAP-70/cbl interaction that is critical for rapid internalization of the TCR/CD3 complex at the CD8 T cell/APC synapse.     

Protein kinase C (PKC) alpha and PKC theta are the major PKC isotypes involved in TCR down-regulation

It is well known that protein kinase C (PKC) plays an important role in regulation of TCR cell surface expression levels. However, eight different PKC isotypes are present in T cells, and to date the particular isotype(s) involved in TCR down-regulation remains to be identified. The aim of this study was to identify the PKC isotype(s) involved in TCR down-regulation and to elucidate the mechanism by which they induce TCR down-regulation. To accomplish this, we studied TCR down-regulation in the human T cell line Jurkat, in primary human T cells, or in the mouse T cell line DO11.10 in which we either overexpressed constitutive active or dominant-negative forms of various PKC isotypes. In addition, we studied TCR down-regulation in PKC knockout mice and by using small interfering RNA-mediated knockdown of specific PKC isotypes. We found that PKCalpha and PKCtheta were the only PKC isotypes able to induce significant TCR down-regulation. Both isotypes mediated TCR down-regulation via the TCR recycling pathway that strictly depends on Ser(126) and the di-leucine-based receptor-sorting motif of the CD3gamma chain. Finally, we found that PKCtheta was mainly implicated in down-regulation of directly engaged TCR, whereas PKCalpha was involved in down-regulation of nonengaged TCR.     

CD3 gamma contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor.

Several cell surface receptors including the T cell receptor (TCR) are phosphorylated and down-regulated following activation of protein kinase C (PKC). Among other substrates the activated PKC in T cells phosphorylates the CD3 gamma subunit of the TCR. To investigate the role of CD3 gamma phosphorylation in PKC-mediated TCR down-regulation, point mutated CD3 gamma cDNA was transfected into the CD3 gamma-negative T cell line JGN and CD3 gamma transfectants were analysed. Phosphorylation at S126 but not S123 in the cytoplasmic tail of CD3 gamma was required for PKC-mediated down-regulation of the TCR. Furthermore, analysis of a series of CD3 gamma truncation mutants indicated that in addition to S126 phosphorylation a motif C-terminal of S126 was required for TCR down-regulation. Point mutation analyses confirmed this observation and demonstrated that a membrane-proximal di-leucine motif (L131 and L132) in the cytoplasmic tail of CD3 gamma was required for PKC-mediated TCR down-regulation in addition to phosphorylation at S126. Incubation of T cells in hypertonic medium known to disrupt normal clathrin lattices severely inhibited PKC-mediated TCR down-regulation in non-mutated T cells, indicating that the TCR was down-regulated by endocytosis via clathrin coated pits. Based on the present results and previously published observations on intracellular receptor sorting, a general model for intracellular sorting of receptors containing di-leucine- or tyrosine-based motifs is proposed.     

Regulation of constitutive TCR internalization by the zeta-chain

The ability of a T cell to be activated is critically regulated by the number of TCRs expressed on the plasma membrane. Cell surface TCR expression is influenced by dynamic processes such as synthesis and transport of newly assembled receptors, endocytosis of surface TCR, and recycling to the plasma membrane of internalized receptors. In this study, the internalization of fluorescently labeled anti-TCR Abs was used to analyze constitutive endocytosis of TCRs on T cells, and to investigate the role of the zeta-chain in this process. We found that cell surface TCRs lacking zeta were endocytosed more rapidly than completely assembled receptors, and that reexpression of full-length zeta led to a dose-dependent decrease in the rate of TCR internalization. Rapid TCR internalization was also observed with CD4(+)CD8(+) thymocytes from zeta-deficient mice, whereas TCR internalization on thymocytes from CD3-delta deficient animals was slow, similar to that of wild-type thymocytes. This identifies a specific role for zeta in the regulation of constitutive receptor internalization. Furthermore, chimeric zeta molecules containing non-native intracellular amino acid sequences also led to high levels of TCR expression and reduced TCR cycling. These effects were dependent solely on the length of the intracellular tail, ruling out a role for intracellular zeta-specific interactions with other molecules as a mechanism for regulating TCR internalization. Rather, these findings strongly support a model in which the zeta-chain stabilizes TCR residency on the cell surface, and functions to maintain cell surface receptor expression by sterically blocking internalization sequences in other TCR components.     

Regulation and Function of the CD3γ DxxxLL Motif: A Binding Site for Adaptor Protein-1 and Adaptor Protein-2 in Vitro

Several receptors are downregulated by internalization after ligand binding. Regulation of T cell receptor (TCR) expression is an important step in T cell activation, desensitization, and tolerance induction. One way T cells regulate TCR expression is by phosphorylation/dephosphorylation of the TCR subunit clusters of differentiation (CD)3γ. Thus, phosphorylation of CD3γ serine 126 (S126) causes a downregulation of the TCR. In this study, we have analyzed the CD3γ internalization motif in three different systems in parallel: in the context of the complete multimeric TCR; in monomeric CD4/CD3γ chimeras; and in vitro by binding CD3γ peptides to clathrin-coated vesicle adaptor proteins (APs). We find that the CD3γ D¹²⁷xxxLL^131/132 sequence represents one united motif for binding of both AP-1 and AP-2, and that this motif functions as an active sorting motif in monomeric CD4/ CD3γ molecules independently of S126. An acidic amino acid is required at position 127 and a leucine (L) is required at position 131, whereas the requirements for position 132 are more relaxed. The spacing between aspartic acid 127 (D127) and L131 is crucial for the function of the motif in vivo and for AP binding in vitro. Furthermore, we provide evidence indicating that phosphorylation of CD3γ S126 in the context of the complete TCR induces a conformational change that exposes the DxxxLL sequence for AP binding. Exposure of the DxxxLL motif causes an increase in the TCR internalization rate and we demonstrate that this leads to an impairment of TCR signaling. On the basis of the present results, we propose the existence of at least three different types of L-based receptor sorting motifs.     

Plasticity and rigidity in adaptor protein-2-mediated internalization of the TCR:CD3 complex

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 (CD3delta > CD3gamma >or= CD3zeta > CD3epsilon). Remarkably, a single, membrane-distal Yxx? motif in CD3delta could mediate approximately 75% of receptor internalization, whereas its removal only reduced internalization by approximately 20%. In contrast, significant rigidity was observed in use of the (D/E)xxxL(L/I) motif in CD3gamma. 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 CD3delta. These contrasting requirements suggest a general principle by which AP-2 may mediate the internalization of large, multichain complexes.     

TCR comodulation of nonengaged TCR takes place by a protein kinase C and CD3 gamma di-leucine-based motif-dependent mechanism

One of the earliest events following TCR triggering is TCR down-regulation. However, the mechanisms behind TCR down-regulation are still not fully known. Some studies have suggested that only directly triggered TCR are internalized, whereas others studies have indicated that, in addition to triggered receptors, nonengaged TCR are also internalized (comodulated). In this study, we used transfected T cells expressing two different TCR to analyze whether comodulation took place. We show that TCR triggering by anti-TCR mAb and peptide-MHC complexes clearly induced internalization of nonengaged TCR. By using a panel of mAb against the Ti beta chain, we demonstrate that the comodulation kinetics depended on the affinity of the ligand. Thus, high-affinity mAb (K(D) = 2.3 nM) induced a rapid but reversible comodulation, whereas low-affinity mAb (K(D) = 6200 nM) induced a slower but more permanent type of comodulation. Like internalization of engaged TCR, comodulation was dependent on protein tyrosine kinase activity. Finally, we found that in contrast to internalization of engaged TCR, comodulation was highly dependent on protein kinase C activity and the CD3 gamma di-leucine-based motif. Based on these observations, a physiological role of comodulation is proposed and the plausibility of the TCR serial triggering model is discussed.     

Ceramide-induced TCR up-regulation

The TCR is a constitutively recycling receptor meaning that a constant fraction of TCR from the plasma membrane is transported inside the cell at the same time as a constant fraction of TCR from the intracellular pool is transported to the plasma membrane. TCR recycling is affected by protein kinase C activity. Thus, an increase in protein kinase C activity affects TCR recycling kinetics leading to a new TCR equilibrium with a reduced level of TCR expressed at the T cell surface. Down-regulation of TCR expression compromises T cell activation. Conversely, TCR up-regulation is expected to increase T cell responsiveness. The purpose of this study was to identify and characterize potential pathways for TCR up-regulation. We found that ceramide affected TCR recycling dynamics and induced TCR up-regulation in a concentration- and time-dependent manner. Experiments applying phosphatase inhibitors indicated that ceramide-induced TCR up-regulation was most probably mediated by serine/threonine protein phosphatase 2A. Analyses of T cell variants demonstrated that TCR up-regulation was dependent on the presence of an intact CD3gamma L-based motif and thus acted on TCR engaged in the recycling pathway. Finally, we showed that TCR up-regulation probably plays a physiological role by increasing T cell responsiveness. Thus, by affecting the TCR recycling kinetics, T cells have the potential both to up- and down-regulate TCR expression and thereby adjust T cell responsiveness.     

Constitutive and ligand-induced TCR degradation

Modulation of TCR expression levels is a central event during T cell development and activation, and it probably plays an important role in adjusting T cell responsiveness. Conflicting data have been published on down-regulation and degradation rates of the individual TCR subunits, and several divergent models for TCR down-regulation and degradation have been suggested. The aims of this study were to determine the rate constants for constitutive and ligand-induced TCR degradation and to determine whether the TCR subunits segregate or are processed as an intact unit during TCR down-regulation and degradation. We found that the TCR subunits in nonstimulated Jurkat cells were degraded with rate constants of approximately 0.0011 min(-1), resulting in a half-life of approximately 10.5 h. Triggering of the TCR by anti-TCR Abs resulted in a 3-fold increase in the degradation rate constants to approximately 0.0033 min(-1), resulting in a half-life of approximately 3.5 h. The subunits of the TCR complex were down-regulated from the cell surface and degraded with identical kinetics, and most likely remained associated during the passage throughout the endocytic pathway from the cell surface to the lysosomes. Similar results were obtained in studies of primary human Vbeta8+ T cells stimulated with superantigen. Based on these results, the simplest model for TCR internalization, sorting, and degradation is proposed.     

The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines

The dopamine transporter (DAT) removes dopamine from the extracellular milieu and is potently inhibited by number of psychoactive drugs, including cocaine, amphetamines, and methylphenidate (Ritalin). Multiple lines of evidence demonstrate that protein kinase C (PKC) down-regulates dopamine transport, primarily by redistributing DAT from the plasma membrane to endosomal compartments, although the mechanisms facilitating transporter sequestration are not defined. Here, we demonstrate that DAT constitutively internalizes and recycles in rat pheochromocytoma (PC12) cells. Temperature blockades demonstrated basal internalization and reliance on recycling to maintain DAT cell surface levels. In contrast, recycling blockade with bafilomycin A1 significantly decreased transferrin receptor (TfR) surface expression but had no effect on DAT surface levels, suggesting that DAT and TfR traffic via distinct endosomal mechanisms. Kinetic analyses reveal robust constitutive DAT cycling to and from the plasma membrane, independent of transporter expression levels. In contrast, phorbol ester-mediated PKC activation accelerated DAT endocytosis and attenuated transporter recycling in a manner sensitive to DAT expression levels. These data demonstrate constitutive DAT trafficking and that PKC-mediated DAT sequestration is achieved by a combination of accelerated internalization and reduced recycling. Additionally, the differential sensitivity to expression level exhibited by constitutive and regulated DAT trafficking suggests that these two processes are mediated by independent cellular mechanisms.     

Receptor engagement transiently diverts the T cell receptor heterodimer from a constitutive degradation pathway

In the absence of ligand, the T cell receptor (TCR)/CD3 complex is continuously internalized and recycled to the cell surface, whereas receptor engagement results in its down-regulation. The present study shows that the TCR and CD3 components follow different fates accompanying their constitutive internalization. Although the CD3 moiety is recycled to the cell surface, the TCR heterodimer is degraded and replaced by newly synthesized chains. Since the TCR heterodimer cannot reach the cell membrane on its own, we propose a model in which recycling CD3 is transported along a retrograde pathway to the endoplasmic reticulum, where it associates with newly made TCR. Interestingly, engagement of the TCR.CD3 complex by superantigen resulted not only in the down-regulation of the TCR and CD3 components but also caused a transient stabilization of the TCR heterodimer. This suggests that TCR engagement diverts the TCR heterodimer from a degradation to a recycling pathway. Contrary to CD3, the intracellular fate of the TCR heterodimer is thus regulated, providing a mechanism for rapidly replacing nonfunctional TCR during intrathymic development of T cells.     

Cutting edge: SIV Nef protein utilizes both leucine- and tyrosine-based protein sorting pathways for down-regulation of CD4.

The Nef protein is unique to primate lentiviruses and is closely linked to accelerated pathogenesis in both human and monkey hosts. Nef acts to down-regulate CD4 and MHC class I, two receptors important for immune function. A recent report demonstrated the presence of two tyrosine motifs in SIV Nef that contribute to its ability to down-regulate CD4 and to associate with clathrin adaptors. These tyrosine motifs are not present in HIV-1 Nef, which instead utilizes a leucine-based motif for its down-regulation of CD4. We now report that SIV Nef also contains a conserved leucine-based motif that contributes to CD4 down-regulation, functions to stimulate internalization, and contributes to the association of SIV Nef with clathrin adaptors AP-1 and AP-2. These results demonstrate that SIV Nef differs from HIV-1 Nef by its ability to use two parallel pathways of the protein-sorting machinery based on either tyrosine or leucine motifs.     

Masking of the CD3 gamma di-leucine-based motif by zeta is required for efficient T-cell receptor expression

The T-cell receptor (TCR) is a multimeric receptor composed of the Ti alpha beta heterodimer and the noncovalently associated CD3 gamma delta epsilon and zeta(2) chains. All of the TCR chains are required for efficient cell surface expression of the TCR. Previous studies on chimeric molecules containing the di-leucine-based endocytosis motif of the TCR subunit CD3 gamma have indicated that the zeta chain can mask this motif. In this study, we show that successive truncations of the cytoplasmic tail of zeta led to reduced surface expression levels of completely assembled TCR complexes. The reduced TCR expression levels were caused by an increase in the TCR endocytic rate constant in combination with an unaffected exocytic rate constant. Furthermore, the TCR degradation rate constant was increased in cells with truncated zeta. Introduction of a CD3 gamma chain with a disrupted di-leucine-based endocytosis motif partially restored TCR expression in cells with truncated zeta chains, indicating that the zeta chain masks the endocytosis motif in CD3 gamma and thereby stabilizes TCR cell surface expression.     

Identification and functional characterization of the zeta-chain dimerization motif for TCR surface expression.

We recognized a common dimerization motif between the transmembrane (TM) domain of zeta-chain family members and glycophorin A. We have shown that a glycine within the zeta-dimerization motif is critical for zeta-homodimerization and also for its association with the TCR/CD3 complex. Similarly, two residues within the CD3 delta gamma TM domains have proven to be critical for their interaction with the zeta-homodimer. A three-dimensional homology model of the zeta-chain TM domain highlights potential residues preferentially involved either in the zeta 2-CD3 or zeta 2-TCR alpha beta association, confirming our experimental findings. These results indicate that, for symmetrical reasons, the zeta-homodimer participates in the TCR/CD3 complex assembly by interacting with CD3 gamma delta TM domains, thereby masking their degradation signals located in the cytoplasmic tails.     

Essential role of NF-kappa B-inducing kinase in T cell activation through the TCR/CD3 pathway

NF-kappa B-inducing kinase (NIK) is involved in lymphoid organogenesis in mice through lymphotoxin-beta receptor signaling. To clarify the roles of NIK in T cell activation through TCR/CD3 and costimulation pathways, we have studied the function of T cells from aly mice, a strain with mutant NIK. NIK mutant T cells showed impaired proliferation and IL-2 production in response to anti-CD3 stimulation, and these effects were caused by impaired NF-kappa B activity in both mature and immature T cells; the impaired NF-kappa B activity in mature T cells was also associated with the failure of maintenance of activated NF-kappa B. In contrast, responses to costimulatory signals were largely retained in aly mice, suggesting that NIK is not uniquely coupled to the costimulatory pathways. When NIK mutant T cells were stimulated in the presence of a protein kinase C (PKC) inhibitor, proliferative responses were abrogated more severely than in control mice, suggesting that both NIK and PKC control T cell activation in a cooperative manner. We also demonstrated that NIK and PKC are involved in distinct NF-kappa B activation pathways downstream of TCR/CD3. These results suggest critical roles for NIK in setting the threshold for T cell activation, and partly account for the immunodeficiency in aly mice.     

The CD3 epsilon subunit of the TCR contains endocytosis signals.

Ligand binding to TCR induces its internalization and cell surface down-modulation. These phenomena contribute to the extinction of activation signals. Due to the multicomponent nature of the TCR-CD3 complex, its internalization may be mediated by one or several of its subunits. Although it has been reported that CD3 gamma and CD3 delta contain endocytosis motifs involved in the internalization of the TCR-CD3 complex, other subunits could also be involved in this process. For instance, CD3 epsilon and CD zeta display amino acid sequences reminiscent of internalization motifs. To investigate whether CD3 epsilon bears endocytosis signals, we have analyzed the internalization capacity of a panel of deletion and point mutants of CD3 epsilon that were expressed on the cell surface independently of other TCR-CD3 subunits. Here we report that CD3 epsilon displays endocytosis determinants. These data indicate that CD3 epsilon could contribute to the internalization and cell surface down-regulation of TCR-CD3 complexes. Moreover, the existence of endocytosis signals in this polypeptide could serve to retrieve unassembled CD3 epsilon subunits or partial CD3 complexes from the plasma membrane, thus restricting the expression on the cell surface to fully functional TCR-CD3 complexes.     

Identification of Grb2 as a novel binding partner of the signaling lymphocytic activation molecule-associated protein binding receptor CD229

Ag recognition by the TCR determines the subsequent fate of the T cell and is regulated by the involvement of other cell surface molecules, termed coreceptors. CD229 is a lymphocyte cell surface molecule that belongs to the CD150 family of receptors. Upon tyrosine phosphorylation, CD229 recruits various signaling molecules to the membrane. One of these molecules is the signaling lymphocytic activation molecule-associated protein, of which a deficiency leads to the X-linked lymphoproliferative syndrome. We report that CD229 interacts in a phosphorylation-dependent manner with Grb2. We mapped this interaction showing that the Src homology 2 domain of Grb2 and the tyrosine residue Y606 in CD229 are required for CD229-Grb2 complex formation. The Grb2 motif in the cytoplasmic tail of CD229 is distinct and independent from the two tyrosines required for efficient signaling lymphocytic activation molecule-associated protein recruitment. CD229, but not other members of the CD150 family, directly bound Grb2. We also demonstrate that CD229 precipitates with Grb2 in T lymphocytes after pervanadate treatment, as well as CD229 or TCR ligation. Interestingly, the CD229 mutant lacking the Grb2 binding site is not internalized after CD229 engagement with specific Abs. Moreover, a dominant negative form of Grb2 (containing only Src homology 2 domain) impaired CD229 endocytosis. Unexpectedly, Erk phosphorylation was partially inhibited after activation of CD229 plus CD3. Consistent with this, CD229 ligation partially inhibited TCR signaling in peripheral blood cells and CD229-Jurkat cells transfected with the 3XNFAT-luciferase reporter construct. Altogether, the data suggest a model whereby CD229 ligation attenuates TCR signaling and Grb2 recruitment to CD229 controls its rate of internalization.