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The CD3 Subunit of the TCR Contains Endocytosis Signals¹

Aldo Borroto^*, Juan Lama^*,, Florence Niedergang, Alice Dautry-Varsat, Balbino Alarcón^* and Andrés Alcover^2,

^* Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas-Universidad Autónoma, Madrid, Spain; and Unité de Biologie des Interactions Cellulaires, Institut Pasteur, Paris, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 and CD3 contain endocytosis motifs involved in the internalization of the TCR-CD3 complex, other subunits could also be involved in this process. For instance, CD3 and CD display amino acid sequences reminiscent of internalization motifs. To investigate whether CD3 bears endocytosis signals, we have analyzed the internalization capacity of a panel of deletion and point mutants of CD3 that were expressed on the cell surface independently of other TCR-CD3 subunits. Here we report that CD3 displays endocytosis determinants. These data indicate that CD3 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 subunits or partial CD3 complexes from the plasma membrane, thus restricting the expression on the cell surface to fully functional TCR-CD3 complexes.

	Introduction

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The TCR expressed in the majority of peripheral T lymphocytes is a complex composed of the hypervariable TCR ß heterodimer noncovalently linked to the monomorphic CD3-, -, -, and -chains (TCR-CD3). The - and ß-chains contain Ig-like variable domains responsible for Ag and superantigen recognition (1), whereas the CD3-, -, -, and -chains are involved in signal transduction (2). Cell surface expression of the TCR-CD3 complex requires the assembly of at least three chain pairs, ß, , and or . Unassembled subunits or partial complexes are not expressed on the plasma membrane, because they are retained along the secretion pathway and eventually degraded (3, 4).

Once on the plasma membrane, TCR-CD3 complexes are continuously internalized and recycled back to the cell surface (5, 6). Moreover, soon after TCR ligation by Ag, superantigen, or mAbs, the cell surface expression of TCR-CD3 is down-modulated. Down-modulation results from increased receptor internalization and degradation (5, 7, 8, 9, 10).

Internalization of cell surface receptors requires the presence of particular sequences, mainly located in their cytosolic regions, which mediate the interaction of receptors with molecular components of the cellular endocytic machinery. These sequences belong mainly to two families, which are the tyrosine-based motif and the di-leucine motif. The tyrosine-based motif involves a critical aromatic residue (usually tyrosine) placed in a context of one or more amino acids with large hydrophobic side chains (Yxx), where x represents any amino acid and a hydrophobic residue. Likewise, sequences composed of two leucines, or leucine and another hydrophobic amino acid, are also involved in receptor endocytosis. Both the tyrosine-based motifs and the di-leucine motifs have been reported to mediate the localization of receptors into clathrin-coated pits (11, 12).

Because the TCR-CD3 complex is composed of multiple subunits, its internalization may depend on sequences present in one or several of its subunits. Interestingly, internalization of TCR-CD3 complexes is determined by one or several CD3 chains, and this depends on the kind of stimulus-inducing internalization. For instance, TCR-CD3 internalization induced by phorbol esters requires the presence of a di-leucine-based motif present in the cytosolic region of CD3 (13, 14). However, this motif is not indispensable for TCR-CD3 internalization induced by enterotoxin superantigens or anti-TCR-CD3 mAbs (9, 15, 16). Internalization of TCR-CD3 induced by an anti-CD3 mAb occurs in the absence of the cytosolic region of either CD3 or CD3, but it is blocked when both cytosolic regions are missing (15). However, the absence of both CD3 and CD3 cytosolic regions still allows TCR-CD3 internalization induced by peptide Ag.³ Therefore, under physiologic conditions, TCR-CD3 internalization likely involves determinants present in TCR-CD3 subunits different from CD3 and CD3. Because TCR and -ß display short cytosolic regions, it appears likely that CD3 could bear determinants involved in TCR-CD3 internalization.

To investigate whether CD3 itself displays endocytosis sequences, we expressed CD3 independently of other TCR-CD3 subunits, and we analyzed its capacity to be internalized. We show here that CD3 contains endocytosis determinants. Therefore, CD3 could potentially contribute to the internalization of TCR-CD3 complexes. Moreover, the presence of endocytosis sequences could allow the retrieval of unassembled CD3 chains that could have escaped retention in the endoplasmic reticulum (ER)⁴ and reached the cell surface independently of a fully formed TCR-CD3 complex.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents

Chemicals were from Sigma Chemical (St. Louis, MO). ¹²⁵I was from Amersham-Pharmacia Biotech (Piscataway, NJ). Human transferrin (Sigma Chemical) was loaded with iron and coupled to lissamine rhodamine (Eastman Kodak Co., Rochester, NY) or to fluorescein isothiocyanate (Molecular Probes, Eugene, OR) as previously described (17, 18). The mouse anti-CD3 mAb (SP34, IgG3) has been previously described (19). F(ab')₂ fluorescein-labeled rabbit anti-mouse Ig was from Dako (Glostrup, Denmark), and F(ab')₂ PE-labeled goat anti-mouse Ig Fc was from Immunotech (Marseille, France). The antiserum to rat growth hormone (GH) for immunocytochemistry (lot AFP4115) was obtained from the National Institute of Diabetes and Digestive and Kidney Diseases (National Institutes of Health, Bethesda, MD). The mouse mAb against human CD8 (OKT8, IgG2a) was from Ortho Diagnostic Systems (Raritan, NJ).

DNA constructs

All chimeras and mutations were constructed by PCR using Taq DNA polymerase (New England Biolabs, Beverly, MA) essentially as previously described (20, 21). Human CD8 and CD3 cDNAs were used as templates, with appropriate oligonucleotides designed to introduce the amino acid changes. All of the PCR products were digested with XhoI and BamHI and cloned in the pSR expression vector (22). The constructs were all checked by complete DNA sequencing.

Cell lines and transfections

COS cells were grown in DMEM containing 4 g/L glucose and 10% FCS. COS cells were transfected by electroporation. Routinely, one 100-mm plate of cells grown to subconfluence was used for each transfection. Cells were resuspended in 200 µl of DMEM supplemented with 10% FCS and 10 mM HEPES buffer (pH 7.2) and gently mixed with the DNA transfection mixture (6 µg of pSR-CD3 vector, 17 µg of pSK plasmid used as carrier DNA, and 5 µl of 1.5 M NaCl) and then electroporated in 0.4-cm cuvettes at 200 V, 900 µF, using an EasyjecT apparatus (Eurogentec, Seraing, Belgium). The cells were carefully resuspended by pipetting and plated in a 100-mm petri dish. After 24 h of culture, the cells were transferred to 24-well plates. Rat basophilic leukemia (RBL) cells were grown in RPMI 1640 supplemented with 10% FCS and 10 mM HEPES buffer (pH 7.2). RBL cells were transfected with 20 µg of pSR-CD3 (R183S) by electroporation at 260 V, 900 µF. Stably transfected cells were selected in 1 mg/ml G418 (Life Technologies, Paisley, U.K.). Positive clones were selected by immunofluorescence and flow cytometry using the anti-CD3 mAb, SP34. Jurkat cells lacking TCR-CD3 surface expression (clone 31-13) (23) were grown in RPMI 1640 supplemented with 10% FCS and 10 mM HEPES buffer (pH 7.2). Cells were transfected with 20 µg of pSR-CD8/ by electroporation at 260 V, 900 µF. Stably transfected cells were selected in 2 mg/ml G418 (Life Technologies). Positive clones were selected by immunofluorescence and flow cytometry using an anti-CD8 mAb, OKT8.

Immunofluorescence labeling, flow cytometry, and confocal microscopy

Experiments were conducted essentially as previously described (8). Fluorochrome-labeled transferrin was used at 150 nM.

Internalization of radiolabeled anti-CD3 mAb

Experiments were conducted 48 h after transfection on COS cells growing in 24-well plates. Cells were cooled on ice for 3 min and washed once at 4°C with DMEM supplemented with 10% FCS and 10 mM HEPES buffer (pH 7.2). The medium was thoroughly removed, and 75 µl of the same cold medium containing ¹²⁵I-labeled anti-CD3 mAb (SP34) at a final concentration of 40 nM was added per well. Cells were incubated for 30 min at 4°C to allow binding of the Ab and then at 37°C for the appropriate times to allow internalization. At the end of each time point, cells were washed three times at 4°C with DMEM. The remaining surface-bound Ab was then removed by two successive acid washes (8 min with 800 µl of DMEM containing 25 mM sodium acetate, pH 1.8). At the end, the cells were lysed in 400 µl of 0.1 M NaOH solution and the wells washed with 800 µl of distilled water. Acid treatment removed the surface-bound Ab with an efficiency of 80–95%. Nonspecific internalization was determined using cells incubated in the presence of a 10-fold excess of unlabeled Ab or using the same amount of mock-transfected COS cells. Both techniques gave similar results. Data were corrected taking into account nonspecific internalization values as well as the efficiency of the acid wash, and the percentage of internalized receptors was calculated as previously described (24).

Down-regulation experiments

Cell surface expression of CD3 was measured by immunofluorescence and flow cytometry at various times after binding of soluble or plastic-bound Abs as previously described (25).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD3 is internalized in the absence of the other TCR-CD3 subunits

To analyze the capacity of CD3 to be internalized, this chain was expressed independently of other TCR-CD3 subunits. Isolated CD3 cannot reach the cell surface due to the presence of an ER retention signal in its cytosolic region (20, 26). We therefore expressed a CD3 form containing a mutation in its ER retention signal (R183S). As shown in Fig. 1A, this mutant was stably expressed on the cell surface of RBL cells. Immunoprecipitation experiments showed that CD3 is mainly expressed in these cells as disulfide-linked dimers, with monomeric and trimeric forms also present (data not shown). Disulfide-linked CD3 homodimers have been reported to exist in T lymphocytes, where they coexist with CD3- and CD3- heterodimers (27). Likewise, homodimeric and trimeric forms of CD3 have been observed in COS cells coexpressing several CD3 subunits (28), as well as in in vitro translated and assembled TCR-CD3 complexes (29). This suggests that the folding and self-assembly of CD3 chains in the presence or absence of other TCR-CD3 subunits are similar. Therefore, these polypeptides are suitable for investigating the presence of internalization determinants in CD3.

View larger version (19K): [in this window] [in a new window]   FIGURE 1. CD3 is internalized in the absence of other TCR-CD3 subunits. A, Cell surface expression of transfected RBL cells stably expressing the R183S CD3 mutant was measured by immunofluorescence and flow cytometry using an anti-CD3 mAb (SP34) and PE-coupled secondary Abs. B, RBL cells expressing CD3 were incubated in the presence of anti-CD3 mAb (SP34) and rhodamine-labeled transferrin for 45 min either at 4°C or at 37°C. Cells were then fixed, permeabilized, stained with fluorescein-coupled secondary Abs, and observed under a confocal microscope. A Z series of optical sections was performed at 0.5-µm increments. Measurements of fluorescein and rhodamine emissions were acquired separately. The image shows a medial optical cut of a representative cell. Arrows show representative vesicles displaying colocalization of transferrin and CD3 labelings. Bar = 10 µm. C, RBL cells expressing CD3 were incubated in conic tubes coated with anti-CD3 mAb (SP34) for various times at 37°C. Cell surface expression of CD3 was then measured by immunofluorescence and flow cytometry using saturating concentrations of the anti-CD3 mAb, SP34, and PE-coupled secondary Abs. The figure represents the average values of three independent experiments ± SD.

CD3 contains endocytosis sequences in its cytosolic region

To localize putative endocytosis sequences in the cytosolic tail of CD3, a panel of mutants encompassing the whole cytosolic region was generated (Fig. 2A). All of these mutants were expressed on the cell surface of transfected COS cells at comparable levels (Fig. 2B). The capacity of these mutants to be internalized was then measured using a ¹²⁵I-labeled anti-CD3 mAb. Consistent with the results shown in Fig. 1, three CD3 mutants containing single-residue changes in the ER retention sequence (L180S, R183K, and R183S) were internalized equally well (Fig. 3A). A panel of truncated CD3 molecules was then tested. As shown in Fig. 3B, the deletion of residues 181–185 (1) did not inhibit CD3 internalization, as compared with single-residue mutants described in Fig. 3A. However, a larger deletion involving residues 166–185 (2) significantly reduced the capacity of CD3 to be internalized. This suggests that some sequences contained in the 166–180 region could be involved in CD3 internalization. Interestingly, this region contains two tyrosine-based motifs (Y166EPI and Y177SGL), which could support CD3 internalization (12). These motifs are part of the ITAM of this subunit that is involved in signal transduction.

View larger version (35K): [in this window] [in a new window]   FIGURE 2. Cell surface expression of a panel of CD3 mutants transiently expressed in transfected COS cells. A, Schematic representation of mutations and deletions performed in the cytosolic region of CD3. The amino acid sequence of the entire cytosolic region of CD3 is shown in single-letter code. Point mutations are underlined by stars. Deletions are shown by lines interrupting the sequence. B, Expression of CD3 on the cell surface was measured 48 h after transfection by immunofluorescence and flow cytometry. Staining was performed with anti-CD3 mAb, SP34, and fluorescein-coupled secondary Abs. The figure shows a representative experiment of three independent experiments conducted.

View larger version (13K): [in this window] [in a new window]   FIGURE 3. CD3 is internalized in transfected COS cells. Internalization was measured, as described in Materials and Methods, using 125I-labeled anti-CD3 mAb (SP34) at a final concentration of 40 nM. Nonspecific internalization was subtracted at each time point. It was determined using cells incubated in the presence of a 10-fold excess of unlabeled Ab (i.e., filled symbols in B) or using the same number of mock-transfected COS cells. The figure represents the average of three independent experiments ± SD in A and B and a representative experiment of two independent experiments in C and D. A, Circles, R183S; squares, L180S; diamonds, R183K. B, Squares, 1; circles, 2. C, Circles, 2; squares, 3; diamonds, 4; upright triangles, 5; inverted triangles, 7. D, Circles, R183S; squares, Y166S, Y177S.

Altogether, these data indicate that no other endocytosis sequences were present in the cytosolic tail of CD3, besides the endocytosis signals present between amino acids 166 and 180. These results suggest that CD3 could also contain endocytic signals outside the cytosolic region.

Mutants of CD3 lacking tyrosine residues of the cytosolic region are internalized

The region between residues 166 and 180 contains two consensus tyrosine-based motifs (Y166EPI and Y177SGL) that could support CD3 internalization (12). To investigate whether any of these motifs is actually a tyrosine-based internalization signal for CD3, we replaced both tyrosine residues by serine. These amino acid substitutions were shown to abolish internalization of other receptors containing tyrosine-based endocytic signals (11). As shown in Fig. 3D, the substitution of both tyrosine residues by serine did not inhibit CD3 internalization. These results indicate that the two tyrosine-based sequences of CD3 do not behave as bona fide tyrosine-based internalization signals for this molecule. Nevertheless, the deletion analysis described above strongly suggests that this region of the molecule is involved in its endocytosis.

The endocytosis signals of CD3 can be transferred to another protein

A further proof for the presence of endocytic signals in a receptor can be obtained by analyzing whether the graft of the region containing the putative endocytic sequence confers to another protein the capacity to be endocytosed. Most of the previously described endocytosis motifs have been localized in the cytosolic region of receptors (12), although several reports have shown that transmembrane regions could also support receptor internalization (32, 33, 34, 35, 36, 37). Therefore, we generated two chimeric molecules in which the extracellular region of CD3 was replaced by the rat GH. These chimeras contained either the transmembrane region alone, or both the transmembrane and the cytosolic region of CD3 (Fig. 4A). The capacity of these chimeras to be internalized was then analyzed in transiently transfected COS cells using anti-GH Abs. As shown in Fig. 4B, both chimeras were internalized, as revealed by down-regulation experiments, although the one lacking the cytosolic region of CD3 was less efficiently down-regulated. Moreover, both chimeras were found in intracellular vesicular compartments, as assessed by immunofluorescence and confocal microscopy (Fig. 5). Double labeling microscopy using rhodamine-coupled secondary Abs to detect the GH chimera and fluorescein-coupled transferrin showed colocalization between GH/CD3 chimera and transferrin labeling (Fig. 5), indicating that the chimeras reached the endocytic organelles. This vesicular staining was not observed in cells incubated with irrelevant Abs, implying that the entry of the anti-GH Ab was mediated by the GH/CD3 chimeras and not by fluid-phase endocytosis (data not shown). These data provide additional proof for the presence of endocytic signals in CD3 and indicate that the transmembrane region of CD3 could also contribute to its internalization.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. The endocytosis signals of CD3 can be transferred to another protein. Chimeric molecules composed of the rat GH polypeptide fused either to the transmembrane region only (GH-CD3TM) or to the transmembrane and cytosolic regions of CD3 (GH-CD3 TM + CYT) (A) were transiently expressed in COS cells. The capacity of internalization of these molecules was then assessed by measuring the percentage of surface molecules down-regulated upon incubation with an anti-rat GH Ab (B). The figure shows a representative experiment of three independent experiments conducted. , GH-CD3TM; •, GH-CD3 TM + CYT.

View larger version (17K): [in this window] [in a new window]   FIGURE 5. Chimeric molecules containing the transmembrane region of CD3 reach the endocytic compartment. COS cells expressing the rat GH polypeptide fused to the transmembrane region of CD3 were incubated with anti-GH Ab for 3 h and fluorescein-coupled transferrin for the last 15 min at 37°C. Cells were then fixed, permeabilized, stained with rhodamine-coupled secondary Ab, and observed under a confocal microscope. Fluorescein and rhodamine emissions were acquired separately. The image shows a medial optical cut of a representative cell. Transferrin labeling is shown in green, and GH-CD3 chimeras in red. Areas of colocalization appear yellow in the computer-generated combined image. Note that surrounding untransfected cells display green transferrin labeling without displaying the red labeling corresponding to CD3 chimeras.

The internalization signals of CD3 are functional in T lymphocytes

Because all of the data described above were obtained in non-T cells, it was necessary to verify whether the internalization signals borne by isolated CD3 molecules are also functional in T lymphocytes. To this end, the same cDNA constructs mutated in the ER retention signal described above were transfected in Jurkat T cells lacking TCR-CD3 cell surface expression. However, CD3 constructs carrying the extracellular and transmembrane regions did not reach the cell surface. This is likely due to its association with other CD3 subunits, which are expressed intracellularly in these cells. This clone lacks TCRß expression but expresses intracellularly all of the other subunits of the complex (23). Although the CD3 constructs transfected lack the ER retention signal, association of these mutants with other CD3 subunits in the ER likely leads to the retention of the complexes via the ER retention signals of other subunits (4). Because the assembly into complexes of TCR-CD3 subunits involves mainly the extracellular and transmembrane regions (4, 28), we made a construct in which the extracellular and transmembrane regions of CD3 were substituted by those of CD8. This chimera was readily expressed in TCR-CD3-negative cells. Moreover, it was internalized as efficiently as CD3 molecules expressed in non-T cells, as assessed by down-modulation experiments (Fig. 6), thus indicating that CD3 carries internalization determinants that are functional in T lymphocytes.

View larger version (19K): [in this window] [in a new window]   FIGURE 6. The internalization signals of CD3 are functional in T lymphocytes. A chimeric protein composed of the extracellular and transmembrane regions of CD8 and the intracellular region of CD3 (A) was expressed in Jurkat T cells lacking TCR-CD3 cell surface expression (clone 31-13). The capacity for internalization of this molecule was assessed, as described in Fig. 1, by measuring the percentage of surface molecules down-regulated after binding of an anti-CD8 (OKT8) mAb (B). The figure shows the average value ± SD of four independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Previously reported data indicated that internalization of TCR-CD3 complexes may involve determinants from one or several of its subunits, depending on the kind of stimulus inducing internalization. For instance, phorbol ester-induced TCR-CD3 internalization depends on the presence of a di-leucine-based motif located in the cytosolic region of CD3 (13, 14). In contrast, TCR-CD3 internalization induced by superantigens or mAbs occurs even in the absence of the entire cytosolic region of CD3 (9, 15, 16). Moreover, TCR-CD3 internalization induced by an anti-CD3 mAb can take place in the absence of the cytosolic region of either CD3 or CD3, but it is blocked when both of these cytosolic regions are deleted (15). However, the absence of the cytosolic regions of CD3 and CD3 still allows TCR-CD3 internalization induced by peptide Ag.³ Therefore, under physiologic conditions, TCR-CD3 internalization likely involves determinants present in TCR-CD3 subunits other than CD3 and CD3. The data we present here demonstrate that CD3 itself bears endocytosis determinants and could therefore support the internalization of TCR-CD3 complexes.

Unlike many other receptors that display only one strong internalization signal in their cytosolic regions (11, 12), our data indicate that CD3 internalization is determined by the additive effect of determinants located in the cytosolic and transmembrane regions of the molecule. One of these is located between amino acids 166 and 180 (Fig. 3). This determinant could be formed by the tyrosine-based motifs (YxxI/L) that constitute the ITAM of CD3. However, these sequences do not seem to behave as typical tyrosine-based motifs described for other receptors (11), because the mutations of both tyrosine residues to serine did not alter CD3 internalization. Rather, this sequence seems to behave as a weak internalization signal, such as those found in the IL-2 receptor ß and subunits (35, 37). The tyrosine-based motifs within the ITAM of other receptors have been shown to be involved in internalization. However, their particular features were different depending on the receptor and on the ligand inducing internalization. For instance, tyrosine-based motifs of the ITAMs of CD3 and CD3, together with a di-leucine motif, mediate the internalization of chimeric molecules containing the cytosolic tail of these CD3 chains (38). Moreover, both tyrosine residues of the ITAM of FcRIII were shown to be required for its internalization, because the mutation of each tyrosine residue to valine or serine abolished multimeric receptor internalization (39). In contrast, although one of the tyrosine-based motifs of the ITAM of Ig is required for B cell receptor constitutive internalization, this mutation does not impair the internalization induced by multivalent ligands (40).

Sequential deletions of the CD3 cytosolic tail between the transmembrane region and aa 166 (Fig. 2A, 3–6) did not inhibit the internalization capacity of the 166–185 (2) deletion mutant (Fig. 3C). This suggests that, within the cytosolic tail of CD3, only the region between amino acids 166 and 180 contains endocytosis signals and that the other internalization determinants could be part of the transmembrane region. In support of this, a chimera containing only the transmembrane region of CD3 fused to the rat GH was internalized, although less efficiently than a chimera displaying both the transmembrane and cytosolic regions of CD3 (Figs. 4 and 5). The presence of signals for sorting in the endocytic pathway in both the cytosolic and transmembrane regions has been reported for other membrane proteins (32, 33, 34, 35, 36, 37), although a transmembrane endocytic motif had not been described.

Our data demonstrate that CD3 has the capacity to be internalized, thus suggesting that this subunit could contribute to TCR-CD3 complex internalization. Moreover, considering, on the one hand, that CD3 and CD3 also carry internalization signals (14, 15, 38) and, on the other hand, that CD3 and CD3 are not the only subunits involved in TCR-CD3 internalization,³ it is likely that TCR-CD3 complex internalization utilizes endocytic signals from all CD3 subunits. This would not be surprising, because all CD3 subunits are involved in TCR signal transduction.

Nevertheless, from our analysis, we cannot determine whether all or only some of the CD3 endocytic determinants shown here are involved in the internalization of the TCR-CD3 complex. In fact, some of these determinants may not be accessible to the endocytic machinery when this subunit is part of a complete TCR-CD3 complex. In this case, endocytosis signals exposed only in isolated CD3 subunits could serve to retrieve CD3 subunits or partial CD3 complexes that would have escaped the ER retention barrier and reached the plasma membrane independently of fully assembled TCR-CD3 complexes. In this regard, it is worth noting that only low amounts of CD3 associated with CD3 or CD3 were found expressed on the surface of certain thymomas as well as on immature CD4^-CD8^- thymocytes but not in mature T cells (41, 42, 43). The removal of isolated CD3 molecules from the plasma membrane may represent an additional control mechanism to prevent the expression of unassembled CD3 subunits on the cell surface.

	Acknowledgments

 
We thank Dr. D. Ojcius for critical reading of the manuscript.

	Footnotes

 
¹ This work was supported by Grant PM95-005 from Comisión Interministerial de Ciencia y Tecnología, Grant 08.2/0009/97 from Comunidad de Madrid, and a France-Spain exchange program (Action Integrée) from the Ministry of Foreign Affairs. J.L. is a recipient of a European Molecular Biology Organization short term fellowship. F.N. is a recipient of a teaching and research assistantship from the University Paris 7.

² Address correspondence and reprint requests to Dr. Andrés Alcover, Unité de Biologie des Interactions Cellulaires, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France. E-mail address:

³ V. Legendre, A. Guimezanes, M. Buferne, M. Barad, A.-M. Schmitt-Verhulst, and C. Boyer. Ag-induced TCR-CD3 down-modulation does not require CD3 or CD3 cytoplasmic domains necessary in response to anti-CD3 antibody. Submitted for publication.

⁴ Abbreviations used in this paper: ER, endoplasmic reticulum; ITAM, immunoreceptor tyrosine-based activation motif; GH, growth hormone; RBL, rat basophilic leukemia.

Received for publication November 25, 1998. Accepted for publication April 14, 1999.

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