©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Distinct Domains of the CD3- Chain Are Involved in Surface Expression and Function of the T Cell Antigen Receptor (*)

(Received for publication, October 18, 1994)

Anne-Marie Karin Wegener (§) Xiaohong Hou Jes Dietrich Carsten Geisler (¶)

From the Institute of Medical Microbiology and Immunology, University of Copenhagen, The Panum Institute, Building 18.3, Blegdamsvej 3C, DK-2200 Copenhagen, Denmark

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The T cell antigen receptor (TcR) is a multisubunit complex that consists of at least six different polypeptides. We have recently demonstrated that the CD3- subunit cannot substitute for the CD3- subunit in TcR cell surface expression, in spite of significant amino acid homology between these two subunits. To identify CD3--specific domains that are required for assembly of the complete TcR and for surface expression and function of the TcR, chimeric CD3-/CD3- molecules were constructed and expressed in T cells devoid of endogenous CD3-. Substitution of the extracellular domain of CD3- with that of CD3- did not allow cell surface expression of the TcR. In contrast, substitution of the transmembrane and/or the intracellular domains of CD3- with those of CD3- did allow TcR cell surface expression. These results conclusively demonstrate that the extracellular domain of CD3- plays a unique role in TcR assembly. Functional analyses of the transfectants demonstrated that the intracellular domain of CD3- is required for protein kinase C-mediated down-regulation of TcR but is dispensable for the pattern of tyrosine phosphorylation observed following activation through TcR.


INTRODUCTION

The T cell antigen receptor (TcR) (^1)is responsible for the specific recognition of antigen-derived peptides in association with polymorphic molecules encoded by the major histocompatibility complex and the translation of that recognition into cellular signals (for reviews see (1) and (2) ). The TcR is a hetero-oligomeric complex assembled from the protein products of at least six different genes (reviewed in (3) ). The polymorphic Ti-alpha and Ti-beta chains form disulfide-linked heterodimers (Ti) and define the antigen specificity of the TcR. The Ti is noncovalently associated with the monomorphic CD3-, CD3-, and CD3- chains and a disulfide-linked homodimer or heterodimer of chains belonging to the family of proteins. The intracellular (IC) domain of each of the CD3 chains encompasses a YXXL-containing motif, first noticed by Reth(4) , which consists of 6 mostly conserved amino acids (D/E-X(7)-D/E-X(2)-Y-X(2)-L/I-X(7)-Y-X(2)-L/I, in the single-letter code for amino acids, where X is any amino acid). It has been suggested that this motif is involved in activating signaling pathways operated by cytosolic protein-tyrosine kinases(5) . The CD3 and family chains are thought to interact with cytoplasmic components, including protein-tyrosine kinases, that are directly involved in signal transduction. The IC domain of and of CD3- can induce protein tyrosine phosphorylation(6, 7) , and the IC domain of CD3- is involved in protein kinase C (PKC)-mediated down-regulation of TcR(8) . However, it is not known whether CD3- and CD3- contribute to the protein tyrosine phosphorylation following TcR-mediated activation of T cells.

Interestingly, the Ti-alpha and Ti-beta transmembrane (TM) domains contain 2 and 1 basic amino acids, respectively, whereas the TM domain of each of the monomorphic chains of the TcR contains a single acidic amino acid. Mutations affecting the charge of both basic residues in the Ti-alpha or the single basic residue in the Ti-beta TM domain(9, 10) prevent proper assembly and surface expression of the TcR in spite of the intracellular formation of disulfide-linked Ti-alpha bullet beta dimers (11, 12) . Likewise, the charged residues of the TM domain of the CD3 chains have been found to be critical for CD3-Ti association but not for the formation of CD3- bullet and CD3- bullet dimers (13, 14, 15) . These studies demonstrate the critical role of the charged amino acids in the TM domain of the TcR components and further indicate that domains other than the TM domain are involved in TcR assembly.

The exact stoichiometry of the TcR is still not known; however, the CD3 chains are thought to be expressed within the TcR as noncovalently associated and dimers(16, 17, 18) . Contradictory results concerning the requirement of CD3- and CD3- for TcR expression have been presented. Some studies have indicated that CD3- and CD3- may substitute for each other and that TcR containing either CD3- or CD3- are expressed at the T cell surface(19, 20) . In contrast to these observations, the TcR was not expressed at the cell surface in a murine T cell line lacking the expression of endogenous CD3-, although all other components of the TcR were present in these cells(21) . Surface expression of the TcR could be reconstituted by the introduction of exogenous CD3- or mutated CD3- lacking most of the IC domain into this CD3--deficient T cell variant. Likewise, in a human T cell line lacking the expression of endogenous CD3-, the TcR was not expressed at the cell surface(22) . Partial complexes of Ti-alpha bullet beta-CD3- bullet were detected within the cells, but these complexes were not normally processed through the Golgi apparatus and did not associate with the chains. When this CD3- negative T cell variant was reconstituted with exogenous CD3-, the TcR surface expression was restored. These two studies demonstrate that despite the high level of amino acid homology between CD3- and CD3-, these chains cannot substitute for each other in TcR expression.

The aim of this study was to define domains of CD3- that account for the uniqueness of this chain, as compared with CD3-, in TcR surface expression and function.


EXPERIMENTAL PROCEDURES

Cells and Medium

JGN cells, a TCR surface-negative variant of the human T cell line Jurkat lacking synthesis of the CD3- chain (22) and cells of the original Jurkat cell line J76 were cultured in RPMI 1640 medium (Life Technologies, Inc., Paisley, UK) supplemented with 0.5 IU/liter penicillin (Leo, Ballerup, Denmark), 500 mg/liter streptomycin (NOVO, Bagsvzrd, Denmark), and 10% (v/v) fetal calf serum (Sera-Lab Ltd., Sussex, UK) at 37 °C in 5% CO(2).

Antibodies and Chemicals

UCHT1 mouse monoclonal antibody (mAb) against human CD3- was obtained purified and phycoerythrin (PE)-conjugated from Dakopatts A/S (Glostrup, Denmark). HMT-3.2 hamster mAb against an epitope in the IC domain of human CD3 was kindly donated by Dr. Ralph Kubo (Cytel Corp., San Diego, CA). F101.01 mouse mAb against a conformational epitope on the TcR was produced in our own laboratory(23) . 4G10 mAb against phosphotyrosine was obtained purified and conjugated to agarose from UBI (Lake Placid, NY). The phorbol ester phorbol 12,13-dibutyrate (PDB) was from Sigma.

Construction of Plasmids

Chimeric CD3-/s were constructed by fusing appropriate cDNA segments as described previously (24) . Wild type human CD3- chain and CD3- chain cDNA containing plasmids pJ6T3--2 (25) and pPGBC9(26) , respectively, were used as templates in a set of polymerase chain reactions together with the appropriate primers to generate the three CD3-/ chimeric constructs shown in Fig. 1A. The primers flanking the 5` and 3` ends of each cDNA construct were designed to encompass a XbaI and a NcoI site, respectively. Chimeric polymerase chain reaction products were digested with XbaI and NcoI, subcloned into the 4.1- kilobase pair XbaI-NcoI fragment of pBluescript-betaWT(27) , and sequenced using the dideoxy chain termination method. The 1.8-kilobase pair XbaI-BamHI fragment of each of the pBluescript clonings were subsequently subcloned into the 5.9-kilobase pair XbaI-BamHI fragment of the expression vector pTbetaFneo (28) and used for transfection.


Figure 1: A, predicted structures of CD3-/ chimers. Chimers were constructed by fusing a cDNA segment encoding the indicated region of the human CD3- polypeptide (represented by an open bar) with a cDNA segment encoding the complementary region of the human CD3- polypeptide (represented by a filled bar). The EC, TM, and IC regions of CD3- correspond to amino acids 1-89, 90-116, and 117-160, respectively (numbering according to (25) ). The EC, TM, and IC regions of CD3- correspond to amino acids 1-79, 80-106, and 107-150, respectively (numbering according to (25) ). B, expression of the CD3- epitope at the surface of transfected JGN cells. The parental and transfected JGN cells were stained with anti-CD3- mAb directly conjugated with PE and subsequently analyzed by flow cytometry. The abscissa represent the fluorescence on a logarithmic scale, and the ordinate represents the relative cell number on a linear scale. The fluorescence histogram of each transfected cell line (filled histograms) is compared with the fluorescence histogram of the parental JGN cell line (open histograms).



Transfection by Electroporation

The different plasmid constructs were separately transfected into JGN cells by electroporation (Bio-Rad Gene Pulser) at 270 V and 960 microfarads using 40 µg of plasmid and 2 times 10^7 cells. After 24 h in RPMI 1640 medium, cells were plated at 1 times 10^4 cells/ml in 96-well tissue culture plates (Greiner GmbH, Frickenhausen, Germany) in medium containing 1 mg/ml G418-sulfate (Geneticin) (Life Technologies, Inc.). After 3-4 weeks of selection, clones were expanded and maintained in medium without G418.

Fluorescence-activated Cell Sorter Analysis

Cytofluorometric analyses of cells stained with PE-conjugated UCHT1 were performed on a FACScan cytometer (Becton Dickinson, Mountain View, CA) as described previously(22) .

Metabolic Labeling, Immunoprecipitation, and SDS-Polyacrylamide Gel Electrophoresis Analysis

Metabolic labeling studies were performed as described previously (22) .[S]Methionine-labeled cells were lysed in 1% digitonin lysis buffer (20 mM Tris-HCl, pH 8.0, 1 mM MgCl(2), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 8 mM iodoacetamide, and 1% digitonin), precipitated with the indicated mAbs, and analyzed by SDS-PAGE.

TcR Down-regulation

Cells were adjusted to 1 times 10^5 cells/ml of medium and incubated at 37 °C with various concentrations of the phorbol ester PDB for 1 h and then transferred to ice-cold phosphate-buffered saline containing 2% fetal calf serum and 0.1% NaN(3) and washed twice. Cells were subsequently stained directly with PE-conjugated UCHT1 and subjected to fluorescence-activates cell sorter analysis. Mean fluorescence intensity (MFI) was recorded and used in the calculation of the percentage of anti-CD3 binding, (MFI of PDB treated cells divided by MFI of untreated cells times 100).

Detection of Tyrosine-phosphorylated Proteins

1.5 times 10^7 cells in 1 ml of RPMI medium were stimulated with 2 µg of anti-TcR antibody (F101.01) at 37 °C for 30 s or 1, 5, or 20 min or left unstimulated. Cells were lysed in modified RIPA buffer (1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, and 50 mM Tris, pH 7.5) containing phosphatase inhibitors (2 mM EDTA and 1 mM vanadate) and protease inhibitor (1 mM phenylmethylsulfonyl fluoride). The postnuclear fractions were precleared with protein A-Sepharose, and phosphotyrosine-containing proteins immunoprecipitated with 4G10 mAb coupled to agarose beads (UBI). The beads were washed three times in modified RIPA buffer without phenylmethylsulfonyl fluoride followed by one wash in TN buffer (50 mM Tris, pH 7.5, and 150 mM NaCl). For immunoblotting analysis, immunoprecipitates were separated by SDS-PAGE on a 10% polyacrylamide gel under non-reducing conditions. The proteins were electrotransferred to nitrocellulose membrane (Hybond-ECL from Amersham Corp.) at 0.8 mA/cm^2 for 1 h and 10 min, blocked with 1% bovine serum albumin and 3% dry skim milk in phosphate-buffered saline, and blotted with 4G10 mAb followed by peroxidase-conjugated rabbit-anti-mouse antibody (Dakopatts A/S). Phosphotyrosine-containing proteins were visualized using the ECL enhanced chemiluminescence kit (Amersham Corp.).


RESULTS

Construction and Transfection of Chimeric CD3-/CD3- Molecules into the CD3- Negative T Cell Variant JGN

To assess the role of the extracellular (EC), TM, and IC domains of CD3- in cell surface expression and function of the TcR, three chimeric CD3-/CD3- cDNA constructs were engineered (Fig. 1A). The CD3- construct corresponds to the EC and TM regions of the human CD3- protein fused with the IC region of the human CD3- protein. The CD3- construct corresponds to the EC region of the CD3- protein fused to the TM and IC regions of the CD3- protein, and the CD3- construct corresponds to the EC region of the CD3- protein fused with the TM and IC region of the CD3- protein. The EC domain of CD3- is 10 amino acids shorter than the EC domain of CD3-, whereas the CD3- and CD3- TM and IC domains contain an equal number of amino acids, in agreement with the relatively faster migration of CD3- protein in SDS-PAGE, compared with CD3-. The predicted CD3- chimeric protein was therefore expected to show a mobility in SDS-PAGE similar to that of CD3-, and the CD3- and the CD3- chimeric proteins were expected to show a mobility similar to that of CD3-. The different constructs, including a CD3- wild type (wt) cDNA, were subcloned into the pTbetaFneo expression vector (28) and separately transfected into the CD3- negative human T cell variant JGN(22) . G418-resistant clones were isolated and the resulting cell lines were designated JGN-, JGN-, JGN-, and JGN-wt, respectively.

Biosynthesis of the Chimeric Proteins

To determine whether introduction of the chimeric cDNA constructs directed the synthesis of corresponding chimeric proteins, each transfectant was metabolically labeled and lysed in digitonin. Aliquots of the lysates were immunoprecipitated with anti-CD3- and anti-CD3- mAb. The precipitates were treated with or without endoglycosidase H (Endo H) and analyzed by SDS-PAGE. The anti-CD3- mAb used in this study is directed against an epitope in the IC region of CD3- and was expected to precipitate chimeric CD3- protein, but not CD3- nor CD3- (Fig. 1A). As expected, anti-CD3- did not precipitate any protein migrating at the size of CD3- from lysates of JGN- or JGN- (Fig. 2, lanes 1, 2, 5, and 6), whereas it precipitated a protein migrating at the size of CD3- from lysates of JGN- (Fig. 2, lanes 9 and 10). The anti-CD3- antibody coprecipitated proteins migrating at the size of CD3- from lysates of both JGN- and JGN- (Fig. 2, lanes 3, 4, 7, and 8) and a protein migrating close to the size of CD3- from lysates of JGN- (Fig. 2, lanes 11 and 12). Based on these results, it was concluded that each transfectant expressed a protein in agreement with the cDNA introduced. Furthermore, each of the chimeric molecules associated with a protein complex containing CD3-. These results also established that none of the transfected chimeric molecules induced the expression of endogenous CD3- protein.


Figure 2: Biosynthesis of CD3-/ chimers in transfected JGN cells. Cells were labeled for 1 h at 37 °C with [S]methionine in methionine-free medium and subsequently lysed in digitonin-containing buffer and immunoprecipitated with an anti-CD3- mAb (lanes 1, 2, 5, 6, 9, and 10) or with an anti-CD3- mAb (lanes 3, 4, 7, 8, 11, and 12). Samples were either treated with Endo H (+) or left untreated(-) before analysis by SDS-PAGE under non-reducing conditions on a 14% polyacrylamide gel. Lanes 1-4, JGN- cells; lanes 5-8, JGN- cells; lanes 9-12, JGN- cells. The positions of the relevant CD3 chains are indicated. fg, fully glycosylated; pg, partially glycosylated; d, deglycosylated).



Cell Surface Expression of Chimeric TcR

To investigate whether the presence of any of the chimeric proteins induced surface expression of TcR, each transfectant was stained with anti-CD3- mAb and analyzed by flow cytometry. CD3- and CD3- did restore surface expression of TcR, and clones expressing the TcR at a level similar to JGN- wt could be isolated (Fig. 1B). In contrast, CD3- did not lead to detectable surface expression of TcR (Fig. 1B). These results demonstrated that the EC domain of CD3- plays a unique role in the cell surface expression of TcR.

Absence of Maturation of Ti in JGN- Cells

We have recently demonstrated the presence of partial Ti-alphabulletbeta, CD3-bullet complexes within the ER of JGN cells. These partial TcR complexes did not transit the Golgi apparatus as judged by the processing of the oligosaccharide side chains but were most probably degraded in the ER(22) . Because the introduction of CD3- into JGN cells did not restore TcR surface expression, we questioned whether the chimeric CD3- formed partial complexes encompassing Ti in JGN- cells and, if this was the case, whether such complexes matured further than the partial complexes found in non-transfected JGN cells. Cells were pulse-chase labeled with [S]methionine, lysed in digitonin, and immunoprecipitated with anti-CD3- mAb. The immunoprecipitates were treated with and without Endo H and analyzed by SDS-PAGE (Fig. 3). Ti dimers could be precipitated from the JGN- lysate, as from J76 after a 30-min pulse (Fig. 3, lanes 2 and 5 and lanes 3 and 6, respectively). As expected, no Ti chains were precipitated from JGN cells by anti-CD3- mAb, because no CD3- protein is synthesized in these cells, and the bands observed may be taken as nonspecific (Fig. 3, lane 1). After a 4-h chase period, very little or no Ti dimer was precipitated from JGN- lysate with anti-CD3- mAb (Fig. 3, lanes 8 and 11) and only immature Ti was precipitated from JGN- lysate with anti-CD3- mAb (data not shown), as seen in non-transfected JGN cells(22) . These results suggest that the partial Ti-CD3 complexes encompassing chimeric CD3- chain detected after the pulse period had dissociated and/or degraded during the chase period, whereas other partial Ti-CD3 complexes encompassing CD3- had not matured.


Figure 3: Biosynthesis and processing of the Ti-alpha bullet beta heterodimer in JGN, JGN-, and J76 cells. Cells were pulse-labeled for 30 min at 37 °C with [S]methionine in methionine-free medium. Cells were then either placed on ice (Pulse) or chased for 4 h at 37 °C (Chase) in complete medium and subsequently lysed in digitonin-containing buffer and immunoprecipitated with an anti-CD3- mAb. Samples were either treated with Endo H (+) or left untreated(-) before analysis by SDS-PAGE under non-reducing conditions on a 10% polyacrylamide gel. Lanes 1, 4, 7, and 10, JGN cells; lanes 2, 5, 8, and 11, JGN- cells; lanes 3, 6, 9, and 12, J76 cells. The positions of the Endo H-resistant mature Ti-alpha bullet beta (alphabeta), the Endo H-sensitive immature Ti-alpha bullet beta (alphabeta), and the deglycosylated Ti-alpha bullet beta (dalphabeta) are indicated.



PKC-mediated TcR Down-regulation Is Impaired in JGN- and in JGN- Cells

We have recently shown that the IC domain of CD3- contains a motif that is involved in TcR down-regulation following PKC activation(8) . To investigate whether the IC domain of CD3- could replace the IC domain of CD3- with respect to this aspect of TcR function, the ability of JGN- and JGN- cells to down regulate the TcR following activation of PKC was analyzed. Both JGN- and JGN- cells showed strongly impaired PKC mediated TcR down-regulation as compared with JGN- wt (Fig. 4).


Figure 4: PKC-mediated down-regulation of the TcR on JGN- wt, JGN-, and JGN- cells. JGN- wt, JGN-, and JGN- cells were incubated with different concentrations of PDB for 1 h at 37 °C and then stained with anti-CD3- mAb directly conjugated with PE and analyzed by flow cytometry. MFI was recorded and used in the calculation of the percentage of anti-CD3 binding (MFI of PDB treated cells divided by MFI of untreated cells times 100).



TcR-mediated Stimulation of J76, JGN-, and JGN- Cells Results in Similar Patterns of Phosphotyrosine-containing Proteins

To assess a possible role of the TM and/or the IC domain of CD3- in the protein-tyrosine kinase pathway activated through TcR, we analyzed the pattern of tyrosine phosphorylation detected following TcR-mediated activation of J76, JGN-, and JGN- cells. The three cell lines showed phosphotyrosine proteins of similar size with comparable kinetics (Fig. 5). This analysis showed that neither the IC nor the TM domain of CD3- contributes to distinct phosphotyrosine proteins upon TcR-mediated activation.


Figure 5: TcR-mediated pattern of phosphotyrosine-containing proteins in J76, JGN-, and JGN- cells. Cells (1.5 times 10^7 cells/lane) were stimulated with 2 µg of anti-TcR-mAb (F101.01) for 30 s () or 1, 5, or 20 min or left unstimulated(-). Phosphotyrosine-containing proteins were immunoprecipitated with 4G10 and resolved on SDS-PAGE, and the phosphotyrosine-containing proteins were detected by 4G10 immunoblot. Molecular size standards are indicated at right in kilodaltons. Lanes 1-5, J76 cells; lanes 6-10, JGN- cells; lanes 11-15, JGN- cells.




DISCUSSION

The four dimers (Ti-alphabeta, CD3-, CD3-, and (2)) defining a minimal model of the TcR have to be coordinately assembled in the ER for efficient transport to and expression at the cell surface. Partially assembled complexes and unassembled subunits are not expressed at the cell surface but undergo degradation either within the ER or in lysosomes, a phenomenon termed architectural editing (reviewed in (29) ). Conflicting data concerning the role of CD3- and CD3- in TcR surface expression have been presented. Recent studies have demonstrated that endogenous CD3- cannot substitute for CD3- in a CD3- negative T cell line (21) and that endogenous CD3- cannot substitute for CD3- in a CD3- negative T cell line (22) in order to obtain surface expression of TcR. Furthermore, the lack of surface expression of the TcR on a HIV-1-infected T cell line has been correlated with a specific defect of the CD3- gene(30) . These studies support a TcR model where both CD3- and CD3- are required for TcR surface expression. This is in agreement with a TcR model proposed by Manolios and co-workers (14) in which CD3- and CD3- pair in an obligatory fashion with Ti-beta and Ti-alpha, respectively. In contrast, other studies of both T cells (19) and non-T cells (20) have led to a TcR model where CD3- and CD3- may substitute for each other. In this model the TcR is expressed as two different isotypes containing either CD3- or CD3-. This model is supported by the description of a human T cell line isolated from an immunodeficient patient. Although this T cell line is deficient in the synthesis of CD3-, it expresses the TcR at the cell surface(31) . These apparently conflicting models may reflect that the constraints controlling surface expression of the TcR are not the same in different cell types (T cells versus non-T cells) and are not the same in different T cells (e.g. immature versus mature T cells and certain malignant T cells versus others). One example of developmentally specific expression is the ability of pre-T cells to express the Ti-beta chain at the cell surface in the absence of the Ti-alpha chain (32, 33) and the inability of mature T cells to do so (34, 35) . A pre-T cell-specific glycoprotein, provisionally named gp33, was shown to be disulfide linked to the Ti-beta chain on the surface of a pre-T cell line(33) . gp33 thus represents a developmentally regulated means of rescuing Ti-beta from intracellular degradation and allowing surface expression. Likewise, certain T cell lines may have acquired means of rescuing TcR surface expression in the absence of one or more of the TcR subunits.

The present study examines the structural and functional role of distinct domains of CD3- in the TcR of the human T cell leukemia Jurkat. Both the TM and the IC domains of CD3- could be substituted by the corresponding domains of CD3- without any detectable loss of TcR surface expression. JGN- and JGN- cells expressed comparable levels of TcR at the cell surface, showing that the efficiency of TcR surface expression was not affected by the substitution of the TM domain of CD3- with the corresponding domain of CD3- despite the limited amino acid homology (37%) between these domains. This observation is consistent with a requirement of the TM domain for conservation of hydrophobicity and a correctly located charged residue (a glutamic acid in CD3- and an aspartic acid in CD3-), rather than amino acid identity(36) . In contrast to JGN- and JGN- cells, JGN- cells did not show detectable surface TcR. These results demonstrate that the EC domain of CD3- possesses a unique role in TcR surface expression, as compared with CD3-, whereas the TM and IC domains do not. In studies of pairwise interactions between single TcR chains, only Ti-beta and not Ti-alpha appeared to interact directly with CD3-(14, 15) . Together with the present results, the specific interaction between Ti-beta and CD3- is suggested to occur via the EC domain of CD3- when these chains are expressed in the context of the full TcR.

Ti, CD3-, and CD3- formed partial complexes within JGN- cells. Due to the similar migration of endogenous CD3- and CD3- proteins, it was not possible to distinguish whether CD3- was also part of CD3- containing partial complexes. From previous studies we know that CD3- does form partial complexes with Ti and CD3- in non-transfected JGN cells(22) . Partial complexes within JGN- may thus be composed of a minimum of either two dimers (Ti and CD3- bullet , or Ti and CD3- bullet ), suggesting that CD3- is alternative to CD3- at this level, or three dimers (Ti, CD3- bullet , and CD3- bullet ) suggesting that CD3- is in addition to CD3-. Irrespective of the composition of the partial complexes, the fate of the Ti dimer in JGN and JGN- could not be distinguished. In both cells partial complexes were formed in a pre-Golgi compartment, probably the ER, but no maturation of Ti could be detected.

The study of the fate of partial complexes in a negative variant of a murine T cell line demonstrated that partial Ti-CD3 complexes may reach the lysosomes in the absence of (reviewed in ref. 29). This implies that partial hexameric complexes can mature in the Golgi apparatus before being sorted to the lysosomes. If this is also true in the Jurkat system, supposing that Ti, CD3- bullet , and CD3- bullet do form partial hexameric complexes, it may be speculated that the substitution of the EC domain of CD3- with that of CD3- results in the loss of a signal patch that otherwise allows such partial complexes to mature in the Golgi apparatus or that such complexes have a short half-life. Alternatively, it may be suggested that the substitution of the EC domain hinders the association of to the partial TcR complexes and that the absence of this association prevents the maturation in the Golgi apparatus.

PKC-mediated phosphorylation of CD3- has been suggested to act as a negative feedback signal that controls the level of surface TcR and/or the function of TcR(37) . Among other substrates, PKC phosphorylates CD3- at serine 126 (S) and possibly also S(38) positioned 10 and 7 amino acids COOH-terminal from the TM domain, respectively (numbering according to (25) ). We have recently demonstrated that the IC domain of CD3- contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the TcR(8) . PKC-mediated down-regulation of the TcR expressed on JGN cells reconstituted with a S126V point-mutated CD3- chain (JGN-S126V) was strongly inhibited as compared with JGN-S123V and JGN- wt cells(8) , and it was concluded that S, but not S, is required for efficient PKC-mediated TcR down-regulation. The IC domain of CD3- also displays a di-leucine motif and has serine (S) at a position corresponding to S, but has alanine (A) at a position corresponding to S. Although CD3- may also be a substrate for PKC(39) , endogenous CD3- does not contribute significantly to PKC-mediated TcR down-regulation(8) . This may be because CD3- differs in amino acid sequence from CD3-, notably by the ``lack'' of S. Alternatively, the geometrical position of CD3- within the TcR may have an impact on the function of the IC domain of CD3-. In the present study, the geometrical positions of the CD3- and the CD3- chimers within the TcR on JGN- and JGN- cells, respectively, are imagined to be similar to that of CD3- within the TcR on JGN- wt cells, as this position is assumed to be directed by the CD3- EC domain. By inference, the CD3- IC domain of either chimer may be positioned within the TcR similar to the CD3- IC domain of a normal TcR. However, JGN- and JGN- cells were functionally similar to JGN-S126V cells, because they showed impaired PKC-mediated down-regulation of TcR. This result suggests that this specialized function of the IC domain of CD3-, as compared with that of CD3-, is a consequence of its specific amino acid sequence and not of its geometrical position within the TcR. The qualitative pattern of tyrosine-phosphorylated proteins following anti-TcR stimulation of J76, JGN-, and JGN- cells was similar and may reflect one of three underlying situations; the IC domain of CD3- and CD3- serve similar functions and either connect to protein-tyrosine kinases with similar substrate specificity or do not connect to protein-tyrosine kinases at all, or CD3-, but not CD3-, connects to protein-tyrosine kinases. Assuming that the YXXL-containing motif in both chains is involved in signal transduction, this would mean that the YXXL motif is redundant or that the YXXL motif may connect to a non-protein-tyrosine kinase signal-transducing pathway (e.g. through association with serine/threonine kinase Raf(40) ). The function of the CD3 chains may be divided in two. First, they play a structural role in controlling the assembly and intracellular transport of the TcR, and second, once expressed on the cell surface, they are involved in ligand-triggered signal transfer across the membrane and in regulating the TcR density at the cell surface. It has been suggested that the CD3- and CD3- arose from a duplicated gene and that these chains have adopted separate functions(25) . This is in agreement with our findings with regard to their respective structural role in TcR assembly. With regard to function, their respective TM and IC domains seem to have adopted both distinct and overlapping functions.


FOOTNOTES

*
This work was supported by the Novo Nordisk Foundation, the Carlsberg Foundation, and the Danish Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a fellowship from the Carlsberg Foundation. To whom correspondence should be addressed. Tel.: 45-3532-7880; Fax: 45-3532-7851.

Member of the Biotechnology Center for Signal Peptide Research.

(^1)
The abbreviations used are: TcR, T cell antigen receptor; Ti, disulfide-linked TcR-alpha bullet beta heterodimer(s); IC, intracellular; PKC, protein kinase C; TM, transmembrane; mAb, monoclonal antibody; PE, phycoerythrin; PDB, phorbol 12,13-dibutyrate; PAGE, polyacrylamide gel electrophoresis; MFI, mean fluorescence intensity; EC, extracellular; wt, wild type; Endo H, endoglycosidase H; ER, endoplasmic reticulum.


ACKNOWLEDGEMENTS

The technical help of Inger Bull Olsen and Bodil Nielsen is gratefully acknowledged.


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