(Received for publication, October 18, 1994)
From the
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.
The T cell antigen receptor (TcR) ()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-
and Ti-
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
-D/E-X
-Y-X
-L/I-X
-Y-X
-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- and
Ti-
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-
or the single basic
residue in the Ti-
TM domain(9, 10) prevent
proper assembly and surface expression of the TcR in spite of the
intracellular formation of disulfide-linked Ti-
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-
and CD3-
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-
-CD3-
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.
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).
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).
Figure 3:
Biosynthesis and processing of the
Ti-
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-
(
), the Endo H-sensitive
immature Ti-
(
), and the deglycosylated
Ti-
(d
) are
indicated.
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
100).
Figure 5:
TcR-mediated pattern of
phosphotyrosine-containing proteins in J76, JGN-, and
JGN-
cells. Cells (1.5
10
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.
The four dimers (Ti-, CD3-
,
CD3-
, and
) 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-
and Ti-
, 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-
chain at the cell
surface in the absence of the Ti-
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-
chain on the surface of a pre-T cell line(33) . gp33
thus represents a developmentally regulated means of rescuing Ti-
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-
and not Ti-
appeared to interact directly with
CD3-
(14, 15) . Together with the present results,
the specific interaction between Ti-
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-
, or Ti and
CD3-
), suggesting that CD3-
is
alternative to CD3-
at this level, or three dimers (Ti,
CD3-
, and CD3-
)
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-
,
and CD3-
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.