From the Laboratorium für Molekulare
Biologie-Genzentrum der Universität München, and the
¶ GSF-Institut für Molekulare Immunologie,
D-81377 München, Germany
Received for publication, October 11, 2000, and in revised form, December 7, 2000
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ABSTRACT |
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CD4 recruitment to T cell receptor
(TCR)-peptide-major histocompatibility class II complexes is
required for stabilization of low affinity antigen recognition by T
lymphocytes. The cytoplasmic portion of CD4 is thought to amplify
TCR-initiated signal transduction via its association with the protein
tyrosine kinase p56lck. Here we describe a novel
functional determinant in the cytosolic tail of CD4 that inhibits
TCR-induced T cell activation. Deletion of two conserved hydrophobic
amino acids from the CD4 carboxyl terminus resulted in a pronounced
enhancement of CD4-mediated T cell costimulation. This effect was
observed in the presence or absence of p56lck, implying
involvement of alternative cytosolic ligands of CD4. A two-hybrid
screen with the intracellular portion of CD4 identified a previously
unknown 33-kDa protein, ACP33 (acidic cluster
protein 33), as a novel intracellular binding
partner of CD4. Since interaction with ACP33 is abolished by deletion
of the hydrophobic CD4 C-terminal amino acids mediating repression of T
cell activation, we propose that ACP33 modulates the stimulatory
activity of CD4. Furthermore, we demonstrate that interaction with CD4
is mediated by the noncatalytic The cell surface glycoprotein CD4 is expressed on subsets of
thymocytes and mature T lymphocytes and, in humans, on monocytes and
macrophages. Early clues to CD4 function came from a strong correlation
between CD4 expression and
MHC1 class II restricted T
helper cell activity that corresponds to the MHC class I-mediated
cytotoxic response of CD8-positive T cells. Subsequent studies using
targeted gene disruption in mice and antigen-dependent
in vitro T cell activation assays implicated an important
role of CD4 in T helper cell development and activation (1, 2).
The molecular mechanism of CD4 function is based on its direct
interaction with invariant regions of MHC class II molecules on ACPs
(3), hence stabilizing low affinity TCR-peptide-MHC complexes.
To attain maximum T cell costimulation, CD4 must be specifically
recruited to antigen-engaged TCR molecules (4). This selective
recruitment is thought to be mediated by the cytoplasmic domain of CD4,
which in turn is associated with the protein-tyrosine kinase
p56lck (5, 6). In this context, the phosphotyrosine-binding
Src homology 2 domain of Lck has been shown to be sufficient
for this interaction (7) and involves phosphorylated and TCR-associated ZAP-70 as a ligand (8). In addition to its adaptor function, p56lck is thought to amplify TCR-dependent signal
transduction by phosphorylation of phosphotyrosine adapter molecules,
immunoreceptor tyrosine-based activation motifs of CD3 chain and
the activation loop of ZAP-70 (9). Thus, both extracellular and
cytosolic domains of CD4 are required for optimal coreceptor function.
This bifunctional activity might account for the variable requirements
for CD4 costimulation by different antigen-dependent T cell
lines (10).
In contrast to its stimulatory role during
antigen-dependent T cell activation, CD4 transmits
inhibitory signals to T cells when engaged independently of the TCR
complex, e.g. by the proposed natural CD4 ligands IL-16 (11)
and gp17 (12). Preincubation with IL-16 has been shown to counteract
mitogen- or TCR-induced activation of human peripheral T lymphocytes
(13, 14). Since gp120 treatment of T lymphocytes leads to induction of
anergy or apoptosis (15), binding of gp120 to CD4 is thought to
contribute to the depletion of CD4-positive T cells in HIV-infected
patients. Similarly, antibody-mediated CD4 clustering results in
inhibitory signals that render T cells unresponsive to subsequent
stimulation by TCR engagement (16-18). The latter finding is
especially relevant for therapeutic approaches involving administration
of anti-CD4 antibodies in vivo for the treatment of
autoimmune syndromes, delayed type hypersensitivity reactions, and
allograft rejection. The molecular basis for the inhibitory CD4
signaling is not known but has recently been shown to be, to some
extent, independent of p56lck under different conditions. For
instance, anti-CD4 antibodies block ongoing antigen-induced T cell
activation in cell lines expressing a CD4 mutant unable to associate
with p56lck, indicating that sequestration of Lck can only
partially account for CD4-mediated inhibition of IL-2 production (19).
Moreover, HIV gp120-induced apoptosis of a lymphoblastoid T cell line
did not require p56lck signaling (20), and in
Lck-deficient cell lines HIV replication was nevertheless
blocked by anti-CD4 antibodies directed against the CDR3-like region
(21, 22). Therefore, it was proposed that inhibitory signal
transduction by CD4 is mediated by alternative ligands of the CD4
cytoplasmic domain (23).
These results prompted us and others to investigate
CD4-dependent signal transduction during T cell activation
in more detail. Interestingly, the phosphotyrosine adaptor molecule
linker for activation of T cells (LAT) has recently been identified as
an alternative CD4 cytoplasmic tail ligand and might contribute to CD4-mediated stimulation of TCR signaling (24). In the present study,
we focused on the hydrophobic C terminus of CD4 and discovered that it
contains a negative regulatory determinant of T cell activation. Moreover, we identified a novel CD4-interacting protein, ACP33, as a
candidate molecule for mediating negative signaling by the hydrophobic
C terminus of CD4.
DNA Constructs--
Full-length cDNA coding for ACP33 was
isolated by a yeast two-hybrid screen using a transactivator fusion
protein cDNA library in conjunction with a LEXA fusion of
the cytoplasmic tail of human CD4 (amino acids 399-433), essentially
as described before (25). The coding region for ACP33 was amplified by
PCR and subcloned into the cIg fusion protein expression vector
p5C7 (26). Mutagenesis of the cIg-ACP33 construct was performed by
polymerase chain reaction to obtain a substitution of serine 109 by alanine.
Full-length cDNAs of human CD4 and p56lck were obtained
from Brian Seed (Harvard University) and subcloned into
HindIII and NotI restriction sites of the
mammalian expression plasmid pN1 (CLONTECH). Truncation mutants of CD4 and a double point mutation changing cysteines 420 and 422 to alanines were generated by replacing the
Bpu1102I-NotI fragment of pCD4-N1 with
polymerase chain reaction products.
For generation of stable transgenic cells, a new vector, pEF-IRESpuro,
was constructed by inserting the CD4 cDNA, an internal ribosome
entry site (IRES) (27) and a puromycin resistance gene into the pEF-BOS
expression cassette (28).
Cell Lines and Antibodies--
Jurkat E6, JCaM1.6, HUT 78, and
HeLa cells were purchased from the American Type Culture Collection and
maintained in RPMI containing 10% fetal calf serum and 10 µg/ml
gentamicin. COS-7 cells were obtained from Brian Seed and grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum and 10 µg/ml gentamicin. The antigen-dependent
murine T cell line 171 was generously provided by Mark Hill and Dan
Littman (New York University). Culture conditions for 171 and the
respective FT7.1 antigen-presenting cells were as described (29).
Monoclonal antibodies directed against ACP33 were generated by
immunizing Lou/C rats with a purified glutathione
S-transferase fusion protein of full-length ACP33 expressed
in bacteria. After an 8-week interval, a final boost was given 3 days
before fusion of the rat spleen cells with the murine myeloma cell line
P3X63-Ag8.653 (30). Hybridoma supernatants were tested in an
enzyme-linked immunosorbent assay using bacterial extracts from
Escherichia coli expressing a fusion protein of ACP33 and
maltose-binding protein (MBP) or a control MBP fusion protein.
Hybridoma supernatants reacting with ACP33-MBP but not with the control
MBP fusion protein were analyzed by Western blots, and clone it1-2D5
(rat IgG2a) was selected for its reaction pattern.
Mouse monoclonal antibodies against p56lck (Lck-01 and
Lck-04) were generously provided by Vaclav Horejsi (Czech Academy of
Science). Monoclonal anti-CD4 antibody MT-151 was donated by Peter
Rieber (University of Dresden, Germany). Polyclonal anti-CD4 antiserum T4-4 (31) was provided by Raymond Sweet (SmithKline Beecham Pharmaceuticals), through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health. All
secondary anti-IgG reagents were purchased from Jackson ImmunoResearch.
T Cell Activation Assays--
Transgenic CD4 expressing 171 murine T cells were generated by electroporation of 10 µg of
pCD4-EF-IRESpuro plasmid or mutant derivatives into 1 × 107 cells. After a 48-h cultivation, transfected cells were
selected for resistance to 2 µg/ml puromycin (Biomol) for 10-12
days. Expression levels of CD4 were monitored by immunofluorescence and
Western blotting. T cell activation assays were performed essentially as described (29). Increasing concentrations of peptide antigen, an
analog of hen egg lysozyme 74-88, were mixed with 106
transgenic 171 T cells and 5 × 105 FT7.1 ACPs in a
final volume of 300 µl of Iscove's modified Dulbecco's medium.
After 24 h, secreted levels of IL-2 were quantified by enzyme-linked immunosorbent assay (OptEIA-Set, BD Pharmingen).
Procedures for transient transfection of JCaM1.6 cells and measurement
of luciferase activity were described before (26). Briefly, 10 µg of
pIL2-GL2 reporter plasmid and 25 µg of pCD4-N1 or mutant derivatives
were cotransfected by electroporation. After 20 h, cells were
stimulated for 8 h with ionophore A23187 (0.5 µg/ml) or phorbol
12-myristate 13-acetate (50 ng/ml) or were left untreated, followed by
the addition of reporter lysis buffer (Promega) and scintillation counting.
Subcellular Fractionation and Immunoprecipitation--
HUT 78 cells were transiently transfected by electroporation using 20 µg of
plasmid expressing cIg-ACP33 or a cIg control. Subcellular
fractionation was performed as previously described (32). Briefly,
cells were lysed by passing through a 26-gauge needle in hypotonic
lysis buffer and centrifuged for 10 min at 20,000 × g.
The supernatant corresponded to the cytoplasmic fraction and the
soluble material from the pellet extracted by detergent lysis buffer,
and further centrifugation was called the particulate fraction.
Immunoprecipitation was performed from lysates of 171 cells,
electroporated HUT 78 cells, and COS-7 cells transfected by
DEAE-dextran as described before (26). Lysis buffer contained 1%
Nonidet P-40, 20 mM Tris (pH 7.5), 150 mM NaCl,
2 mM EDTA, 10 µg/ml leupeptin, 5 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride. After 30 min on ice,
lysates were cleared at 20,000 × g and the supernatant was incubated for 1 h with 1 µl of antiserum for
immunoprecipitation of CD4 or, in case of precipitation of cIg fusion
proteins, was directly incubated for 30 min with protein A immobilized
on Sepharose 6MB (Sigma). Beads were washed three times in lysis
buffer, and bound proteins were released by boiling in SDS-loading
buffer. Samples were resolved by SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting.
Immunofluorescence--
To detect intracellular antigens, HUT 78 cells were placed on poly-L-lysine-coated coverslips in PBS
for 30 min at 4 °C and fixed for 15 min with 2% (w/v) freshly
prepared paraformaldehyde in PBS at 25 °C. Fixed cells were blocked
by washing three times for 5 min with 2% (w/v) glycine in PBS and
permeabilized for 15 min in 0.2% SDS, which was found to increase
reactivity of anti-ACP33 mAb 2D5. Unspecific binding was blocked by
washing three times for 5 min with 2% fetal calf serum in PBS.
Endogenous ACP33 was stained by incubation with undiluted 2D5 hybridoma
supernatant for 60 min and washing three times for 5 min with 2% fetal
calf serum in PBS followed by detection with FITC-conjugated anti-rat IgG. An isotype-matched control antibody directed against a non-T cell
protein did not result in significant fluorescence.
Where indicated, cells were incubated with rhodamine-conjugated wheat
germ agglutinin, transferrin, or DAMP (all from Molecular Probes, Inc.,
Eugene, OR). Endogenous CD4 was detected using T4-4 antiserum and
rhodamine-conjugated anti-rabbit IgG. Total rabbit IgG was used as a
control. Staining of cIg fusion proteins was achieved using
FITC-conjugated anti-human IgG. Microscopical imaging was done on a
Leica TCS confocal laser microscope. FITC and rhodamine signals for a
single confocal section were recorded separately and overlaid using
Adobe Photoshop software.
For extracellular CD4 staining, cells were incubated with anti-CD4 mAb
MT-151 and FITC-conjugated anti-mouse IgG and analyzed by flow
cytometry (Coulter Epics XL).
Truncation of the Hydrophobic COOH Terminus of CD4 Results in a
Gain-of-function Phenotype--
The CD4 cytoplasmic domain is highly
homologous among its mammalian orthologues (Fig.
1A). Conserved residues
include the membrane-proximal cysteines 394 and 397, which can be
palmitoylated (33), a short
To analyze the functional role of the conserved hydrophobic C-terminal
residues of CD4 with respect to antigen-dependent T cell
activation, we used the CD4-negative murine T cell line 171, previously
established as a model system for CD4 function (7, 29). Oligoclonal
pools of 171 cells expressing either native human CD4 or truncated
mutant derivatives (Fig. 1B) were selected and found to
display comparable amounts of CD4 on the cell surface (Fig.
2A). T cell activation was
induced by adding increasing concentrations of peptide antigen, and
secreted IL-2 was quantified by enzyme-linked immunosorbent assay.
Consistent with earlier findings (29), expression of CD4 was required
for measurable IL-2 production by 171 cells (Fig. 2B).
Deletion mutants of CD4 clearly showed different influences on
costimulatory function. Truncation of only two COOH-terminal amino
acids (431*) led to a pronounced gain-of-function phenotype. However,
further deletion (419*, 398*/Pal
Since gain-of-function mutants of CD4 have not been described before,
we were interested in further characterizing this effect. One potential
explanation for the 431* mutant phenotype implicates increased
p56lck activity. However, association of p56lck with
the CD4 mutant 431* was unaltered compared with native CD4 as analyzed
by coimmunoprecipitation (Fig. 2C). As expected from previous reports (35), no anti-p56lck reactive band was
detected in CD4 precipitates of 419*-, 398*/Pal The Phenotype of the 431* Truncation Mutant Is Independent of
p56lck--
The JCaM1 cells we used here to study
Lck-independent CD4 function did not express detectable levels of
p56lck, in contrast to normal Jurkat cells (data not shown).
Expression vectors for native CD4 or several CD4 mutants were
transiently transfected into JCaM1 cells together with a luciferase
reporter plasmid that allowed quantification of IL-2 promoter activity. Expression levels were monitored by flow cytometry and found to be
within a comparable range (Fig.
3A). To our surprise,
transfection of native CD4 was sufficient to significantly induce IL-2
promoter activity in phorbol ester-costimulated JCaM1 cells without
further cross-linking (Fig. 3B). The Lck
Taken together, removal of the hydrophobic amino acids proline and
isoleucine from the COOH terminus of the CD4 cytoplasmic domain results
in enhanced T cell costimulation in two independent assay systems. The
presence of p56lck is neither required nor inhibitory for the
gain-of-function phenotype of CD4 431*, suggesting the existence of an
alternative mechanism involving novel mediators of CD4 function.
Cloning of ACP33 as a CD4-binding Protein--
A yeast two-hybrid
screen of a Jurkat T cell cDNA library was performed to identify
alternative CD4 cytoplasmic tail binding partners. Several selected
clones were analyzed, and all were found to contain an identical
cDNA insert. To identify its translation initiation site, we
isolated a further 250 nucleotides of this cDNA by polymerase chain
reaction from a plasmid library. This revealed that the two-hybrid
isolate contained the complete open reading frame for a protein of 308 amino acids. The deduced protein does not contain any known subcellular
localization signals, leader sequence, or transmembrane helix. However,
a cluster of four acidic amino acids is located close to the N terminus
(Fig. 4A). Blast homology
searches (40) of protein data banks revealed identity with the recent
GenBankTM entry NP_057714, a hypothetical protein encoded
by a bone marrow-derived mRNA. A very limited sequence similarity
to bacterial enzymes containing an
Multiple tissue Northern blot hybridization with a cDNA fragment
from the CD4-binding protein detected a single mRNA species of 2.1 kilobases in length (Fig. 4B). All human tissues analyzed contained detectable and comparable amounts of this transcript. To
address protein expression and localization of the novel CD4 binding
factor, we generated monoclonal antibodies against the complete
polypeptide expressed in Escherichia coli. One clone, mAb
2D5, specifically detected a protein of 33 kDa in immunoblotted detergent lysates of a variety of human and murine cell lines (Fig.
4C). Furthermore, the antibody reacts with several fusion proteins of the novel CD4-binding protein, indicating that it specifically recognizes an epitope of this protein (data not shown). We
propose the term ACP33 (acidic cluster
protein of 33 kDa) to denote this new factor.
Subcellular Localization of ACP33--
To test if subcellular
distribution of ACP33 is compatible with a functional interaction with
CD4, we analyzed the intracellular localization of the mAb 2D5 epitope
in a CD4-positive T cell line, HUT 78. As an additional control, we
expressed an ACP33 fusion protein (cIg-ACP33) by transient
transfection, the two NH2-terminal immunoglobulin domains
from human IgG1 serving as an epitope for standard anti-human IgG
reagents (Fig. 5A). Chimeric
proteins containing Ig domains have been successfully used to study
subcellular localization (43, 44), protein-protein interaction (26), and protein function (25). We employed a simple biochemical cell
fractionation protocol with hypotonic cell lysates and analyzed ACP33
distribution in the soluble cytosol and the particulate fraction. The
latter primarily consists of membrane- and cytoskeleton-associated proteins. As described previously (32), the cIg control protein is
localized in the cytosolic fraction (Fig. 5B). In contrast, a membrane-resident derivative of the Ig fusion domain (sIg
control) containing a leader sequence and transmembrane helix is
totally confined to the particulate fraction. Both endogenous and
transiently expressed cIg-ACP33 was evenly distributed with
approximately equimolar amounts in both fractions, indicating that
ACP33 is a cytosolic protein, which partially associates with
particulate cellular structures.
To characterize these particulate structures, we studied the in
situ subcellular localization of ACP33 by immunofluorescence in
HUT 78 cells. Endogenous ACP33 was analyzed using mAb 2D5 and secondary
anti-rat IgG fluorescein conjugates, while anti-human IgG reagents were
used to detect cIg-ACP33 and cIg control proteins. Both endogenous and
transfected cIg-ACP33 were partially localized in the cytosol but also
accumulated on an intracellular vesicular compartment (Fig.
5C), whereas the control protein was uniformly distributed
throughout the cell interior.
ACP33 Colocalizes with CD4 on the Endosomal/trans-Golgi
Network--
Using immunofluorescence, we compared localization of
endogenous ACP33 relative to established cellular markers in HUT 78 cells. Wheat germ agglutinin is a lectin that preferentially stains the
Golgi apparatus in most cell types (45). Overlay of the wheat germ
agglutinin-rhodamine signal with the anti-ACP33 mAb 2D5 staining showed
only very limited overlapping immunofluorescence (Fig.
6c). In contrast, the
vesicular structures marked by ACP33 partially coincided with both
rhodamine-labeled transferrin (Fig. 6f), which is confined
to the early endosomal recycling pathway (46), and with acidic
organelles (Fig. 6i) stained by the acidotropic dye DAMP
(47). We therefore conclude that endogenous ACP33 is partitioned
between the cytosol and vesicles of the
endosomal/trans-Golgi network.
In unstimulated T cells, CD4 is distributed between the plasma membrane
and the endosomal recycling pathway. T cell activation, however,
induces rapid endocytosis and rerouting of CD4 to lysosomes. HUT 78 represents a partially activated T cell line expressing the activation
molecules Ia and the IL-2 receptor (48). When analyzed by
immunofluorescence using a highly specific antiserum against human CD4,
the majority of CD4 molecules were localized to intracellular vesicles,
and only a minor fraction resided in the plasma membrane (Fig.
6k). Importantly, the intracellular fraction of CD4
molecules colocalized to a large extent with endogenous ACP33 (Fig.
6l), supporting a potential physical and functional interaction of these molecules.
CD4 Coprecipitates with cIg-ACP33--
Since it was found that
anti-ACP33 monoclonal antibody mAb 2D5 precipitates endogenous ACP33
poorly, the Ig fusion protein cIg-ACP33 was used for
immunoprecipitations to analyze interactions with endogenous CD4. Ig
fusion proteins were purified from detergent lysates of transfected HUT
78 cells and analyzed by immunoblotting using a polyclonal human CD4
antiserum. Endogenous CD4 specifically coprecipitated with cIg-ACP33
but not with cIg control protein (Fig.
7A). However, the
stoichiometry of the interaction seems rather low with respect to the
CD4 levels present in the total cell lysate (Fig. 7A,
lane 3). This low stoichiometry might reflect physiological levels of a transiently formed complex or might be due to
a high off-rate during precipitation.
Mutational Mapping of the CD4-ACP33 Complex--
To determine the
molecular requirements for CD4-ACP33 complex formation, we analyzed
ACP33 binding to the various truncation mutants of CD4 described above.
Moreover, we investigated the CD4 binding potential of a point mutant
of ACP33 generated by replacing serine 109 by alanine (Fig.
7B). Serine 109 is likely to be part of the structural
element resembling the nucleophile elbow of Simultaneous Interaction of ACP33 and p56lck with
CD4--
To determine whether ACP33 interferes with binding of
p56lck to the CD4 cytoplasmic tail or possibly interacts with
p56lck directly, we coexpressed CD4, p56lck, and
cIg-ACP33 in COS cells. Lck efficiently coprecipitated with ACP33 via
the cIg-ACP33·CD4 complex (Fig.
8A, lane
1), but no direct interaction between p56lck
and cIg-ACP33 could be detected. As expected, neither p56lck
nor the truncated CD4 molecules were detected in cIg-ACP33
immunoprecipitates from cells expressing the CD4 431* C-terminal
deletion mutant (lane 2). Equally, interaction
with the p56lck-CD4 complex was undetectable when cIg-ACP33 was
replaced by the S109A substitution mutant (lane
4). Moreover, comparable amounts of CD4 coprecipitated with
cIg-ACP33 in the presence or absence of p56lck
(lanes 1 and 3, second
panel), suggesting that ACP33 and p56lck can
interact independently and simultaneously with CD4 (Fig. 8B). We therefore conclude that ACP33 is a likely candidate
for the observed modulation of CD4 function via an Lck-independent mechanism.
CD4 has been previously shown to exhibit a complex pattern of
signal transduction, i.e. to mediate stimulatory or
inhibitory signals after extracellular ligand binding (2). In this
report, we identified a negative regulatory determinant in the
cytoplasmic tail of CD4, which specifically interacts with a novel
CD4-binding protein, ACP33. Using a well established
antigen-dependent model system, we analyzed C-terminal
truncation mutants of CD4 (Fig. 1) and showed that the negative
regulatory determinant in the CD4 cytoplasmic tail depends on the
integrity of two hydrophobic amino acids at its C-terminal end (Fig.
2). This finding was confirmed in an independent functional assay for
CD4 employing the JCaM1 cell line, which is deficient in p56lck expression.
Unexpectedly, CD4 exhibited a costimulatory effect on JCaM1 T cell
activation in the absence of p56lck, suggesting the involvement
of a previously unknown positive regulatory determinant. This
determinant resides between amino acids 431 and 419 of the CD4
cytoplasmic tail but is only partially dependent on cysteines 420 and
422, since the Lck Irrespective of the mode of CD4-mediated IL-2 promoter induction in
these two functional assays, the stimulatory signal was negatively
modulated by the hydrophobic carboxyl terminus of CD4. Therefore, its
deletion resulted in both cases in a gain-of-function phenotype. Since
the 431* mutant of CD4 completely lacks detectable association with
ACP33 (Figs. 7 and 8), its gain-of-function phenotype clearly
correlates with association of ACP33 but not with that of
p56lck. Moreover, ACP33 and CD4 colocalize on intracellular
vesicles (Figs. 5 and 6), supporting the plausibility of a functional interaction.
As yet, we cannot discriminate between whether ACP33 actively
transduces an independent negative regulatory signal or modulates a
positive signal, i.e. by increasing CD4 internalization and degradation. In the latter case, deletion of the hydrophobic C-terminal amino acids would lead to a prolonged duration of signal transduction, resulting in the gain-of-function phenotype of the 431* mutant CD4.
This possibility is supported by the subcellular localization of ACP33,
the majority of which is recruited to the cytoplasmic face of
endosomal/trans-Golgi vesicles (Fig. 6). CD4 internalization and/or degradation was closely monitored in phorbol 12-myristate 13-acetate-stimulated, stably transfected 171 cells, but so far we were
unable to detect any difference between localization or half-life of
CD4 and the 431* mutant. However, it is possible that
binding of ACP33 is required for subtle changes in subcellular
trafficking of CD4. Interestingly, HIV Nef-induced degradation
of CD4 has recently been shown to depend on a diacidic motif in the Nef
protein (53). Moreover, several other proteins contain clusters of
acidic amino acids that are involved in protein sorting from endosomes
to various cellular compartments, e.g. to lysosomes or to
the plasma membrane. By interacting with the acidic clusters of furin
and the cation-independent mannose 6-phosphate receptor, a family of
cytosolic sorting proteins, termed PACS, mediate their cellular routing
(54, 55). Since ACP33 contains a cluster of four aspartic acid
residues, it might function by connecting CD4 to related cellular
sorting factors, thus regulating the fate of internalized CD4.
Endosomal CD4 is targeted to lysosomes (34), to glycolipid-enriched
membrane compartments (56-58), or back to the plasma membrane,
depending on the cellular activation status (59).
Interestingly, inhibition of T cell activation by CD4 antibodies (19)
or HIV gp120 (60) was recently found to be p56lck-independent,
which implies that alternative CD4-associated proteins are actively
transducing inhibitory signals under these conditions. We tried to
analyze a potential inhibitory signaling function of ACP33 by
overexpression studies, but we were unable to obtain cells that
overexpress cIg-ACP33 more than 2-fold above the level of endogenous
ACP33. Therefore, the mechanism of ACP33 mediated modulation of CD4
function could not be analyzed in detail.
Binding of ACP33 to CD4 depends on the ACP33 serine residue 109 (Fig.
7). This residue is predicted to be part of a structural element
conserved in ACP33 is a ubiquitously expressed protein (Fig. 4), which implies that
it interacts with alternative ligands in CD4-negative cell types. In
the present study, however, we focused on a functional interaction of
ACP33 and CD4 in T cells. We have evidence that the minimal interaction
motif for ACP33 binding consists of certain combinations of paired
hydrophobic, nonaromatic amino acid residues at the carboxyl terminus
of polypeptides,2 consistent
with the motif conserved in mammalian CD4 cytoplasmic domains (Fig.
1).
Taken together, the presence of dissectable positive and negative
regulatory determinants in the CD4 cytoplasmic tail confirms recently
proposed models for T cell activation, in other words that integration
of both stimulatory and repressive signals determine the outcome of T
cell receptor stimulation (2, 10, 63). Since binding of ACP33
correlates with inhibition of CD4 function, we propose that ACP33 is a
novel negative regulatory factor involved in CD4-dependent
T cell activation.
/
hydrolase fold domain of
ACP33. This suggests a previously unrecognized function for
/
hydrolase fold domains as a peptide binding module mediating
protein-protein interactions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helix between amino acid 409 and 414, which mediates binding of the AP-2 and AP-1 adaptor protein complexes (34), and a double cysteine motif (position 420 and 422) required for
interaction with p56lck (35). We noticed an additional
conserved motif consisting of two hydrophobic amino acids at the
carboxyl terminus: either proline and isoleucine, leucine and
isoleucine, or two leucines. Interestingly, hydrophobic COOH termini of
transmembrane receptors have been implicated in mediating
protein-protein interactions, e.g. with PDZ
domain-containing proteins involved in signal transduction, multimerization, and subcellular sorting events (36, 37).
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Fig. 1.
Alignment and mutants of the CD4 cytoplasmic
domain. A, amino acid alignment of mammalian CD4
cytoplasmic domains. Residues identical to human CD4 are dashed. In the
consensus sequence, conserved positively charged residues are indicated
by +, alcoholic residues by , hydrophobic residues by
, and
nonconserved residues by x. B, schematic
representation of CD4 cytoplasmic domain mutants. Homo s.,
Homo sapiens; Pan t., troglodytes;
Macaca n., Macaca nemestrina; Mus m.,
Mus musculus; Rattus n., Rattus
norvegicus; Felis c., Felis catus;
Canis f., Canis familiaris; Oryctolagus
c., Oryctolagus cuniculus.
) or double point
mutations in the cysteine motif required for Lck binding
(Lck
) clearly resulted in reduced IL-2 secretion by the
respective transfectants, confirming that p56lck makes a
significant contribution to stimulatory CD4 signal transduction (29).
These results were highly reproducible; experiments with three
independently derived oligoclonal cell populations yielded very similar
functional data (not shown). In contrast to results obtained by
Glaichenhaus et al. (29), expression of a truncation mutant
removing the entire cytoplasmic domain of CD4 (398*/Pal
)
resulted in measurable IL-2 secretion. This difference might be due to
higher CD4 expression levels in our system. It has been shown that
surface expression of tailless CD4 at high levels is able to circumvent
the T cell development requirement for signal transduction by the
cytoplasmic domain of CD4 (38).
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Fig. 2.
Functional analysis of CD4 mutants in
antigen-dependent T cell activation. A,
expression of CD4 mutants in transgenic pools of the CD4 negative
murine T cell line 171 analyzed by flow cytometry. B,
antigen-induced IL-2 production by 171 cell lines expressing mutated
CD4 molecules. Indicated are average values of three independent
experiments with S.D. less than 15%. Removal of two hydrophobic amino
acids from the COOH terminus of CD4 (431*) results in a
gain-of-function phenotype compared with native CD4 (wt),
whereas further truncations impair IL-2 production. C,
coprecipitation of p56lck with CD4 mutants from stably
transfected 171 cells. The indicated band represents p56lck,
and the lower band present in all
lanes presumably results from cross reactivity with the
precipitated anti-CD4 rabbit antiserum. Amounts of p56lck
coprecipitating with native CD4 or the 431* mutant are
comparable.
-,
and Lck
-expressing cell lysates. Nevertheless, the
gain-of-function phenotype of 431* could still be mediated by
p56lck activity, so to exclude this possibility we analyzed CD4
function in the Lck-deficient Jurkat E6 derived cell line JCaM1 (39). Moreover, since 171 T cells are of murine origin, we also wanted to
confirm the 431* phenotype in a human assay system.
CD4
mutant had a slightly reduced effect on phorbol 12-myristate 13-acetate-induced IL-2 promoter induction, whereas the 419* and 398*/Pal
mutants were completely ineffective. These
results indicate the existence of Lck-independent CD4 signal
transduction in JCaM1 cells, presumably mediated by alternative ligands
for the CD4 cytoplasmic domain, as discussed below. Strikingly,
cotransfection of the 431* truncation mutant resulted in a moderate but
consistent increase in IL-2 promoter activity compared with native CD4,
resembling the gain-of-function phenotype in
antigen-dependent T cell activation described above.
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Fig. 3.
Transient expression of CD4 induces
stimulation of the IL-2 promoter in p56lck-deficient JCaM1
cells. A, CD4 surface expression in transiently
transfected JCaM1 cells. B, IL-2 promoter induction in JCaM1
cells expressing CD4 was quantified by luciferase activity. Indicated
are average luciferase activities relative to unstimulated, native CD4
expressing transfectants. Results are from triplicate experiments with
S.D. values less than 20%. Expression of the truncation mutant 431*
results in gain of function.
/
hydrolase fold was also
detected (41). Similarity is most significant within the region
surrounding the catalytic nucleophilic residue of this class of
hydrolases. In
/
fold hydrolases, the nucleophilic residue is
part of a structural element known as the nucleophile elbow (42), which
comprises a
strand, a
-like turn containing the nucleophilic
residue, and an
helix. Secondary structure predictions for the
region of similarity in the novel CD4-interacting protein indicate that
a motif surrounding serine 109 might adopt a similar structural fold.
Furthermore, homology searches in protein domain consensus data bases
(e.g. Pfam, Prosite profiles, ESTHER) detected significant
similarity between the newly identified protein and the family of
/
hydrolase fold proteins.
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Fig. 4.
Sequence and expression pattern of
ACP33. A, amino acid sequence of human ACP33. The
acidic cluster at the N terminus is underlined. A predicted structural
element conserved in /
fold bacterial hydrolases surrounding
serine 109 is highlighted. B, human multiple tissue Northern
blot analysis of ACP33 mRNA expression. Lane
1, poly(A)+ RNA prepared from heart;
lane 2, brain; lane 3,
placenta; lane 4, lung; lane
5, liver; lane 6, skeletal muscle;
lane 7, kidney; lane 8,
pancreas. The amount of poly(A)+ RNA has been adjusted by
the manufacturer (CLONTECH Laboratories) to obtain
an equal
-actin signal in each lane. kb,
kilobases. C, Western blot analysis of ACP33 protein
expressed in various human and murine cell lines. For detection,
hybridoma supernatant of the anti-ACP33 rat mAb 2D5 and a secondary
goat anti-rat IgG peroxidase conjugate were used. Jurkat E6, JCaM1.6,
and HUT 78 are human lymphoblastoid T cell lines, HeLa is a human
epitheloid, COS-7 a simian fibroblastoid, 171 a murine T cell
line, and FT7.1 a murine L cell fibroblast derivate.
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Fig. 5.
Subcellular distribution of endogenous and
transiently expressed chimeric ACP33 in HUT 78 T cells.
A, schematic drawing of Ig fusion proteins. B,
fractionation of hypotonic lysates of transfected HUT 78 cells into the
soluble cytosol and the particulate (i.e. membrane and
cytoskeletal) fraction. Endogenous and transfected cIg-ACP33 is
distributed between both fractions. C, immunofluorescence detection of
endogenous ACP33 (stained by mAb 2D5 and FITC anti-rat IgG), chimeric
cIg-ACP33, and cIg control (detected by FITC anti-human IgG) in HUT 78 cells. ACP33 is detected in the cytosol and on intracellular vesicles,
whereas cIg is evenly distributed throughout the cell.
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Fig. 6.
ACP33 partially colocalizes with CD4 on
TGN/endosomal vesicles. Endogenous ACP33 was detected by
immunofluorescence in HUT 78 cells using mAb 2D5 and FITC anti-rat IgG
(panels a, d, g, and
j). Golgi vesicles were marked by wheat germ
agglutinin-rhodamine (b), early endosomal vesicles by
transferrin-rhodamine (e), late endosomal/lysosomal vesicles
by DAMP and anti-DNP-rhodamine (h), and CD4 by anti-CD4
antiserum and rhodamine-conjugated anti-rabbit IgG (k).
Overlays of the two preceding panels are shown in
c, f, i, and l,
respectively.
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Fig. 7.
Mutational analysis of CD4-ACP33 interaction
by coprecipitation. A, endogenous CD4 coprecipitates
with cIg-ACP33 but not with cIg control from lysates of transiently
transfected HUT 78 cells. B, amino acid sequence surrounding
serine 109 in cIg-ACP33 and cIg-ACP33 S109A. C, CD4
coprecipitates with cIg-ACP33 from lysates of transfected COS-7 cells.
Coprecipitation is prevented by the S109A mutation in cIg-ACP33 and by
truncation of two amino acids (431*) or more from the CD4 COOH
terminus. In the second panel, expression of CD4
mutants is shown to be comparable; the third
panel documents even expression levels of cIg-ACP33 and
cIg-ACP33 S109A.
/
fold hydrolases
(42). Mutated proteins were expressed in transiently transfected COS-7
cells, and any physical interaction was determined by coprecipitation.
Similar to the results obtained in T cells, native CD4 specifically
coprecipitated with cIg-ACP33 but not the cIg control protein (Fig.
7C, lanes 1 and 3). Under these conditions, however, the stoichiometry of the CD4 coprecipitation with cIg-ACP33 was markedly higher than in T cell lysates.
Interestingly, the S109A point mutation in cIg-ACP33 completely
abolished detectable interaction with CD4 (lane
2). On the other hand, removal of the last two amino acids
from the C terminus of CD4 (431*) was sufficient to prevent
coprecipitation with cIg-ACP33 (lane 6).
Logically, further C-terminal truncation mutants of CD4 also failed to
interact with cIg-ACP33 (lanes 4 and
5). This indicates that the integrity of the hydrophobic
amino acids proline and isoleucine at the COOH terminus of CD4 are
essential for ACP33 binding. This interaction also requires a critical
serine residue in ACP33. Therefore, these findings demonstrate a
noncatalytic peptide-binding function for the
/
hydrolase fold
domain of ACP33. A nonenzymatic protein recognition function has
previously only been suggested for
/
hydrolase fold domains that
are part of the extracellular portion of cell-cell-adhesion receptors,
e.g. neuroligin (49-51).
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Fig. 8.
cIg-ACP33 and p56lck simultaneously
interact with CD4. A, COS cells were cotransfected with
expression vectors for cIg-ACP33, CD4, and p56lck. Following
immunoprecipitation (IP) of cIg-ACP33, p56lck
is detectable if coexpressed with native CD4, but not with the 431* CD4
truncation mutant unable to associate with ACP33. B,
schematic drawing of the complex formed by cIg-ACP33, CD4, and
p56lck.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
mutant retained ~50% of native CD4
activity (Fig. 3). It has been previously suggested that CD4 associates
with alternative binding partners in Lck-negative cell types,
e.g. with other Src family kinases in monocytes (52).
A recently described alternative ligand for the CD4 and CD8 cytoplasmic
tails is the phosphotyrosine adaptor protein LAT, which competes with
p56lck for CD4 and CD8 binding in T cells (24). Intriguingly,
substitution of the double cysteine motif in CD8 reduced but did not
abrogate association of LAT, implicating LAT as a candidate molecule
responsible for the observed CD4-mediated signal transduction in JCaM1 cells.
/
fold hydrolases, suggesting that a protein-protein interaction module in ACP33 has evolved from an ancient enzymatic domain during molecular evolution. Examples for noncatalytic members of
the
/
hydrolase fold-containing protein family have been described before (49). Neuroligin and related proteins, for example,
contain a noncatalytic, acetylcholinesterase-like
/
hydrolase
fold domain, which has been implicated in mediating heterophilic
cell-cell adhesion (50, 51). However, our data demonstrate for the
first time that this structural domain also serves as an intracellular
protein-protein interaction module. Similar evolutionary processes have
been proposed for transcription factors (61) and phosphatase-like
domains (62).
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ACKNOWLEDGEMENTS |
---|
We thank Rudi Grosschedl and Ernst-L. Winnacker for general support; Brian Seed, Vaclav Horejsi, Peter Rieber, Mark Hill, and Dan Littman for generous donation of materials; and Avril Arthur-Goettig (BioScript) for proofreading the manuscript.
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FOOTNOTES |
---|
* This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Sander-Stiftung.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ The first two authors contributed equally to this work.
To whom correspondence should be addressed: Genzentrum der
Universität München, Feodor-Lynen-Str. 25, D-81377
München, Germany. Tel.: 49-89-2180-6878; Fax: 49-89-2180-6999;
E-mail: kolanus@lmb.uni-muenchen.de.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M009270200
2 L. Zeitlmann, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: MHC, major histocompatibility complex; TCR, T cell receptor; IL, interleukin; HIV, human immunodeficiency virus; IRES, internal ribosomal entry sequence; MBP, maltose-binding protein; DAMP, N-(3-((2,4-dinitrophenyl)amino)propyl)-N-(3-aminopropyl)methylamine dihydrochloride; LAT, linker for activation of T cells; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; cIg, cytoplasmic Immunoglobulin; sIg, surface Immunoglobulin.
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