(Received for publication, June 9, 1995; and in revised form, August 21, 1995)
From the
CD28 and CTLA-4, T cell receptors for B7-1 (CD80) and
B7-2 (CD86) molecules on antigen-presenting cells, transmit
costimulatory signals important for optimal T cell activation. Despite
sharing sequence homology and common ligands, these receptors have
distinct binding properties and patterns of expression. The function of
CTLA-4 during T cell activation is not well understood, although an
important role is suggested by complete amino acid sequence
conservation of its cytoplasmic tail in all species studied to date. We
report here a role of the cytoplasmic tail of CTLA-4 in regulating its
subcellular localization and cell surface expression. In activated
human peripheral blood T cells, or in several transfected or transduced
cell types, CTLA-4 is not primarily a cell surface protein, but rather
is localized intracellularly in a region which overlaps the Golgi
apparatus. Transfer of 11 cytoplasmic residues, TTGVYVKMPPT, from the CTLA-4 cytoplasmic tail to the
homologous position in CD28 was sufficient to confer intracellular
localization. Mutation of the tyrosine residue (Tyr
) in
this motif to phenylalanine resulted in increased surface expression of
CTLA-4. Thus, the subcellular localization of CTLA-4 is controlled by a
tyrosine-containing motif within its cytoplasmic domain. Contained
within this motif is a binding site for SH2 domains of the p85 subunit
of phosphatidylinositol 3-kinase.
Full activation of T cells requires engagement of the TCR-CD3
complex and ligation of costimulatory receptor(s)(1) . T cells
activated in the absence of costimulatory signals may enter a state of
hyporesponsiveness or anergy(2) . Recent studies have shown
that B7-1 (CD80) and B7-2 (CD86) molecules on antigen-presenting cells
(APC) ()provide a major T cell costimulatory signal in
vitro and in vivo(3, 4) . T lymphocyte
receptors for CD80 and CD86 are CD28 and CTLA-4, homologous members of
the immunoglobulin superfamily (5) which are encoded by closely
linked genes on human chromosome 2(6) . CD28 is a low avidity
receptor for both CD80 and CD86, whereas CTLA-4 is a high avidity
receptor(7) . A highly conserved motif (MYPPPY) in the
CDR3-like region of both CD28 and CTLA-4 is involved in binding of
these receptors to CD80(8) . CD28 and CTLA-4 have different
patterns of expression during an immune response, with the former being
expressed in both resting and activated cells and the latter, only in
activated cells(9, 10) .
A major function of CD28
costimulation is to increase production of interleukin-2 and other T
cell cytokines(11, 12) . The role of CTLA-4 is less
clear, although there is complete sequence conservation in the
cytoplasmic domains of mouse(13) , human(5) , and
rabbit ()CTLA-4, which suggests conserved function(s).
Studies with mAbs against CTLA-4 have led to two different and distinct
models for the role of CTLA-4 in T cell activation(14) , either
to cooperatively up-regulate T cell activation (9, 15) or to antagonize CD28 and down-regulate T cell
activation(16) .
Studies on CTLA-4 function have been
hampered by its low level of expression. Very low levels of CTLA-4 were
detected on the cell surface of activated T cells, although RNA
transcripts were readily detectable (9, 15) . We have
also found very low levels of CTLA-4 expressed on the cell surface
following its transfection or transduction into numerous cell types. ()In contrast, cell surface expression of CD28 is easily
observed in peripheral blood T cells or in transfected or transduced
cells. These observations led us to hypothesize that expression of
CTLA-4 was subject to more complex regulation than CD28. Here we show
that one reason for the low level of CTLA-4 cell surface expression is
that it is localized primarily on intracellular membranes. Furthermore,
we show that the cytoplasmic domain of CTLA-4 contains an intracellular
localization motif which targets the protein to a perinuclear Golgi or
post-Golgi compartment in transfected cells, consequently reducing its
cell surface expression. This motif may regulate CTLA-4 surface
expression and function during T cell activation.
For transient COS cell transfection, cDNAs were excised from the retroviral constructs with HindIII and ClaI, blunt-ended with Klenow DNA polymerase, and ligated into the EcoRV site of the expression vector pcDNA1 (Invitrogen, San Diego, CA). Truncation, homologue swap and point mutations were constructed using polymerase chain reaction (8) and were cloned into pcDNA1. The amino acid sequences surrounding the replacement regions of R1-R5 are shown in Table 1. All mutations were confirmed by DNA sequencing.
For immunoprecipitation of cell surface CTLA-4 or CD28 molecules (surface immunoprecipitation), cells were resuspended in 0.5 ml of phosphate-buffered saline containing 10 µg of CD80Ig or anti-CD28 mAb 9.3 and incubated on ice for 30 min. After two washes with ice-cold phosphate-buffered saline, cells were dissolved in 0.5 ml of lysis buffer (17) containing 5 µg of CTLA-4t or CD28t to saturate unoccupied antibody binding sites. Immune complexes were immediately precipitated with immobilized protein A. For immunoprecipitation of total CTLA-4 or CD28 molecules (totalintraperitoneal), samples were directly lysed and immunoprecipitation was performed with CD80Ig or anti-CD28 mAb 9.3, followed by immobilized protein A. Immunoprecipitates were treated with endoglycosidase H (endo H, Boehringer Mannheim) as suggested by the manufacturer. Immunoprecipitates were analyzed by SDS-PAGE.
Figure 1:
Posttranscriptional regulation of
CTLA-4 cell surface expression on activated T cells. A,
activated T cells contain equivalent amounts of mRNA for CD28 and
CTLA-4. Peripheral blood T cells were activated by anti-CD3 mAb
stimulation for 3 days. Cells were then harvested, total RNA was
extracted, and the indicated amounts were analyzed by blot
hybridization analysis using P-labeled probes for CD28 and
CTLA-4. The blot was hybridized first with the CD28 probe, stripped,
and then hybridized with the CTLA-4 probe. B, cell surface
expression of CD28 is much higher than cell surface expression of
CTLA-4 on activated T cells. T cells activated as in A were
analyzed for cell surface expression of CD28 or CTLA-4 by staining with
biotinylated anti-CD28 mAb 9.3 or anti-CTLA-4 mAb 11D4, respectively,
followed by PE-SA. Stained cells were then analyzed by flow cytometry. Solid lines, histograms obtained with mAbs 9.3 or 11D4; dotted lines, histograms obtained with isotype control
mAbs.
Figure 2: Intracellular localization of CTLA-4 in activated T cells. Peripheral blood T cells were activated with anti-CD3 mAb and treated with or without saponin. Intact (-Saponin) or permeabilized (+Saponin) cells were stained with an IgG2a isotype control mAb (an IgG1 mAb gave identical results), or with anti-CD28 mAb 9.3, or anti-CTLA-4 mAb 11D4, followed by FITC-GAM. Stained cells were then fixed with paraformaldehyde and analyzed by confocal microscopy.
The increase in CTLA-4 staining following saponin
treatment was quantitated by flow cytometry. In five experiments with T
cell blasts activated by anti-CD3 treatment for 3 days, CTLA-4
expression was consistently increased by treatment with saponin prior
to staining (mean increase of 5.4 ± 1.7-fold). Excess unlabeled
CTLA4Ig blocked the intracellular staining, indicating that it was
specific (data not shown). The increase in CTLA-4 staining intensity
following saponin treatment was also observed when T cells were
activated for 3 days with phytohemaglutinin. In comparison, the
intensity of CD28 staining was not consistently affected by saponin
treatment (mean increase 1.5 ± 1.1-fold). Staining intensities
obtained with anti-CD2, anti-CD4, and anti-CD7 mAbs decreased or
remained constant following saponin treatment (data not shown). Thus,
CTLA-4 staining was increased 5-fold by mild detergent treatment
of activated T cells, whereas staining of other T cell surface antigens
was much less affected.
We also examined the subcellular
distribution of CD28 and CTLA-4 by comparing their accessibilities with
lactoperoxidase-mediated iodination. Similar levels of I-labeled CD28 were immunoprecipitated following
iodination of intact cells or isolated membranes, but
7-fold more
I-labeled CTLA-4 was immunoprecipitated from isolated
membranes than from intact cells. Therefore, CTLA-4 was more accessible
to lactoperoxidase-catalyzed iodination when the cell surface membrane
was disrupted. This difference could not be explained by exposure of
new sites for iodination in the predicted intracellular portion of
CTLA-4, since only two of the nine tyrosine residues are contained in
this region. Identical results were obtained whether CTLA-4 was
immunoprecipitated with mAb 11D4 or CD80Ig, indicating that CTLA-4
exposed by membrane disruption could bind to a physiological ligand(s).
Taken together, the data suggest that most CTLA-4 in activated T cells
is associated with intracellular membranes, rather than the cell
surface.
Figure 3: The cytoplasmic tail of CTLA-4 inhibits its own surface expression. Left column, the cytoplasmic tail of CTLA-4 inhibits cell surface expression of CD28. The indicated cells were transfected or transduced with full-length CD28 cDNA (solid lines) or the chimeric cDNA C28-4 (dashed lines) and stained with biotinylated anti-CD28 mAb 9.3, followed by PE-streptavidin. Wild type (C8.A3, 3T3) or mock-transfected (COS) cells were used as negative controls (dotted lines). Right column, the cytoplasmic tail of CD28 increases cell surface expression of CTLA-4. The indicated cells were transfected or transduced with full-length CTLA-4 cDNA (solid line) or chimeric cDNA C4-28 (dashed line) and stained with biotinylated anti-CTLA-4 mAb 11D4, followed by PE-streptavidin. Wild type (C8.A3, 3T3) or mock-transfected (COS) cells were used as negative controls (dotted lines).
In other experiments, we tested by flow cytometry whether the reduced surface expression of constructs containing the intracellular region of CTLA-4 was associated with intracellular accumulation of these proteins. 3T3 cells transduced with CD28 showed similar levels of CD28 expression before or after saponin treatment, but expression of CTLA-4 was markedly increased following saponin treatment. Specificity of intracellular staining following saponin treatment was indicated by the ability of CD28Ig or CTLA4Ig to inhibit staining with PE-conjugated specific mAbs. Similar results were obtained when CTLA-4 was expressed in C8.A3 and COS cells. In all cases, saponin treatment increased staining with anti-CTLA-4 mAb 11D4, but not staining with anti-CD28 mAb 9.3. The cytoplasmic tail of CTLA-4 therefore is responsible for its retention in an intracellular compartment in both lymphoid and non-lymphoid cells.
We further characterized the intracellular compartment in which CTLA-4 was retained in 3T3 cells using confocal microscopy (Fig. 4). 3T3-transduced cells were fixed and permeabilized with methanol and stained with anti-CTLA-4 mAb 11D4. The same cells were also stained with LcL, a marker for the Golgi apparatus(23) . This lectin recognizes fucosylated N-linked carbohydrate side chains that terminate with galactose, N-acetylglucosamine or sialic acid(24) , structures found in the Golgi apparatus, but not the endoplasmic reticulum(25) . With CTLA-4-transduced cells, the staining pattern obtained with anti-CTLA-4 mAb 11D4 overlapped with staining obtained with LcL. In both cases, intracellular perinuclear vesicular staining was seen. With C4-28-transduced cells, CTLA-4 staining was primarily cell surface-associated, although intracellular perinuclear vesicular staining was also seen. These findings demonstrate that the CTLA-4 cytoplasmic tail directs its localization to an intracellular compartment which overlaps with the Golgi. Control experiments established that the coincidence of the two staining patterns was not caused by ``spillover'' of the emission of either chromaphore into the detection system for the other.
Figure 4: The cytoplasmic tail of CTLA-4 directs its expression to a subcellular compartment which overlaps the Golgi apparatus. Wild type 3T3 cells, or 3T3 cells transduced with full-length CTLA-4 or chimeric (C4-28) CTLA-4 cDNAs, were permeabilized with methanol, and stained with biotinylated LcL, anti-CTLA-4 mAb 11D4, or a combination of the two. Cells were then incubated with Texas Red-conjugated streptavidin and FITC-GAM and analyzed by confocal microscopy. Left column, LcL binding as detected by Texas Red fluorescence; middle column, mAb 11D4 binding as detected by FITC fluorescence; and right column, overlay of red and green fluorescence patterns.
Figure 5: Identification of a motif in the CTLA-4 cytoplasmic tail that inhibits surface expression in COS cells. A, comparison of the CD28 and CTLA-4 cytoplasmic tails. Numbering refers to the mature polypeptides. The ends of the C28-4 carboxyl-terminal truncations are indicated below the CTLA-4 cytoplasmic tail (&cjs1219;). The underlined region of CTLA-4 represents the residues transferred in mutant R1. B, schematic representation of mutations used to localize the CTLA-4 intracellular localization motif. Expression plasmids directing expression of CD28, CTLA-4, and various mutants of these were constructed and expressed in COS cells as described under ``Materials and Methods'' and Table 1. The extracellular and transmembrane (TM) domains of the constructs are listed, and the cytoplasmic tails are diagrammed schematically. Open bars represent CD28 sequences, while solid bars represent CTLA-4 sequences. The consensus PI3K binding sites (YMNM and YVKM) are indicated above. On the right are given MFI values determined after staining transfected COS cells with biotinylated mAbs to the corresponding extracellular domains (anti-CD28 mAb 9.3 or anti-CTLA-4 mAb 11D4), followed by PE-SA. Values have been corrected for background by subtracting the MFI of mock transfected COS cells (4 units).
Figure 6: Immunofluorescence patterns of CD28, mutant R1, CTLA-4, and mutant Y165F. COS cells were transfected with expression plasmids encoding the indicated proteins. Forty-eight hours following transfection, cells were fixed with methanol and stained with biotinylated anti-CD28 mAb 9.3 or anti-CTLA-4 mAb 11D4 followed by PE-SA (red fluorescence). Nuclei were stained with YO-PRO-1 (green fluorescence).
Analysis of carboxyl-terminal
truncations of C28-4 showed that up to 16 terminal residues could be
deleted from the cytoplasmic tail of C28-4 without significantly
changing surface expression levels (compare D8 and D16). When 20
residues were deleted (D20), a modest increase in surface expression
was detected by flow cytometry. When 23 residues were deleted from
C28-4 (D23), surface expression increased further. These results
suggested that the carboxyl terminus of a localization motif was
between Met and Thr
. We then tested
homologue swap mutants in which small regions of the cytoplasmic tail
of CD28 were replaced by the corresponding residues from CTLA-4 (see Table 1). As shown in Fig. 5B, COS cells
transfected with mutant R1 had the greatest reduction in surface
expression (similar to C28-4). Mutants R2-R4 gave partial
reductions in surface expression, whereas mutant R5 had surface
expression similar to CD28. Immunofluorescence microscopy experiments
showed that mutant R1 had primarily intracellular localization (Fig. 6), whereas mutants R2-R5 were primarily cell
surface-associated (data not shown). Mutant R1 therefore contains the
smallest CTLA-4 region which inhibits surface expression when
transferred to CD28.
We next introduced into CTLA-4 mutations at
each of the 11 amino acid residues contained in R1. Substitution of
alanine for each residue (except for Tyr) did not
increase surface expression of CTLA-4 (data not shown). To test the
involvement of Tyr
in regulation of cell surface
expression, we introduced a tyrosine to phenylalanine mutation into
this position (mutant Y165F). This mutation increased surface staining
for CTLA-4 (Fig. 5B and Fig. 6), indicating that
Tyr
regulates cell surface expression.
We also tested by flow cytometry whether mutant R1 accumulated in an intracellular compartment(s). When COS cells transfected with R1 were treated with saponin prior to staining with PE-conjugated anti-CD28 mAb 9.3, the fraction of cells staining was clearly increased. The MFI of the total population (after correction for background) increased noticeably from 3 to 11 following saponin treatment, whereas the MFI of CD28-transfected cells decreased slightly from 55 to 38. Staining was specific because it was decreased down to background levels by addition of CD28Ig. These results are in agreement with immunofluorescence experiments (Fig. 6) showing that mutant R1 was primarily localized in an intracellular compartment.
Mutant R1 also was expressed at lower levels than CD28. We reproducibly detected 3-8-fold lower amounts of mutant R1 in transfected COS cells, as judged by specific immunoassays (data not shown). The basis for this reduced expression is not currently known, although all molecules containing the CTLA-4 cytoplasmic tail showed a similar reduction in expression. With C28-4, protein expression was reduced when compared with CD28 observed, even though similar levels of RNA transcripts were detected. This suggested that as yet unidentified posttranscriptional mechanisms may regulate the levels of CTLA-4 expression.
Figure 7:
Mutant R1 is preferentially retained
inside COS cells. COS cells were transfected with expression plasmids
containing full-length CD28 cDNA or mutant R1. Transfected cells were
metabolically labeled with [S]methionine for 15
min at 37 °C and then incubated for varying periods of time at 37
°C in medium containing excess unlabeled methionine. At each of the
indicated chase times, equal numbers of cells were subjected to surface
immunoprecipitation (surface i.p.) or total immunoprecipitation (total
i.p.) with anti-CD28 mAb 9.3 as described under ``Materials and
Methods.'' Top and middle, autoradiograms of 16%
SDS-PAGE gels run under reducing conditions. Bottom, the
amounts of radioactivity precipitated at each time by surface and total
immunoprecipitation were estimated by densitometry of the
autoradiograms shown above. The ratio of surface radioactivity to total
radioactivity at each time was plotted. Open circles,
CD28-transfected cells; closed circles, mutant R1-transfected
cells.
We also used pulse-chase analysis to compare the intracellular
localization of CTLA-4 and mutant Y165F (Fig. 8). During the
90-min chase period, very little wild type CTLA-4 accumulated at the
cell surface, as detected by surface immunoprecipitation, whereas
mutant Y165F rapidly appeared at the cell surface, reaching a maximum
at 40 min. Very similar amounts of radioactive CTLA-4 and Y165F
were obtained by total immunoprecipitation, in agreement with
immunoassays showing that these proteins were expressed at similar
levels (data not shown). Normalization of the amount of radioactivity
in surface immunoprecipitation for the amount in total
immunoprecipitation indicated that
4-fold more mutant Y165F
accumulated at the cell surface than wild type CTLA-4. Moreover,
accumulation of surface CTLA-4 was steady and slow, suggesting that
newly synthesized CTLA-4 molecules were blocked during surface
translocation, rather than being rapidly internalized after reaching
the surface. In other experiments, later time points were studied, but
the fraction of CTLA-4 and Y165F at the cell surface did not increase
further beyond
90 min. CD80Ig was used for immunoprecipitation
analysis in Fig. 9. Since similar amounts of CTLA-4 and mutant
Y165F were purified by total immunoprecipitation, intracellular CTLA-4
was able to bind its physiological ligand, and hence, was biologically
active.
Figure 8: Tyrosine 165 controls intracellular retention of CTLA-4 in COS cells. COS cells were transfected with expression plasmids containing full-length CTLA-4 cDNA or mutant Y165F, metabolically labeled, and subjected to immunoprecipitation analysis with CD80Ig as described in the legend to Fig. 8. Top and middle, autoradiograms of 12% SDS-PAGE gels run under reducing conditions. Bottom, the amounts of radioactivity precipitated at each time by surface and total immunoprecipitation were estimated by densitometry of the autoradiograms shown above. The ratio of surface radioactivity to total radioactivity at each time was plotted. Open circles, CTLA-4-transfected cells; closed circles, mutant Y165F- transfected cells.
Figure 9:
CTLA-4 and mutant Y165F acquire resistance
to endo H with similar kinetics. COS cells were transfected with
expression plasmids encoding either wild type CTLA-4 or mutant Y165F
and metabolically labeled with [S]methionine as
in Fig. 8. After the indicated times of chase with unlabeled
methionine, equal numbers of cells were subjected to
immunoprecipitation analysis. Identical immunoprecipitates were treated
without(-) or with (+) Endo H before analysis by SDS-PAGE
(12% gel) under reducing conditions. The positions of molecular weight
standards are indicated on the right.
To further characterize the intracellular compartment where
CTLA-4 was retained, we performed another pulse-chase analysis and
tested the sensitivity of CTLA-4 and mutant Y165F to endo H during the
chase period (Fig. 9). Both CTLA-4 and mutant Y165F were
initially endo H-sensitive but became resistant during the chase
period. The kinetics of acquisition of resistance to endo H were
similar for both proteins (t
30 min). This
indicates that their transit times were similar through the endoplasmic
reticulum into the middle to trans- Golgi, where
-mannosidases
responsible for processing of N-linked carbohydrates are
localized(26) .
CD28 and CTLA-4 are homologous molecules with features
typical of cell surface membrane receptors(5, 6) . We
have shown here that in activated human peripheral blood T cells CTLA-4
was primarily (80%) localized on intracellular membranes rather
than at the cell surface. Intracellular localization of CTLA-4 was also
observed in several cell types transduced or transfected with CTLA-4
cDNA, indicating that intracellular localization is an inherent
property of the CTLA-4 protein and not the particular cell type in
which it is expressed. In contrast, CD28 was primarily expressed at the
cell surface, in agreement with numerous studies implicating it as a T
cell surface receptor(11, 12) . Thus, in addition to
having different avidities for B7 ligands(7) , and different
patterns of expression(9, 10) , CD28 and CTLA-4 have
different subcellular localization. This further argues that these
molecules have different functions during T cell activation (14) .
Using mutational analysis, we identified an
intracellular localization motif in the CTLA-4 cytoplasmic domain which
limits its cell surface expression in COS cells and localizes it to an
intracellular region. Mutant R1 showed reduced cell surface expression
and primarily intracellular localization. In contrast, mutants R2, R3,
and R4, which contain only some of the CTLA-4 residues present in R1,
showed greater cell surface expression and less intracellular
localization. Therefore, the 11 residues, TTGVYVKMPPT,
represent the minimal region in the CTLA-4 cytoplasmic domain to confer
full intracellular localization function when transferred to the
analogous positions of CD28.
Mutant Y165F showed greater cell
surface expression and less intracellular expression than wild type
CTLA-4. This indicates that Tyr is critical for
intracellular localization function. Several other tyrosine-containing
intracellular internalization/localization motifs have been identified,
such as in transferrin receptor, low density lipoprotein receptor,
cation-independent mannose 6-phosphate receptor, and the trans-Golgi
protein TGN38(27, 28) . However, none of these motifs
bears obvious homology with the CTLA-4 localization motif. Moreover, in
transferrin, low density lipoprotein, and cation-independent mannose
6-phosphate receptors, phenylalalnine can substitute for the tyrosine
with no loss of activity(27) . Intracellular localization of
CTLA-4 could be due to the lack of transport proteins required to
target CTLA-4 to the cell surface or to retention of CTLA-4 by proteins
interacting with the intracellular localization motif. Since disruption
of the CTLA-4 intracellular localization motif increased cell surface
expression, we favor the latter possibility. Although intracellular
localization motifs have been identified in many proteins, less is
known about protein(s) interacting with these motifs (reviewed in (29) ).
Intracellular staining of CTLA-4 overlapped with the
staining pattern of LcL, a marker for the Golgi apparatus(23) .
Further evidence as to the site of intracellular localization of CTLA-4
comes from the kinetics of acquisition of resistance to endo H. CTLA-4
became endo H-resistant with identical kinetics as mutant Y165F, which
is transported 4-fold more efficiently to the cell surface. Thus,
the block in transport of CTLA-4 to the cell surface occurs subsequent
to processing of high mannose N-linked carbohydrate chains,
which occurs in the middle to trans-Golgi(25, 26) .
Taken together, these data suggest that CTLA-4 is retained in a trans-
or post-Golgi compartment.
The CTLA-4 intracellular localization motif contains a consensus binding site (-YVKM-) for the Src homology 2 (SH2) domains of the p85 subunit of phosphatidylinositol 3-kinase (PI3K)(30) . Previous studies showed that anti-CTLA-4 mAbs triggered binding of p85 to this site in the cytoplasmic domain of CTLA-4 in an HTLV I-transformed T cell line (31) . The CD28 cytoplasmic domain also contains a similar motif (YMNM) which binds p85 following engagement of CD28 with mAbs or its CD80 ligand(32, 33, 34, 35, 36, 37) . Thus, the intracellular localization motif of CTLA-4 and the analogous region of CD28 contain sequence(s) involved in signaling through these receptors. However, our data provide evidence that these sites may not be functionally equivalent in CD28 and CTLA-4, since the site in CTLA-4 has intracellular localization function, whereas that in CD28 does not. Another difference in cytoplasmic domains of CTLA-4 and CD28 was reported by Stein et al.(33) , who showed that CD8 chimeras containing the CD28 cytoplasmic tail bound PI3K and triggered interleukin-2 production, but that CD8/CTLA-4- cytoplasmic tail chimeras did not. Taken together, these studies suggest that the YXXM-containing motif in CTLA-4 and CD28 may share some functions, but differ in others.
The overlap between the CTLA-4
internal localization motif and the binding site for p85 PI3K suggests
the possibility of PI3K involvement in the intracellular localization
of CTLA-4. Recent evidence has implicated PI3K in regulation of
cellular protein trafficking. A yeast PI3K homologue functions in
protein sorting of vacuolar hydrolases(38, 39) . Other
studies have suggested a role for PI3K in internalization of
platelet-derived growth factor receptor following ligand
binding(40) . If PI3K binding to the cytoplasmic domain of
CTLA-4 requires phosphorylation of Tyr(31) , it
seems likely that involvement of PI3K in intracellular localization
would also require phosphorylation of this residue. The disruption of
intracellular localization of CTLA-4 by mutation of Tyr
to phenylalanine would be consistent with such a requirement.
However, other roles for Tyr
independent of its
phosphorylation are also possible. Conclusive demonstration of a role
for PI3K in intracellular localization of CTLA-4 will require direct
biochemical demonstration of interaction between these proteins.
The
intracellular localization of CTLA-4 was unexpected, and the reason(s)
for it are currently unclear. Several possible explanations can be
envisioned. Intracellular CTLA-4 could function in an autocrine
fashion. Activated T cells express
B7-1(41, 42, 43) , so it is possible that
intracellular interaction between B7-1 and CTLA-4 can occur in
activated T cells and that CTLA-4 in fact functions inside the cell.
Alternatively, intracellular retention of CTLA-4 may delay surface
transport until it is assembled into a multisubunit complex. Parallels
of this scenario can be found in the expression of CD3 , major
histocompatability complex class I and class II molecules, where newly
synthesized molecules are retained in the endoplasmic reticulum by the
chaperonin
calnexin(44, 45, 46, 47) . Possibly,
such a complex might function at a particular stage of T cell
development and/or activation and the cells we tested may lack other
components of such a complex.
Finally, intracellular localization of CTLA-4 may indicate a mechanism to achieve subtle control of its expression on the T cell surface. Since CTLA-4 has such high avidity for B7 molecules, its ability to bind B7 molecules is disproportionate to its levels of expression(9) . It may therefore be necessary to tightly regulate and focus the small number of CTLA-4 molecules on the cell surface following T cell activation. The pattern of intracellular localization of CTLA-4 overlaps that of the Golgi complex, i.e. it is polarized to one side of the cell. Following cell-cell contact, the Golgi apparatus is typically reoriented such that it faces the site of cell contact (48) . This results in the directional release of secretory proteins such as cytokines proteins toward sites of cell-cell contact(49, 50) . Since secretory and membrane proteins share common pathway(s) of protein export, directional release of membrane proteins may also occur. Polarization of the Golgi complex and/or intracellular CTLA-4 during activated T cell-APC interactions could lead to preferential organization of intracellular CTLA-4 facing toward sites of APC contact. This could localize CTLA-4 at sites of APC contact and regulate the ability of CTLA-4 to function during an immune response (14) . The existence of such a mechanism to control surface expression of CTLA-4 would further argue for the importance of its role during T cell activation.