From the Laboratory of Molecular Tumor Biology,
Division of Cellular and Gene Therapies, and § Retroviral
Immunology Section, Division of Viral Products, Center for Biologics
Evaluation and Research, Food and Drug Administration,
Bethesda, Maryland 20892
Received for publication, January 31, 2001, and in revised form, May 11, 2001
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ABSTRACT |
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Interleukin (IL)-13 receptor Unlike receptors for the related cytokine
IL-4,1 the receptors for
IL-13 (IL-13R) have not been well characterized. We have been studying
the structure of IL-13R in various cell types (1-6). We reported that
IL-13 binds to two isoforms of 65-kDa proteins in human renal cell
carcinoma cells, and one of these proteins also binds IL-4 (1). On the
basis of binding characteristics, cross-linking, and displacement of
radiolabeled IL-4 and IL-13 in various cell types, we hypothesized
that, similar to the IL-4R system, IL-13R may also exist as three
different types (1-6). Two different chains of the IL-13 receptor,
IL-13R Cells selectively internalize specific surface ligand-receptor
complexes through receptor-mediated endocytosis. This process of
endocytosis begins when receptors are selectively sequestered into
specialized structures on the plasma membrane, termed clathrin-coated pits. These pits are able to recognize receptors through short structures of amino acids in the cytoplasmic domains (12-15). These domains contain specific targeting information. The most common internalization signals described are the tyrosine-based motif and the
dileucine motif. The tyrosine-based motif contains a tyrosine residue
usually composed of 4-6 amino acids and is generally formed of
NPXY or YXXØ (where X is any amino
acid and Ø is a hydrophobic residue; Refs. 16-19). There are various
examples that utilize NPXY or YXXØ motifs for
endocytosis. Although the precise mechanism for the sequestration of
surface receptors in coated pits is unknown, low density lipoprotein
receptors are shown to be endocytosed via their NPXY motif
(20). Similarly, numerous other cell surface proteins including
epidermal growth factor receptor, insulin receptor family, the
Recently, we have demonstrated that the IL-13R Recombinant Cytokine, Toxin, and Cell Culture--
Recombinant
human IL-13 was produced and purified in our laboratory (39).
Recombinant IL13-PE38QQR was also produced and purified in our
laboratory.2 Monkey kidney
fibroblast (COS-7) cell line was purchased from the American Type
Culture Collection (Manassas, VA). Cells were cultured in DMEM
containing 10% fetal bovine serum (BioWhittaker, Walkersville, MD), 1 mM HEPES, 1 mM L-glutamine, 100 µg/ml penicillin, and 100 µg/ml streptomycin (BioWhittaker).
Mutagenesis and Transient Transfection of DNA--
cDNAs of
the human IL-13R
Plasmid DNAs (12 µg/100-mm culture dish) were transfected into
semiconfluent cells using GenePORTER transfection reagent (Gene Therapy
Systems, San Diego, CA) according to the manufacturer's instructions.
Briefly, cells (2 × 106/100-mm dish) were incubated
with the DNA-GenePORTER mixture for 5 h in DMEM (BioWhittaker).
Then DMEM containing 20% FBS was added, and incubation was continued.
24 h after transfection, the medium was changed to DMEM with 10%
fetal bovine serum, and the cells were incubated for an additional
24 h.
RT-PCR--
To detect the mRNA expression of the IL-13R Radioreceptor Binding Assay--
Recombinant human IL-13 was
labeled with 125I (Amersham Pharmacia Corp.) using IODO-GEN
reagent (Pierce) as previously described (1). The specific activity of
the radiolabeled IL-13 was estimated to be 6.0 µCi/µg of protein.
For binding experiments, 5 × 105 cells in 100 µl of
binding buffer (RPMI 1640 containing 0.2% human serum albumin and 10 mM HEPES) were incubated with 200 pM 125I-IL-13 with or without 40 nM unlabeled
IL-13 at 4 °C for 2 h. Cell-bound 125I-IL-13 was
separated from unbound by centrifugation through a phthalate oil
gradient, and radioactivity was determined with a Internalization Assay--
Internalization assays were performed
as described before (10, 41). COS-7 cells transfected with the
IL-13R
To determine if proteasome-mediated proteolysis is involved in the
expression and internalization of IL-13R Protein Synthesis Inhibition Assay--
The cytotoxic activity
of IL-13 toxin was tested as previously described (38). Typically,
104 cells/well were cultured in leucine-free medium with or
without various concentrations of IL13-PE38QQR for 20-22 h at
37 °C. Then 1 µCi of [3H]leucine (PerkinElmer Life
Sciences) was added to each well and incubated for an additional 4 h. Cells were harvested, and radioactivity incorporated into cells was
measured by a Construction of IL-13R 125I-IL-13 Binding to IL-13R Dileucine Motif-mediated Internalization of IL-13R Tyrosine Motif-mediated Internalization of IL-13R
To further study this phenomenon and determine whether both dileucine
motif and tyrosine motif mutatation in the IL-13R Effect of Co-expression of Wild Type IL-13R Cytotoxicity of IL13-PE38QQR to IL-13R Proteasome Inhibitor MG132 Does Not Alter Internalization or
Stabilize the Ligand Dissociation from IL-13R In this study, we have characterized the molecular basis for
IL-13-mediated internalization of the IL-13R Although mRNA expression was confirmed by RT-PCR, a truncation
mutation, To characterize the role of the dileucine motif in a trileucine region
in the internalization process, eight IL-13R When all three leucine residues were converted to isoleucine
(L335I/L336I/L337I), no diminution of internalization was observed. This is in contrast to IL-6R gp130, in which mutation of the first leucine (Leu145) to isoleucine resulted into diminished
internalization function (23). However, in the wild type leukemia
inhibitory factor receptor (LIFR), a leucine-isoleucine internalization
motif exists naturally (43). Furthermore, in IL-6R, GLUT4, and CD4, the
dileucine motif acts in cooperation with an upstream serine for
internalization (23, 44, 45). Similarly, CD3 It is of interest to note that when double or triple leucine mutants
(L335A/L337A and L335A/L336A/L337A) were transfected with the wild type
IL-13R Because the IL-13R Several studies have suggested that covalent modification of proteins
such as ubiquitination can modulate receptor internalization and
ligand-induced signal transduction (48-50). Generally, target proteins
are tagged with multiple small protein ubiquitin, which are then
destroyed by the proteasome complex. Ubiquitination has been shown to
regulate IL-2-induced signal transduction through stabilization of
STAT5 activation (49) and IFN- In summary, we have characterized the internalization motifs in the
IL-13R2 (IL-13R
2)
chain is an essential binding component for IL-13-mediated ligand
binding. Recently, we have demonstrated that this receptor chain also
plays an important role in the internalization of IL-13. To study the
mechanism of IL-13 internalization, we generated mutated IL-13R
2
chains that targeted trileucine residues (Leu335,
Leu336, and Leu337) in the transmembrane domain
and a tyrosine motif (Tyr343) in the intracellular
domain and transfected these cDNAs in COS-7 cells. Cells that
expressed a C-terminally truncated IL-13R
2 chain (
335) did not
bind IL-13, suggesting that the trileucine region modulates IL-13
binding. Truncation of IL-13R
2 chain with a mutation in the
trileucine region resulted in significantly decreased internalization
compared with wild type IL-13R
2 chain transfected cells. COS-7 cells
transfected with tyrosine motif mutants exhibited a similar
internalization level compared with wild type IL-13R
2 chain
transfected cells; however, dissociation of cell surface IL-13 was
faster compared with wild type IL-13R
2 transfectants. These results
were further confirmed by determining the cytotoxicity of a chimeric
protein composed of IL-13 and a mutated form of Pseudomonas
exotoxin (IL13-PE38QQR) to cells that expressed IL-13R
2 chain
mutants. We further demonstrate that the IL-13R
2 chain is not
ubiquitinated and that internalization of IL-13R
2 did not depend on
ubiquitination. Together, our findings suggest that the dileucine motif
in the trileucine region and tyrosine motif participate in IL-13R
2
internalization in distinct manners.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and IL-13R
2 (also known as IL-13R
' and IL-13R
,
respectively), have been cloned and correspond to the two 65-kDa
isoforms as we originally proposed (1). The murine and human IL-13R
1
chains were cloned first (7, 8). This chain binds IL-13 at low levels,
but when coupled with the IL-4R
chain (also known as IL-4R
) it
binds IL-13 with higher affinity and mediates IL-13-induced signaling (9, 10). The second chain of IL-13R, termed IL-13R
2, has been cloned
from a human renal cell carcinoma cell line (Caki-1). This chain has
50% identity to the IL-5R at the DNA level, has a short intracellular
domain, and binds IL-13 with high affinity (11).
-subunits of three integrin receptors, and the amyloid A4 precursor
protein utilize the NPXY motif for internalization (20). On
the other hand, the transferrin receptor and the asialoglycoprotein receptor endocytose via a YXXØ motif (16, 21). It has been demonstrated that a dileucine motif in the intracellular domain of
various receptor systems (e.g. interleukin-6 receptor
(IL-6R) gp130, granulocyte colony-stimulating factor receptor,
epidermal growth factor receptor, growth hormone receptor, human
insulin receptor,
2-adrenergic receptor,
lutropin/choriogonadotropin receptor, and erythropoietin receptor)
plays an essential role in the internalization of ligand (22-30).
2 chain plays a
critical role in ligand binding and internalization (10, 31). After
binding to its receptor, IL-13 can signal through the c-Jun-activated
kinase/STAT signal transduction pathway (4-6, 32-35). Although we and
others have reported that IL-13R
2 does not participate in the signal
transduction pathway, it can bind and rapidly internalize IL-13 (10,
31, 36). However, the mechanism of how the IL-13R
2 chain mediates
internalization is unknown (10). To address this issue, we generated
IL-13R
2 chain mutants that were transfected in COS-7 cells. The
roles of the trileucine motif (positions Leu335,
Leu336, and Leu337) in the C terminus of the
transmembrane domain and the tyrosine motif (position
Tyr343) in the intracellular domain of the IL-13R
2 chain
were studied in internalization assays using 125I-IL-13.
Internalization assays were also performed by determining the
cytotoxicity of a chimeric protein composed of IL-13 and a mutated form
of Pseudomonas exotoxin (IL13-PE38QQR) (31, 37, 38) to cells
that were transfected with IL-13R
2 chain mutants. IL13-PE38QQR binds
to IL-13R and is internalized by endocytosis, subsequently causing cell
death through the inhibition of new protein synthesis. Thus,
cytotoxicity observed in transfected cells indicates receptor
internalization. Here we demonstrate that the dileucine motif in a
trileucine region is critical for ligand binding and internalization,
while the tyrosine motif is not responsible for internalization.
Instead, the tyrosine motif appears to be responsible for cell surface
IL-13 binding characteristics. We further demonstrate that the
IL-13R
2 chain is not ubiquitinated.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 chain (wild type; Ref. 11) were cloned into a
pCI-neo mammalian expression vector (Promega, Madison, WI). The
IL-13R
2 deletion mutants
335,
338,
343, and Y343F were
constructed by polymerase chain reactions (PCRs) using
Taq Gold DNA polymerase (PerkinElmer Life Sciences) and pME18 s-IL13R
2 as a template (Ref. 31) with the primer
5'-CCGCTCGAGATGGCTTTCGTTTGCTTGGCTATCGG-3' and 3'-
GCTCTAGATCAACCGGTTACAAATATAACTAATATTAAG-5' (
335) or
3'-GCTCTAGATCACAAAAGCAGACCGGTTACAAATATAAC-5' (
338) or
3'-GCTCTAGATCAGGTGTTTGGCTTACGCAAAAG-5' (
343) or
3'-GCTCTAGATCATGTATCACAGAAAAATTCTGGAATCATTTTTGGGAAGGTG-5' (Y343F), each
containing an in-frame stop codon. For the other IL-13R
2 mutants,
PCR was performed using the primer
5'-CCGCTCGAGATGGCTTTCGTTTGCTTGGCTATCGG-3' and
3'-GCACCGGTTACAAATATAACTAATAAGATGAAACC-5' containing an AgeI restriction site, and 5'-primers containing an AgeI
restriction site and 3'-GAGCTCGGTACCCGGGGATCCAGAC-5'. Other 5'-primers
with an AgeI restriction site were as follows:
5'-GTAACCGGTGCGCTTTTGCGTAAGCCAAACACCTACCCAAAAATG-3' (L335A),
5'-GTAACCGGTCTGGCTTTGCGTAAGCCAAACACCTACCCAAAAATG-3' (L336A), 5'-GTAACCGGTCTGCTTGCGCGTAAGCCAAACACCTACCCAAAAATG-3' (L337A),
5'-GTAACCGGTGCGCTTGCGCGTAAGCCAAACACCTACCCAAAAATG-3' (L335A/L337A),
5'-GTAACCGGTGCGGCTGCGCGTAAGCCAAACACCTACCCAAAAATG-3' (L335A/L336A/L337A), or
5'-GTAACCGGTATTATTATTCGTAAGCCAAACACCTACCCAAAAATG-3' (L335I/L336I/L337I). These PCR products were digested with
AgeI restriction enzyme and ligated with the DNA Ligation
Kit version 1 (TAKARA Shuzo, Shiga, Japan). Using these ligation
reaction mixtures as template, PCRs were performed with the primer
5'-CCGCTCGAGATGGCTTTCGTTTGCTTGGCTATCGG-3' and
3'-GCTCTAGATCATGTATCACAGAAAAATTCTGG-5' or
3'-GCTCTAGATCATGTATCACAGAAAAATTCTGGAATCATTTTTGGGAAGGTG-5' (L335A/L336A/L337A/Y343F) containing an in-frame stop codon. Finally, the mutant cDNAs for the IL-13R
2 were subcloned into the
expression vector pCI-neo using the XhoI and XbaI
sites. All constructs were verified for sequence by ABI Prism 310 (PerkinElmer Life Sciences).
2
chain in DNAs-transfected COS-7 cells, total RNA was isolated using
TRIZOL reagent (Life Technologies, Inc.), and then RT-PCR analysis was
performed. 2 µg of total RNA was incubated for 30 min at 42 °C in
20 µl of reaction buffer containing 10 mM Tris-HCl (pH
8.3), 5 mM MgCl2, 50 mM KCl, a 1 mM concentration of each dNTP, 1 unit/µl RNase inhibitor, 2.5 µM random hexamer, and 2.5 unit/µl of
Moloney murine leukemia virus reverse transcriptase (PerkinElmer Life
Sciences). A 10-µl aliquot of reverse transcription reaction was
amplified in a 100-µl final volume of PCR mixture containing 10 mM Tris-HCl (pH 8.3), 2 mM MgCl2,
50 mM KCl, 1 unit of AmpliTaq Gold DNA polymerase (PerkinElmer Life Sciences), and 0.1 µg of specific primer
(5'-AATGGCTTTCGTTTGCTTGG-3' and 5'-ACGCAATCCATATCCTGAAC-3') (40). The
PCR product (20 µl) was run on a 2% agarose gel for UV analysis.
counter (Wallac,
Gaithersburg, MD).
2 chain were incubated in binding buffer containing 0.2 nM chloroquine at 37 °C for 5 min to prevent degradation
of internalized 125I-IL-13. The cells were then washed, and
2 × 107 cells were incubated with 0.5 nM
125I-IL-13 at 4 °C for 2 h. After removing free
125I-IL-13, cell pellets were resuspended in 2 ml of
binding buffer and incubated at 37 °C. At various time intervals,
two duplicate sets of 50-µl aliquots were taken. One set was
incubated with glycine buffer (final pH 2.0) for 10 min on ice. The
suspension was then centrifuged through a mixture of phthalate oils,
and the radioactivity in the cell pellet (acid-resistant or
internalized) and in the supernatant (surface-bound plus dissociated)
was determined. The other set of 50-µl aliquots was directly
centrifuged through phthalate oils, and the radioactivity observed in
the supernatants was used for dissociated 125I-IL-13
values. Surface-bound 125I-IL-13 was determined by
subtracting dissociated 125I-IL-13 values from
surface-bound plus dissociated values.
2 chain, we pretreated COS-7
cells with 0.1% Me2SO or 50 µM proteasome
inhibitor, MG132 (Sigma) for 30 min at 37 °C. During binding and
internalization assays, Me2SO or MG132 continued to be
present in the binding buffer.
plate counter (Wallac).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 Mutants--
Single, double, or triple
amino acid substitutions in the trileucine region of IL-13R
2 chain
were performed by site-directed mutagenesis. As shown in Fig.
1, trileucine residues resided in the
transmembrane region of the IL-13R
2 chain. Leucine residues were
either changed to alanine or to isoleucine without any other modifications or combined with the substitution of tyrosine at position
343 by phenylalanine of the IL-13R
2 chain. However, in some cases
leucine residues were unchanged, while the intracellular domain of
IL-13R
2 chain was either completely or partly deleted or only one
tyrosine residue was changed to phenylalanine at position 343. Finally,
one mutant lacked all three leucine residues and the complete
intracellular domain.
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Fig. 1.
Schematic representation of the wild type and
mutant IL-13R 2 chains. EC,
extracellular domain; TM, transmembrane domain;
IC, intracellular domain of the IL-13R
2 chain. Leucines
335-337, alanines 335-337, tyrosine 343, and phenylalanine 343 are
indicated.
2 Mutants--
To
confirm the successful transfection of plasmid DNAs for IL-13R
2
mutants in COS-7 cells, total RNA was extracted from the transfectants,
and RT-PCR analysis was performed using primers that can detect part of
the extracellular domain of the IL-13R
2 chain (40). As shown in Fig.
2A, wild type IL-13R
2 and
all of the mutants showed high expression of mRNA for the
extracellular domain of the IL-13R
2 chain. In naive and vector only
(mock) transfected COS-7 cells, very faint expression of this chain was observed as we previously demonstrated (40). These data suggest that
all IL-13R
2 mutants were successfully transfected although COS-7
cells seemed to express very faint IL-13R
2 mRNA. To determine the amount of protein for each expressed plasmid,
125I-IL-13 binding assays were performed on various
transfectants, since specific antibody to IL-13R
2 chain is not
commercially available for Western blot analysis. Mutated IL-13R
2
transfected cells were incubated with 125I-IL-13 in the
absence or presence of a 200-fold molar excess of IL-13. As shown in
Fig. 2B, 125I-IL-13 bound to all receptor
mutant-transfected COS-7 cells at similar levels with the exception of
naive cells, mock-transfected cells, or cells transfected with the
335 construct. Excess unlabeled IL-13 displaced the binding of
125I-IL-13, indicating specific IL-13 binding.
Interestingly, in
335-transfected COS-7 cells,
125I-IL-13 did not bind although these cells expressed
mRNA for this chain.
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Fig. 2.
Expression of wild type and mutant
IL-13 2 chains in COS-7 cells.
A, 2 days after transfection, total RNA was extracted from
COS-7 cells, which were transfected with vector only, wild type, or
mutant IL-13R
2 chains, and examined for IL-13R
2 chain
extracellular domain expression by RT-PCR analysis. B,
binding of 125I-labeled IL-13 was performed as described
under "Experimental Procedures." Cells (5 × 105)
were incubated at 4 °C for 2 h with 200 pM
125I-IL-13 with or without 40 nM unlabeled
IL-13. Data represent the mean of duplicate determinations, and the
experiment was repeated three times with similar results.
Bars, S.D.
2
Chain--
To investigate the role of the dileucine motif in the
trileucine region in the C terminus of the transmembrane domain of the IL-13R
2 chain, mutated IL-13R
2 genes,
338, L335A, L336A,
L337A, L335/L337A, L335A/L336A/L337A, or L335I/L336I/L337I, were
transfected in COS-7 cells, and internalization assays were performed.
We also performed internalization assays using COS-7 cells transfected with vector only that served as a mock control; however, their binding to radiolabeled IL-13 was too low to detect significant internalization (data not shown). As shown in Fig.
3, dileucine motif conserved (two of
three leucines unchanged) mutants,
338, L335A, and L337A, showed
similar internalization levels (up to 80% at 120 min) as IL-13R
2
when transfected in COS-7 cells. However, in COS-7 cells
transfected with L336A or L335A/L337A, which has no continuous leucine
residues, the internalization level was decreased to 65% (L336A) or
49% (L335A/L337A) at 120 min. Furthermore, in COS-7 cells
transfected with L335A/L336A/L337A in which all three leucine residues
were converted into alanine, the internalization level decreased to
40%, although the maximum plateau internalization level was observed
between 90 and 120 min, whereas L335I/L336I/L337I transfectants showed
the same internalization level (up to 79% at 120 min) as IL-13R
2.
These results demonstrate that the dileucine motif in the trileucine
region of the IL-13R
2 chain is necessary for efficient IL-13
internalization.
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Fig. 3.
Internalization of 125I-IL-13 by
the trileucine region targeted mutants. 2 days after transfection,
COS-7 cells were preincubated in binding buffer containing 0.2 nM chloroquine at 37 °C, followed by incubation with 0.5 nM 125I-IL-13 at 4 °C for 2 h. Then the
temperature was raised to 37 °C, and internalization assays were
performed. Data are expressed as a percentage of total IL-13 bound at
time 0. Open squares, surface IL-13 bound on the cells;
closed diamonds, internalization in the cells. Values are
the mean of two independent experiments. When not shown, S.D. bars are
smaller than the symbol.
2
Chain--
In the intracellular domain of IL-13R
2 chain, there is
one tyrosine residue at amino acid position 343. To determine whether this tyrosine plays a role in internalization, mutated cDNAs of IL-13R
2,
343, and Y343F were transfected in COS-7 cells, and internalization assays were performed. As shown in Fig.
4, IL-13R
2-transfected COS-7 cells
internalized 125I-IL-13 in a time-dependent
manner, and the internalization level increased up to 81% in 120 min.
In
343- or Y343F-transfected COS-7 cells, the 125I-IL-13
internalization level was found to be similar to IL-13R
2 transfectants (80% in 120 min). However, dissociation of surface-bound 125I-IL-13 in
338,
343, and Y343F transfectants was
faster compared with IL-13R
2. The half-life
(t1/2) of the dissociation of cell surface
125I-IL-13 binding in IL-13R
2 transfectants was
estimated to be 31 ± 2 min compared with 11 to 13 min in
tyrosine-mutated IL-13R
2 chain transfectants (Table
I). These results suggest that although Tyr343 does not participate directly in the internalization
process, it plays an important role in maintaining cell surface IL-13
binding to its receptor.
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Fig. 4.
Internalization of 125I-IL-13 by
the tyrosine motif targeted mutants. 2 days after transfection,
COS-7 cells were harvested, and internalization assays were performed
as described in Fig. 3 legend. Data are expressed as a percentage of
total IL-13 bound at time 0. Open squares, surface IL-13
bound on the cells; closed diamonds, internalization in the
cells. Values are the mean of two independent experiments. When not
shown, S.D. bars are smaller than the symbol.
Ligand-induced dissociation of cell surface 125I-IL-13 binding
capacity
2 chain modulated
endocytosis, internalization assays were performed using
L335A/L336A/L337A/Y343F-transfected cells. As shown in Fig. 4, the
125I-IL-13 internalization level was similar (32% in 120 min) to that of the L335A/L336A/L337A mutant (Fig. 3). However, this
internalization level was lower than that seen in Y343F transfectants.
On the other hand, the ligand dissociation was faster in
L335A/L336A/L337A/Y343F compared with L335A/L336A/L337A transfectants.
These results suggest that the L335A/L336A/L337A phenotype dominates
over the Y343F phenotype as far as endocytosis is concerned; however,
the Y343F phenotype dominates over the L335A/L336A/L337A phenotype for
ligand dissociation. These data also suggest that Tyr343 in
the intracellular domain of IL-13R
2 chain is required to maintain
cell surface 125I-IL-13 binding at physiological temperature.
2 Chain with
Dileucine Targeted Mutants on 125I-IL-13 Internalization in
COS-7 Cells--
To further study how leucines affect receptor
internalization, we co-transfected COS-7 cells with equal amounts of
DNA (6 µg/each) for the IL-13R
2 chain and either L335A/L337A or
L335A/L336A/L337A receptor mutants, and internalization assays were
performed. As shown in Fig. 5, the
maximum internalization level was lower in both types of transfectants
(50% in 120 min) compared with wild type IL-13R
2 transfectants
(80% in 120 min; Fig. 3). However, IL-13R
2 + L335A/L337A or
IL-13R
2 + L335A/L336A/L337A transfectants still showed similar or
slightly better internalization compared with cells transfected with
L335A/L337A or L335A/L336A/L337A alone (Fig. 3; 50% versus
49% in IL-13R
2 + L335A/L337A and L335A/L337A transfectants,
respectively, and 51% versus 40% in IL-13R
2 + L335A/L336A/L337A and L335A/L336A/L337A transfectants, respectively). In contrast, the dissociation rate of surface-bound
125I-IL-13 appeared to be similar to that of wild type
IL-13R
2 transfectants. These results further confirmed our findings
that the IL-13R
2 chain utilizes the dileucine motif for ligand
internalization.
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Fig. 5.
Internalization of 125I-IL-13 in
the co-existence of wild type IL-13R 2 and the
trileucine region targeted mutants. 2 days after co-transfection
with IL-13R
2 and either L335A/L337A or L335A/L336A/L337A mutants,
COS-7 cells were harvested, and internalization assays were performed
as described in the legend to Fig. 3. Data are expressed as a
percentage of total IL-13 bound at time 0. Open squares,
surface IL-13 bound on the cells; closed diamonds,
internalization in the cells. Values are the mean of two independent
experiments. When not shown, S.D. bars are smaller than the
symbol.
2 Mutant-transfected COS-7
Cells--
To further confirm the results obtained by internalization
assays, the cytotoxicity of recombinant IL13-PE38QQR, which targets IL-13R, was assessed. IL13-PE38QQR binds to IL-13R and is internalized by endocytosis, subsequently causing cell death through the inhibition of new protein synthesis. Thus, cytotoxicity observed in transfected cells indicates receptor internalization (10, 31, 37, 38). COS-7 cells
were transfected with the IL-13R
2 chain or its mutants, and
sensitivity to IL13-PE38QQR was determined (Fig.
6). The IC50 values (IL-13
toxin concentration causing 50% inhibition of protein synthesis) was
calculated from the cytotoxicity data (Table
II). When COS-7 cells were transfected
with IL-13R
2, the cytotoxicity of IL13-PE38QQR increased in these
cells. The IC50 value in IL-13R
2 transfected cells was
10-fold lower compared with cells transfected with vector only (from
200 versus 20 ng/ml). In COS-7 cells transfected with Y343F
or L335I/L336I/L337I, sensitivity to IL-13 toxin was similar to that
seen in IL-13R
2 transfectants (Fig. 6, C and F). On the other hand, when COS-7 cells were transfected
with dileucine motif-deleted or substitution mutants (
335,
L335A/L337A, or L335A/L336A/L337A), sensitivity to IL-13 toxin did not
change compared with cells transfected with vector only (Fig. 6,
B, D, and E). These data further
confirm the findings that the dileucine motif mediates internalization
and the tyrosine motif does not play a direct role in this process.
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Fig. 6.
Cytotoxicity of IL-13 toxin to COS-7 cells
transfected with IL-13R 2 chain mutants.
COS-7 cells were transfected with wild type or mutant IL-13R
2
chains, and then IL13-PE38QQR-mediated cytotoxicity was determined by a
protein synthesis inhibition assay. COS-7 cells were transfected with
vector only (open circles) or the wild type or mutant
IL-13R
2 chains (closed squares). The results are
represented as means ± S.D. of quadruplicate
determinations.
Cytotoxicity of IL-13 toxin to COS-7 cells transfected with wild type
or mutant IL-13R2 chains
2 Chain--
To assess
whether proteasome-mediated proteolysis is involved in the expression
and endocytosis of IL-13R
2 chain, COS-7 cells were transfected with
cDNA for the IL-13R
2 chain and then incubated with a proteasome
inhibitor (MG132), and internalization assays were performed. MG132 did
not affect the binding of 125I-IL-13 in IL-13R
2
transfectants (data not shown). Similarly, as shown in Fig.
7, both internalization and surface-bound
125I-IL-13 levels in IL-13R
2 transfectants were
identical in both control (incubated with Me2SO) and MG132
treatment groups. In addition, there was no significant difference in
the dissociation rate in both groups. The concentration of MG132 used
(50 µM) has been shown to stabilize IL-2-induced STAT5
activation in CTLL-2 cells (49). These results suggest that the
IL-13R
2 chain is not ubiquitinated and that the internalization
process does not depend on ubiquitination.
View larger version (12K):
[in a new window]
Fig. 7.
Internalization and dissociation of
125I-IL-13 is not prolonged by the proteasome inhibitor
MG132. 2 days after transfection with cDNA for the IL-13R 2
chain, COS-7 cells were harvested and pretreated with 0.1%
Me2SO (DMSO) or 50 µM MG132 for 30 min at 37 °C. These cells were then utilized in internalization and
dissociation assays as described in the legend to Fig. 3. Data are
expressed as a percentage of total IL-13 bound at time 0. Open
squares, surface IL-13 bound on the cells; closed
diamonds, internalization in the cells. Values are mean of two
independent experiments. When not shown, S.D. bars are smaller than the
symbol.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 chain. By generating IL-13R
2 mutants targeted to the dileucine motif in a trileucine region in the C terminus of the transmembrane domain and a tyrosine motif in the intracellular domain of the IL-13R
2 chain, we performed binding assays and internalization assays to investigate the mechanism of IL-13 internalization by the IL-13R
2 chain.
335, which deletes three leucine residues in the transmembrane domain and the whole intracellular domain, did not demonstrate a great deal of IL-13R on the cell surface of COS-7 cells
as assessed by radiolabeled binding assays. This is because naive COS-7
cells express IL-13R
1 and IL-4R
chains along with very faint
mRNA for the IL-13R
2 chain by RT-PCR as we have previously reported (40). These cells expressed IL-13R at a levels similar to
naive cells and cells transfected with vector only. The lack of IL-13
binding in
335-transfected cells may be due to the deletion of
hydrophobic amino acid residues
Leu335/Leu336/Leu337 in the C
terminus of the transmembrane domain and/or deletion of the
intracellular domain. This modification in the IL-13R
2 chain may
provide no anchor for cell surface receptor expression and/or proper
folding of extracellular domain necessary for IL-13 binding. These
results suggest that the trileucine residues and/or intracellular
domain of the IL-13R
2 chain may be essential for IL-13 binding.
2 mutants were generated
in which one, two, or all three leucine residues were changed to
alanine or isoleucine and cDNAs were transiently transfected in
COS-7 cells. Conversion of all three leucine residues (Leu335/Leu336/Leu337) to alanine
decreased the internalization level to half of the wild type IL-13R
2
transfectants; however, when only one leucine at position 335 (L335A)
or 337 (L337A) was converted to alanine, no diminution in
internalization was observed. When leucine 336 was converted to alanine
(L336A) or two discontinuous leucines, Leu335 and
Leu337, were converted to alanine (L335A/L337A), a decrease
in the internalization rate was observed. These results suggest that
the dileucine motif is required for IL-13-IL-13R
2 chain complex
internalization. Thus, our results confirm previous observations that
the dileucine motif is necessary for internalization of various
cytokine-receptor complexes (22-30).
and invariant chains
are internalized by a dileucine motif and an upstream aspartic acid
(46, 47). These results suggest that the dileucine motif may not be
solely responsible for receptor internalization. Although the dileucine motif in the trileucine region of the IL-13R
2 chain does not have
upstream serine or aspartic acid, our findings suggest that the
trileucine residue or the dileucine motif by itself plays an essential
role in IL-13 internalization.
2 chain, a significant inhibition of internalization was
observed compared with IL-13R
2 transfectants. However, the
internalization level was slightly higher compared with that caused by
L335A/L337A or L335A/L336A/L337A transfectants. These results suggest
that mutant IL-13R
2 chains may form a complex with the wild type
IL-13R
2 chain, resulting in diminished internalization without
affecting the dissociation of surface-bound 125I-IL-13.
2 chain has a YPKM motif at amino acids 343-346
and this motif is equivalent to the YXXØ (where
X represents any amino acid and Ø is a hydrophobic motif
(11, 16-19)), we generated two mutants targeting this motif, and its
role in the internalization process was investigated. Interestingly,
343 or Y343F did not change internalization level compared with wild type IL-13R
2 when mutant cDNAs were transfected in COS-7.
However, the dissociation of surface-bound 125I-IL-13 was
faster in
343 and Y343F transfectants compared with IL-13R
2
transfectants. This mechanism of faster dissociation of IL-13 in
343
or Y343F mutant is not clear. It is possible that the tyrosine residue
at position 343 forms a tight
-turn in the secondary structure of
the IL-13R
2 chain that would retain ligand for a longer period of
time on the cell surface (16-19).
-induced STAT1 activation (51). We
examined whether the IL-13R
2 chain was ubiquitinated and whether the
internalization process was modulated by ubiquitination. We found that
the IL-13R
2 chain was not ubiquitinated and that the internalization
process did not depend on ubiquitination. However, whether
ubiquitination modulated the IL-13-induced signal transduction pathway
is unknown and is the subject of investigations in our laboratory.
2 chain. Although the interaction between a dileucine motif
and a tyrosine motif is not the same in different receptor types, in
the case of the IL-13R
2 chain the dileucine motif in the trileucine
region was found to play an essential role in internalization, and the
tyrosine motif was found to play an indirect role in ligand binding and internalization.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Bharat H. Joshi for the IL-13 and IL13-PE38QQR, Dr. Mariko Kawakami for technical assistance, Pamela Dover and Dr. S. Rafat Husain for helpful suggestions and reading the manuscript, and Dr. Gibbes Johnson for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* 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.
¶ To whom correspondence should be addressed: Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, NIH Bldg. 29B, Rm. 2NN10, 29 Lincoln Dr. MSC 4555, Bethesda, MD 20892. Tel.: 301-827-0471; Fax: 301-827-0449; E-mail: puri@cber.fda.gov.
Published, JBC Papers in Press, May 11, 2001, DOI 10.1074/jbc.M100936200
2 B. H. Joshi and R. K. Puri, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
IL, interleukin;
IL-6R and IL-13R, IL-6 and IL-13 receptor, respectively;
IL-13R2, interleukin-13 receptor
2 chain;
DMEM, Dulbecco's modified Eagle's
medium;
IL13-PE38QQR, a recombinant fusion protein composed of IL-13
and a truncated form of Pseudomonas exotoxin A;
STAT, signal
transducers and activators of transcription;
PCR, polymerase chain
reaction;
RT-PCR, reverse transcriptase-PCR.
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