Identification of Distinct Roles for a Dileucine and a Tyrosine Internalization Motif in the Interleukin (IL)-13 Binding Component IL-13 Receptor α2 Chain*

Interleukin (IL)-13 receptor α2 (IL-13Rα2) 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-13Rα2 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-13Rα2 chain (Δ335) did not bind IL-13, suggesting that the trileucine region modulates IL-13 binding. Truncation of IL-13Rα2 chain with a mutation in the trileucine region resulted in significantly decreased internalization compared with wild type IL-13Rα2 chain transfected cells. COS-7 cells transfected with tyrosine motif mutants exhibited a similar internalization level compared with wild type IL-13Rα2 chain transfected cells; however, dissociation of cell surface IL-13 was faster compared with wild type IL-13Rα2 transfectants. These results were further confirmed by determining the cytotoxicity of a chimeric protein composed of IL-13 and a mutated form of Pseudomonasexotoxin (IL13-PE38QQR) to cells that expressed IL-13Rα2 chain mutants. We further demonstrate that the IL-13Rα2 chain is not ubiquitinated and that internalization of IL-13Rα2 did not depend on ubiquitination. Together, our findings suggest that the dileucine motif in the trileucine region and tyrosine motif participate in IL-13Rα2 internalization in distinct manners.

(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)(2)(3)(4)(5)(6). Two different chains of the IL-13 receptor, IL-13R␣1 and IL-13R␣2 (also known as IL-13R␣Ј 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-13induced 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).
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)(13)(14)(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 ␤-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, ␤ 2adrenergic receptor, lutropin/choriogonadotropin receptor, and erythropoietin receptor) plays an essential role in the internalization of ligand (22)(23)(24)(25)(26)(27)(28)(29)(30).
Recently, we have demonstrated that the IL-13R␣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 Leu 335 , Leu 336 , and Leu 337 ) in the C terminus of the transmembrane domain and the tyrosine motif (position Tyr 343 ) in the intracellular domain of the IL-13R␣2 chain were studied in internalization assays using 125 I-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.
Plasmid DNAs (12 g/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 ϫ 10 6 /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␣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 g of total RNA was incubated for 30 min at 42°C in 20 l of reaction buffer containing 10 mM Tris-HCl (pH 8.3), 5 mM MgCl 2 , 50 mM KCl, a 1 mM concentration of each dNTP, 1 unit/l RNase inhibitor, 2.5 M random hexamer, and 2.5 unit/l of Moloney murine leukemia virus reverse transcriptase (PerkinElmer Life Sciences). A 10-l aliquot of reverse transcription reaction was amplified in a 100-l final volume of PCR mixture containing 10 mM Tris-HCl (pH 8.3), 2 mM MgCl 2 , 50 mM KCl, 1 unit of AmpliTaq Gold DNA polymerase (PerkinElmer Life Sciences), and 0.1 g of specific primer (5Ј-AATGGCTTTCGTTTGCT-TGG-3Ј and 5Ј-ACGCAATCCATATCCTGAAC-3Ј) (40). The PCR product (20 l) was run on a 2% agarose gel for UV analysis.
Radioreceptor Binding Assay-Recombinant human IL-13 was labeled with 125 I (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 Ci/g of protein.
For binding experiments, 5 ϫ 10 5 cells in 100 l of binding buffer (RPMI 1640 containing 0.2% human serum albumin and 10 mM HEPES) were incubated with 200 pM 125 I-IL-13 with or without 40 nM unlabeled IL-13 at 4°C for 2 h. Cell-bound 125 I-IL-13 was separated from unbound by centrifugation through a phthalate oil gradient, and radioactivity was determined with a ␥ counter (Wallac, Gaithersburg, MD).
Internalization Assay-Internalization assays were performed as described before (10,41). COS-7 cells transfected with the IL-13R␣2 chain were incubated in binding buffer containing 0.2 nM chloroquine at 37°C for 5 min to prevent degradation of internalized 125 I-IL-13. The cells were then washed, and 2 ϫ 10 7 cells were incubated with 0.5 nM 125 I-IL-13 at 4°C for 2 h. After removing free 125 I-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-l 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-l aliquots was directly centrifuged through phthalate oils, and the radioactivity observed in the supernatants was used for dissociated 125 I-IL-13 values. Surface-bound 125 I-IL-13 was determined by subtracting dissociated 125 I-IL-13 values from surface-bound plus dissociated values.
To determine if proteasome-mediated proteolysis is involved in the expression and internalization of IL-13R␣2 chain, we pretreated COS-7 cells with 0.1% Me 2 SO or 50 M proteasome inhibitor, MG132 (Sigma) for 30 min at 37°C. During binding and internalization assays, Me 2 SO or MG132 continued to be present in the binding buffer.
Protein Synthesis Inhibition Assay-The cytotoxic activity of IL-13 toxin was tested as previously described (38). Typically, 10 4 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 Ci of [ 3 H]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 ␤ plate counter (Wallac).

Construction of IL-13R␣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.
125 I-IL-13 Binding to IL-13R␣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 Dileucine Motif-mediated Internalization of IL-13R␣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.
Tyrosine Motif-mediated Internalization of IL-13R␣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 125 I-IL-13 in a time-dependent manner, and the internalization level increased up to 81% in 120 min. In ⌬343or Y343Ftransfected COS-7 cells, the 125 I-IL-13 internalization level was found to be similar to IL-13R␣2 transfectants (80% in 120 min). However, dissociation of surface-bound 125 I-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 125 I-IL-13 binding in IL-13R␣2 transfectants was estimated to be 31 Ϯ 2 min compared with 11 to 13 min in tyrosine-mutated IL-13R␣2 chain transfectants (Table I). These results suggest that although Tyr 343 does not participate directly in the internalization process, it plays an important role in maintaining cell surface IL-13 binding to its receptor.
To further study this phenomenon and determine whether both dileucine motif and tyrosine motif mutatation in the IL-13R␣2 chain modulated endocytosis, internalization assays were performed using L335A/L336A/L337A/Y343F-transfected

TABLE I Ligand-induced dissociation of cell surface 125 I-IL-13 binding capacity
Half-lives (t 1/2 ) of the dissociation of 125 I-IL-13 from the cell surface were determined from the internalization data in Figs. 3 and 4. t 1/2 and maximum internalization rate represent mean Ϯ S.D. of four determinations.
11 Ϯ 3 7 9 Ϯ 9 Y343F 11 Ϯ 1 8 0 Ϯ 7 cells. As shown in Fig. 4, the 125 I-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 Tyr 343 in the intracellular domain of IL-13R␣2 chain is required to maintain cell surface 125 I-IL-13 binding at physiological temperature.

FIG. 5. Internalization of 125 I-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. 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.
Cytotoxicity of IL13-PE38QQR to IL-13R␣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 IC 50 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 IC 50 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.
Proteasome Inhibitor MG132 Does Not Alter Internalization or Stabilize the Ligand Dissociation from IL-13R␣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 125 I-IL-13 in IL-13R␣2 transfectants (data not shown). Similarly, as shown in Fig. 7, both internalization and surfacebound 125 I-IL-13 levels in IL-13R␣2 transfectants were identical in both control (incubated with Me 2 SO) and MG132 treatment groups. In addition, there was no significant difference in the dissociation rate in both groups. The concentration of MG132 used (50 M) 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. DISCUSSION In this study, we have characterized the molecular basis for IL-13-mediated internalization of the IL-13R␣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.
Although mRNA expression was confirmed by RT-PCR, a truncation mutation, ⌬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 Leu 335 /Leu 336 /Leu 337 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.
To characterize the role of the dileucine motif in a trileucine region in the internalization process, eight IL-13R␣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 (Leu 335 /Leu 336 /Leu 337 ) 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, Leu 335 and Leu 337 , 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)(23)(24)(25)(26)(27)(28)(29)(30).
When all three leucine residues were converted to isoleucine (L335I/L336I/L337I), no diminution of internalization was ob- served. This is in contrast to IL-6R gp130, in which mutation of the first leucine (Leu 145 ) 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␥ 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.
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␣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 125 I-IL-13.
Because the IL-13R␣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 surfacebound 125 I-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).
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-␥-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.
In summary, we have characterized the internalization motifs in the IL-13R␣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.