High-affinity interleukin 2 receptor alpha and beta chains are internalized and remain associated inside the cells after interleukin 2 endocytosis.

High-affinity interleukin 2 (IL2) receptors on human T lymphocytes are multimeric complexes containing two IL2-binding polypeptides, alpha and beta chains of 50-55 and 70-75 kDa, respectively, associated by noncovalent bonds. IL2 binds to high-affinity IL2 receptors on the surface of T lymphocytes, mediates cell growth, and is internalized. In this paper, we used a biochemical method to directly identify the receptors components internalized together with the ligand. 125I-IL2-receptor complexes were solubilized with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate, and IL2-binding polypeptides were identified by cross-linking with disuccinimidyl suberate. Under such conditions, the noncovalent association between alpha and beta is maintained. After IL2 internalization, two complexes of about 70 and 90 kDa, IL2 crosslinked to alpha and beta, respectively, were found inside the cells. Both components were immunoprecipitated with either anti-alpha or anti-beta monoclonal antibodies. This shows that the alpha and beta chains are found in an intracellular compartment after IL2 endocytosis, and remain associated as a ternary complex with IL2.

Interleukin 2 (IL2),' a growth factor for T lymphocytes, transduces the growth signal through a specific receptor complex that binds IL2 with high affinity ( K d , 10-100 PM) (1)(2)(3). The high-affinity receptor complex consists of at least two distinct receptor components, the a chain of 50-55 kDa (4- 8), and the p chain of 70-75 kDa (9-13). a and p polypeptides, when expressed on the cell surface without / 3 and a , respectively, bind IL2 with a lower affinity. They form low and intermediate affinity receptors, with dissociation constants of 10 and 1 nM, respectively. The very high affinity of functional receptors for the ligand is due to the combination of two properties of individual chains, the high association rate of IL2 to a chains, and the slow dissociation of IL2 to ( 3 chains (14, 15). a and ( I subunits are bound by noncovalent association to form high-affinity receptors (16), the structure of which has not been further determined. In addition to a and p, the ~5 6 ' '~ tyrosine kinase is associated to the p chain and * This work was supported by the Association pour la Recherche sur le Cancer, the Agence Nationale de Recherche sur le Sida, and the Ligue Nationale Contre le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The may participate in 112-mediated signal transduction (17). Furthermore, other chains may be associated to this complex After IL2 binding to surface high-affinity receptors, IL2 is internalized and degraded (26)(27)(28)(29)(30). Surface high-affinity receptors occupied with IL2 disappear from the cell surface at the same rate as IL2 is internalized (27,31). This suggests that receptors, or some of their components, are internalized and degraded and do not recycle to the cell surface. The p chain, without a, is sufficient for IL2 endocytosis, since IL2 bound to intermediate affinity receptors is internalized as rapidly as when it is bound to high-affinity receptors (20,(32)(33)(34). a alone does not appear to mediate endocytosis of IL2 (28,29,35).
We have developed a new method to directly identify highaffinity IL2 receptor chains internalized in the human T cell line IARC 301.5 (31,36). The IL2-receptor complex was solubilized in conditions such that the a and / 3 chains remain associated, and IL2 binding components were identified by chemical cross-linking. We show that both a and ( 3 are internalized, that the association between a and p is maintained, and that IL2 is still bound to both chains inside the cells.
Thus, the ternary complex a . p . IL2, which constitutes highaffinity receptors on the cell surface, is also present inside the cells after IL2 endocytosis. (18-25).

MATERIALS AND METHODS
Cell Lines and Antibodies-The human tumor T cell clone IARC 301.5 (31) was cultured in RPMI supplemented with 10% fetal calf serum, 2 mM L-glutamine, 20 mM Hepes, pH 7.2. The rat IgG,. monoclonal antibody against IL2 receptor cy chain 2734.6 (18 pg/ml) and control IgGI. anti-HLA antibody 35A1.6 (ascitic fluid 1/500) (37) were gifts from D. Olive (INSERM, U119, Marseille, France) and purified from ascites on a DEAE-52 column. The mouse IgG,, anti$ receptor chain 561 (2 Fg/ml) was a kind gift from Dr R. Robb  was removed by acid pH treatment as described previously (31). Chemical Cross-linking of "'I-IL2 to Intact Cells and Cell Lysates-When intact cells were used, cells (1.5 X lo') labeled with 12s11-IL2 were resuspended in 1 ml of phosphate-buffered saline. DSS was added to the cell suspension at 2 mM final concentration for 15 min at 4 "C. After centrifugation at 800 X g, the cells were solubilized for 30 min at 4 "C with 100 pl of lysis buffer (10 mM sodium potassium phosphate, pH 7.6, 10 pg/ml leupeptin, 0.1 unit/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride, and 5 mM CHAPS). The insoluble fraction was separated by centrifugation at 13,000 X g, 10 min at 4 "C, and the supernatant was analyzed by gel electrophoresis, or immunoprecipitated prior to gel electrophoresis. When cell lysates were used, after lZ5I-IL2 binding, the cell pellet (1.5 X 10' cells) was solubilized with 100 pl of lysis buffer. The insoluble fraction was eliminated by centrifugation at 13,000 X g and cross-linking was performed by addition of 2 mM DSS and incubation for 15 min at 4 "C. The reaction was quenched with 10 mM ammonium acetate. In some experiments, cross-linked material was centrifuged a t 100,000 X g in an airfuge (Beckman Corp.), rotor A100/18,10 min a t room temperature. Cross-linked 1ZsI-IL2-receptor complexes were analyzed by gel electrophoresis.
Precipitation of 1251-IL2-Receptor Complex with PEG-Precipitation with PEG 8000 was performed at 4 "C in Eppendorf tubes, in 50 mM Tris-HC1, pH 7.2. To 100 pl of solubilized 'Z'II-IL2-receptor complex, 100 pl of human IgG, 0.5 mg/ml final concentration, and 300 pl of PEG, 0-18% final concentration, were sequentially added. After vortexing the tubes, PEG precipitates were separated from the soluble fraction by centrifugation for 4 min at 13,000 X g at 4 "C.
Immunoprecipitation-Immunoprecipitation was performed as previously described (38). One hundred pl of soluble material was incubated for 1 h at 4 "C with anti-a, anti-& or control antibodies in the presence of 0.5% ovalbumin. When rat Ig were used, the incubation was persued for 1 h at 4 "C in the presence of 10 pg of rabbit anti-rat IgG. Fifty pl of protein A-Sepharose CL-4B (Pharmacia LKB Biotechnology Inc.) (v/v) in lysis buffer was added and the incubation was continued for another hour at 4 "C. Immunoprecipitates were washed 3 times with 0.5 ml of lysis buffer and stored at -20 "C.
Gel Electrophoresis-Cell lysates or immunoprecipitates were boiled in the presence of 5% 2-mercaptoethanol and 2% sodium dodecyl sulfate and analyzed by sodium dodecyl sulfate-polyacrylamide (7.5%) gel electrophoresis. The gels were stained with Coomassie Blue and exposed from 5 days to 2 weeks either on hyperfilm MP (Amersham Corp.) or preflashed XAR film (Kodak Corp.). The amount of material present in the bands of the autoradiogram on preflashed films was quantified by computer-assisted image analysis.

RESULTS
Solubilization of High-affinity lZ5I-IL2-receptor Complexes with CHAPS-The zwitterionic detergent CHAPS (39) was used to solubilize receptors such as opiate receptor (40), prolactin receptor (41), IgE receptor (42), and IL1 receptor (43), and was shown to preserve the integrity of the tetrameric complex of IgE receptor (42). We have used this detergent to solubilize high-affinity IL2 receptors complexed with their ligand. lZ5I-IL2 was bound on IARC 301.5 cells at 37 "C for 30 min at a concentration of 25 PM, such that only high-affinity sites are occupied (36). After washing unbound ligand, the cells were solubilized 30 min at 4 "C with various CHAPS concentrations. The radioactivity present in the soluble fraction, after elimination of insoluble material, is shown in Fig. lA. The lower CHAPS concentration which gives maximum solubilization, i.e. 5 mM, was used in subsequent experiments unless otherwise stated. The soluble fraction was then precipitated with various PEG concentrations (Fig. 1B). Ninety percent of the radioactivity was precipitated with 12% PEG, a concentration at which only 12% of free lZ5I-IL2 is found in the precipitate. Thus lZ5I-IL2 in the CHAPS-soluble fraction is associated with proteins that form a complex precipitated with PEG.
To characterize the lZ5I-IL2 binding components that have been solubilized in CHAPS, surface high-affinity receptors    1 and 4, and 1 X lo6 in lanes 2 and 5. B, cross-linked material from A , lane 1, was ultracentrifuged at 100,000 x g and the supernatant was analyzed by gel electrophoresis.
were saturated with lZ5I-IL2 at 4 "C and unbound ligand was washed away. The soluble fraction obtained after solubilization with 5 mM CHAPS and centrifugation at 13,000 X g, was cross-linked with DSS (2 mM) as described under "Materials and Methods," and IL2-binding proteins were identified after migration on a 7.5% polyacrylamide gel under reducing conditions and autoradiography. In controls, cross-linking with DSS was performed on intact cells prior to solubilization: in such conditions, the two IL2 binding chains, (Y and p, which constitute high-affinity IL2 receptors, are detected (9,10,12,38) . When cross-linked to lZ5I-IL2 they form two complexes of about 70 and 90 kDa, respectively ( Fig. 2A, lanes 3-5). The 90-kDa complex is usually seen as a doublet in IARC 301.5 cells as well as in other cell lines (9,11,12). This doublet is better separated on long polyacrylamide gels than on minigels. In CHAPS extracts treated with 2 mM DSS, two IL2-binding components with similar migrations, 70 and 90 kDa, were identified ( Fig. 2 A , lanes 1 and 2). Storage at -20 'C of crosslinked material before electrophoresis did not modify these results. To confirm that IL2-receptor complexes detected in the 13,000 X g supernatant are in a soluble form, the crosslinker was added, then the material was centrifuged for 10 min at 100,000 x g. In such conditions, plasma membranes sediment (sedimentation coefficient = 72 S), and 80% of the radioactivity was found in the 100,000 x g supernatant. When the high speed supernatant was analyzed by gel electrophoresis both the 90-and 70-kDa complexes were identified (Fig.  2B). This shows that a soluble form of IL2-receptor complexes is indeed present in the 13,000 x g supernatant. In conclusion, solubilization of high-affinity IL2-receptor complexes with 5 mM CHAPS maintains the association between IL2 and the a chain, and IL2 and the @ chain, respectively. a and p Chains Remain Associated in CHAPS-solubilized ZL2-Receptor Complexes-The identification of the two 70and 90-kDa components in solubilized cell fractions suggests that the noncovalent association between a and @ is maintained after cell solubilization with 5 mM CHAPS. Next we performed experiments to rule out the possibility that IL2 is bound to a and / 3, but that CY-@ association is not maintained in the detergent. If a-@ were dissociated, IL2 would be released from a in a few seconds (15), and if IL2 concentration were sufficient, IL2 would rebind to free a chain within seconds (15). This type of hypothesis for p is unlikely because of the slow off-rate of IL2 bound to @, t2,, = 4-5 h at 4 "C (15). Two protocols were used. In the first protocol, IARC 301.5 cells were incubated with 1251-IL2 at concentrations varying from 25 to 200 PM followed by 5 mM CHAPS solubilization at 2 X lo8 cells/ml (Fig. 3, lanes 1-4). In the second protocol, 1251-IL2 was bound at 200 PM, and cell concentration in lysis buffer varied from 0.25 to 2 X 10' cells/ml (Fig. 3, lanes 5-8).
If '251-IL2 had dissociated from a, its concentration in the lysate would be 110 and 80 PM in lanes 1 and 8, respectively. At such concentrations, less than 1% low affinity receptors can be occupied (Kd = 10 nM (44)): Since the 70-kDa complex was still detected after cross-linking of the cell lysate, a-p dissociation can be excluded.
The association of a and @ in the complex was also shown directly by immunoprecipitation (Fig. 4). In this experiment, high-affinity IL2 receptors were saturated with lZ5I-IL2 and cross-linking was performed on intact cells. The cells were V. Duprez, M. Ferrer, and A. Dautry-Varsat, unpublished data. then solubilized with 5 mM CHAPS and the soluble fraction was immunoprecipitated with anti-a antibody 2734.6, or anti-@ antibody 561. Both the 90-and 70-kDa protein complexes were identified with either anti-a or anti-@ antibody (lanes 2 and 4 ) , while control antibodies did not precipitate these components (lanes 3 and 5 ) .
Evidence for Internalization of a and @ Chains of Highaffinity IL2 Receptors-When IL2 is bound to high-affinity receptors on the cell surface, it is rapidly internalized at 37 "C (31, 36). The method of solubilization of the IL2-receptor complex described below was used to identify IL2 binding chains internalized with IL2.
Cell surface high-affinity receptors were saturated with lz5I-IL2 at 4 "C. The cells were washed, and either kept at 4 "C, or incubated at 37 "C to allow endocytosis to proceed. Surface bound IL2 was removed by acid wash, then the cells were solubilized with 5 mM CHAPS, cross-linked with DSS, and internalized IL2-receptor complexes were identified by gel electrophoresis (Fig. 5). After 20 min of internalization, both the 90-and 70-kDa complexes were detected on the autoradiogram (lane 3 ) . They represent intracellular components, since they were absent in control cells, which had not internalized IL2 and had been treated with acid pH (lane 1 ). Thus both a and @ chains are internalized and still bound to IL2 after IL2 endocytosis via high-affinity receptors. Since the solubilization conditions preserve the noncovalent association between a and @, the presence of the two bands in Fig. 5  were followed after various times of IL2 internalization at 37 "C ( Fig. 6). A maximal amount of a bound to IL2 and j3 bound to IL2 was found inside the cells after about 40 min of internalization. Thereafter, the intensities of the bands corresponding to the complexes between IL2 and the a or j3 chain decrease in a parallel manner. This indicates that IL2 dissociates simultaneously from a and j 3 .

DISCUSSION
A biochemical method was developed to assess the fate of a and j3 chains of high-affinity IL2 receptor after IL2 internalization. lZ5I-IL2 was bound on the surface of IARC 301.5 human T cells, and the IL2-receptor complex was solubilized with the detergent CHAPS and identified by chemical crosslinking with DSS. Two components of 70 and 90 kDa were detected, IL2 cross-linked to a and j3 high-affinity receptor chains. Thus, in CHAPS detergent the association between IL2 and a and between IL2 and j3 is preserved. The noncovalent association between a and j3 chains is also maintained in the detergent: indeed, under the solubilization conditions that excluded a possible binding of free IL2 to isolated OL and j3 in solution, both components were still detected. In addition an anti-a monoclonal antibody, or an a n t i 6 monoclonal antibody, immunoprecipitated both the 70-and 90-kDa complexes. Finally identical results were obtained whether or not an excess of unlabeled IL2 (20 nM) was added in the solubilization buffer (data not shown). A higher molecular weight complex was often observed. It migrates in gel electrophoresis as a diffuse band and may represent the ternary complex of IL2 with a and j3 chains solubilized in CHAPS.
Various studies have been performed with monoclonal antibodies against a and j3 receptor chains to try to crossprecipitate the two chains of high-affinity receptors on the cell surface. In murine cells, some anti-a antibodies were able to precipitate IL2 cross-linked to j3 receptors on the cell surface (16), or to precipitate a chain cross-linked to j3 chain (19), which confirms a and j3 association in the soluble form of high-affinity receptors. In human cells similar observations were made with anti-j3 (24, 45) and anti-IL2 antibodies (46). In other reports, anti-a, failed to immunoprecipitate the second chain (10, 11, 13). The conflicting data concerning the human IL2 receptor could be related to the detergent used in these experiments (Nonidet P-40 or Triton X-loo), or the possible effect of the antibody on the complex stability. The approach developed here, namely cross-linking IL2-receptor complexes solubilized with CHAPS, may be used to study the parameters that influence the stability of a and j 3 association.
To identify the chains of the receptor that are internalized, 1251-IL2 was endocytosed at 37 "C, and the surface-bound lZ5I-IL2 removed by acid pH wash. When the cells were solubilized, and IL2 binding components identified by cross-linking, both the 70-and 90-kDa complexes were detected. Therefore a and j3 chains of IL2 receptors are found intracellularly after IL2 binding to surface high-affinity receptors. Fung et al. (47) have reported, in YT cells stimulated with forskolin, that an anti-a monoclonal antibody, 7G7.B6, is internalized only when IL2 is present at concentrations such as to saturate high-affinity receptors. Our results are in good agreement with their observation. A maximum amount of IL2 bound to intracellular j3 and a was found inside the cells after about 40 min of internalization. The subsequent decrease in both the 90-and 70-kDa components is likely to reflect the dissociation of internalized IL2 from a and j 3 . Indeed IL2 internalization is followed by its degradation in acidic compartments (27). Since the method we used maintains the association between a and j3, our data also show that the ternary complex aej3. IL2 which constitutes high-affinity receptors on the cell surface, is found inside the cells after IL2 internalization. This was confirmed by the ability of anti-a antibody to coprecipitate intracellular j3 chain and of anti-j3 antibody to coprecipitate a chain (data not shown). In addition, the presence of a and j3 in intracellular endocytic compartments after IL2 internalization was shown, by confocal microscopy, using monoclonal antibodies against both polypeptides?
The molecular mechanisms that mediate IL2-induced proliferation through high-affinity receptors are poorly understood. The j3 chain is thought to be responsible for delivering the IL2 induced growth signal intracellularly (48-50). The usual pathways known to transduce hormone signals, activation of protein kinase C and phosphoinositides metabolism, may not be essential for IL2-induced proliferation. Several proteins are phosphorylated on tyrosines in response to IL2, including the / 3 chain of IL2 receptor, but a or j3 do not seem to account for this kinase activity (51, 52). The tyrosine protein kinase p56Ick may be involved in this process (17). Selective internalization of IL2 bound to high or intermediate affinity receptors suggests that receptor-mediated endocytosis might be necessary for IL2 biological activity. The role of internalization in signal transduction is also suggested by the ability of anti-a antibodies, cross-linked with second antibodies, to inhibit both IL2 internalization and IL2-dependent growth in IARC 301. 5

T cells (38).
The intracellular fate of high-affinity IL2-receptor complexes after endocytosis is not completely understood. The half-life of surface high-affinity receptors on the cell surface is very short, about 15-25 min when the receptors are saturated with ligand (31). In such conditions the a chain halflife, more than 40 h (53), is much longer than expected if a chains associated with j3 and internalized were then degraded (about 3-6 h). Taken together with the present data, showing that a is internalized, this suggests that after co-internalization of a and j3 chains, the two polypeptides may follow different intracellular routes, j3 being degraded while a recycles back to the cell surface.