©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Solution Assembly of a Soluble, Heteromeric, High Affinity Interleukin-2 Receptor Complex (*)

Zining Wu (1), Kirk W. Johnson (2), Byron Goldstein (3), Yoon Choi (2), Steven F. Eaton (4), Thomas M. Laue (4), Thomas L. Ciardelli (1)(§)

From the (1)Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755 and The Veterans Administrations Hospital, White River Junction, Vermont 05009, the (2)Chiron Corporation, Emeryville, California 94608-2916, (3)Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, and the (4)Department of Biochemistry, University of New Hampshire, Durham, New Hampshire 03867

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In this study, we report the use of coiled-coil (leucine zipper) molecular recognition for the solution assembly of stable, high affinity, heteromeric interleukin-2 receptor complexes. Co-expression of interleukin-2 receptor and extracellular domains (ectodomains), each fused to seven coiled-coil heptad repeats, resulted in the formation of heteromeric complexes that bound interleukin-2 in a cooperative fashion and with much higher affinity than similar homomeric complexes. The dissociation constants for these solution complexes are within the range of values reported for the comparable cell surface ``pseudo high affinity'' interleukin-2 receptor. Ligand-induced cross-linking of homomeric or heteromeric receptor subunits is the common signal transmission mechanism employed by hematopoietin receptors. Individual receptor ectodomains, however, often do not bind ligand with measurable affinity. This is the first study to demonstrate the feasibility of coiled-coil mediated preassembly of cytokine receptor complexes.


INTRODUCTION

The hematopoietin receptor family shares several conserved structural features as well as a common signal transmission mechanism, that of ligand-induced receptor subunit cross-linking(1) . One of the first cytokine systems to be classified among this group was interleukin-2. The high affinity interleukin-2 receptor (IL-2R)()results from the noncovalent interaction of three cell surface proteins, all of which are capable of independently binding IL-2 with much lower affinity. The 55-kDa -subunit binds IL-2 with a dissociation constant (K) of 1 10M(2) . The larger 75-kDa -subunit was originally reported to have a K = 1 10M(3) ; however, the identification of the 64-kDa -subunit (4) demonstrated that it also participated in the ligand-induced formation of 1 nM intermediate affinity site. Cooperative binding of IL-2 by these three proteins ultimately results in cross-linking of the - and -subunits and signaling via association of their cytoplasmic domains(5) . In addition, recent reports indicate that the -subunit is shared by the IL-4 and IL-7 receptors(6) , while both - and -subunits participate in the formation of the IL-15 receptor(7) .

Although the high affinity IL-2R employs three subunits, heterodimers of IL-2R subunits also play physiologically important roles. We have previously determined that the (/) ``pseudo high affinity'' heterodimer exists, preformed, on the surface of activated T-cells and serves to capture ligand(8) . This observation was in accordance with theoretical analyses of ligand binding data(9) . Furthermore, in the presence of IL-2, a second heterodimer (/) is formed. This signaling complex exists on the majority of natural killer cells in the absence of the -subunit(10) . Therefore, the physiologically functional IL-2R exists in several multimeric forms.

The ligand recognition properties of the individual ectodomains of these subunits have been studied. The soluble -subunit ectodomain binds IL-2 with an affinity similar to the cell surface low affinity site(11) . We have demonstrated that the -subunit ectodomain is also capable of binding IL-2 in solution(12) . Although the -subunit ectodomain binds IL-2 very weakly in solution, our studies have demonstrated that it can function cooperatively with the -subunit ectodomain to form a stable /-ectodomainIL-2 complex(13) .

In an effort to better understand the cooperativity of cytokine receptor ectodomains in ligand binding, we have evaluated the addition of coiled-coil recognition sequences to the receptor ectodomains as a means of generating stable solution complexes that mimic cell surface ligand binding. The coiled-coil interaction was first described 40 years ago by Crick (14) and has been studied in detail by Hodges as the structural element present in tropomyosin(15, 16, 17) . It is now recognized that the coiled-coil motif or ``leucine zipper'' is a recognition element common to a variety of macromolecular protein complexes(18) .

Recently, we have prepared and characterized an IL-2R ectodomain-coiled-coil fusion protein (19) and demonstrated the stable formation of a homotrimeric ectodomain complex (cc) capable of binding ligand. In this report, we describe the first solution assembly of a heteromeric IL-2R complex using idealized coiled-coil recognition sequences. This trimeric heterocomplex binds IL-2 with an affinity much greater than both the individual subunit ectodomains or their homomeric complexes and in a manner that indicates subunit cooperativity. In addition, this complex binds IL-2 with an affinity similar to that reported for the pseudo high affinity cell surface site.


MATERIALS AND METHODS

Design of the Coiled-coil Sequences

The designs of the coiled-coil heptads were based on previous studies of the stability of peptide coiled-coils(16, 20) . Seven repeats of the sequence LEALKEK were employed as the recognition sequence for the coiled-coil-modified -subunit ectodomain (IL-2Rcc), while seven repeats of LKALEKE were chosen for the -subunit ectodomain (IL-2Rcc). These sequences (Fig. 1) employed Leu residues at both the a and d positions of the heptad, with like-charged residues at the e and g positions of each helix in order to favor heteromeric over homomeric association based on electrostatic interactions(21) . In one version of the -subunit coiled-coil construct (IL-2Rcc), a DNA sequence encoding the peptide epitope tag EYMPME (13) was added 3` to the heptad repeat sequence to facilitate purification of the protein.


Figure 1: Helical wheel representations. A, the heterotrimeric association of the coiled-coil sequences fused to the IL-2R and ectodomains in the cc complex (arrows indicate potential electrostatic interactions); B, the homotrimeric association of the coiled-coil sequences fused to the IL-2R ectodomain in the cc complex.



Preparation of the Fusion Proteins

The cDNA for the -subunit fusion protein possessing the LKALEKE heptad repeat was constructed in pUC-19. The first double-stranded oligonucleotide cassette encoding approximately one-half of the seven-heptad repeat segment, a 5` NheI site and two DsaI sites for directional cloning of the -subunit ectodomain, was inserted at the BamHI site in pUC-19. After verification of sequence and orientation, the second double-stranded cassette completing the coiled-coil cDNA was inserted between AflII and BstBI sites of cassette I. The cDNA encoding the -subunit ectodomain, obtained as described previously(19) , was directionally inserted between the two DsaI sites. The resulting IL-2Rcc sequence was removed from pUC-19 (NheI and BamHI digestion) and subcloned into the pBlueBac II baculovirus expression vector (Invitrogen) and verified by sequencing.

Similarly, the cDNA encoding the -subunit fused to the LEALKEK coiled-coil repeat was constructed in pUC-8. The first cassette encoding a 5` XbaI site in addition to the coiled-coil cDNA was inserted at the BamHI site in pUC-8. The second cassette with the remaining coiled-coil cDNA was inserted between XhoI and AflII sites in the first cassette. The cDNA encoding the -subunit ectodomain was directionally cloned between the BamHI and XbaI sites. The complete fusion protein cDNA was then removed from pUC-8 (BamHI and BglII) and subcloned into the BamHI site of the pBlueBac II baculovirus expression vector (Invitrogen). The sequence and orientation were verified by sequencing. High titer recombinant virus was then prepared as described previously(19) . Trichoplusia ni (High-Five) insect cells were infected with either a single virus or simultaneously co-infected with IL-2Rcc and IL-2Rcc recombinant virus, and the cell-free culture supernatants were collected 72 h postinfection.

Purification of IL-2Rcc was carried out by passing the harvested cell supernatant over an epitope tag immunoaffinity column. The bound fraction was eluted from the column with a solution of epitope peptide, and the protein-containing fractions were pooled and dialyzed against phosphate-buffered saline.

After co-infection, preliminary isolation of IL-2Rcc from the cell-free supernatant was performed in a similar fashion using the tag epitope column. In order to obtain the heterocomplex containing both receptor ectodomains, the bound fraction eluted from the epitope immunoaffinity column (-subunit specific) was applied to an immunoaffinity column specific for the -subunit ectodomain (TIC antibody,(12) ). The complex containing both domains was eluted with 0.2 M HOAc, 0.2 M NaCl and dialyzed against phosphate-buffered saline.

Characterization of the Protein Complexes

The composition of the purified complexes was analyzed by SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue staining. The IL-2Rcc complex was also subjected to automated N-terminal Edman sequencing. Ultracentrifugation analysis of the complexes was performed at the centrifugation facility at the University of New Hampshire on a Beckman XL-A analytical ultracentrifuge equipped with Rayleigh interference optics (22) using the methods described previously for the cc complex(19) . The partial specific volume for the complexes was calculated assuming a protein molecular weight of 31,194 for the -subunit ( 0.73 ml/g) and 30,447 for the -subunit ( 0.74 ml/g) and a carbohydrate molecular weight ( 0.63 ml/g) of 12,200 g/mol and 8,400 g/mol for the - and -subunit, respectively (see Ref. 19). Sedimentation velocity was conducted at 50,000 rpm, and the sedimentation coefficient distributions (g(s*)) were determined from the time derivative of the concentration profile as was described for the cc complex(19) .

Competitive Radioligand Binding

Competitive displacement of radiolabeled IL-2 from the low affinity IL-2R on the human leukemic T-cell line MT-1(2) was carried out as described previously(19) . The assay was performed at 37 °C under conditions where the depletion of free I-IL-2 is negligible. Thus, the fraction of maximum bound I-IL-2 (b = c (1 + K*C* )/(1 + cK*C*), where C* is the total concentration of I-IL-2 (500 pM), C* is the concentration of I-IL-2 in the presence of soluble receptor, c = C*/C*, and K* is the equilibrium constant for I-IL-2 binding to the cell surface -subunit on MT-1 cells. In the nonlinear least-squares fits to the data, we took the concentration of I-IL-2 bound to soluble receptor to be KC*R/(1 + KC*), where R is the total concentration of soluble receptor, and K is an equilibrium binding constant for the binding of I-IL-2 to the soluble receptor. Since the total concentration of I-IL-2 remains unchanged, C* = C* + KC*R/(1 + KC*), can be solved for c, and c can then be substituted into the expression for b. The following equation for total radioactivity bound, cpm, was used to fit the data cpm = ns + (cpm(0) - ns)b, where ns is the amount of nonspecifically bound I-IL-2. In control experiments where unlabeled IL-2 was used to compete I-IL-2 (not shown), we determined that the K for unlabeled binding to the -subunit on MT-1 cells was 2.0 10M (three separate experiments). When fitting the soluble receptor data, we took K* equal to this value. Estimates of the standard errors of K were obtained using a bootstrap method (23) where 150 simulations were performed for each estimate.

Neutralization of IL-2 Bioactivity

The ability of the complexes to neutralize IL-2 bioactivity was determined as described previously (8) but in the presence of an EC (10 pM) IL-2. The assay was carried out on normal human peripheral blood lymphocytes, the IL-2-dependent human T-cell line Kit 225(13) , or the murine IL-2-dependent T-cell line CTLL-2(24) .


RESULTS AND DISCUSSION

The levels of expression of cc and cc fusion protein complexes in insect cells were comparable with those previously observed for the expression of the individual IL-2R ectodomains (12, 13) and for the cc homomeric complex(19) . The initial attempts at co-expression of both and - receptor subunit fusion proteins were performed employing IL-2Rcc virus. Therefore, preliminary isolation was carried out by passing the harvested supernatant over an epitope tag immunoaffinity column. The bound fraction was recovered and tested for its ability to inhibit IL-2 activity in a competitive bioassay(13) . In preliminary experiments (data not shown), this preparation was 50-100-fold more potent than a complex of IL-2Rcc alone (prepared and isolated in a similar fashion). Although not designed to be quantitative, these experiments suggested the presence of a mixed subunit complex with greatly increased binding affinity.

Since the tag affinity column was specific only for the -subunit fusion protein, it was possible that this fraction also contained homomeric cc complex. In order to isolate the cc heterocomplex from this mixture, the fraction that bound to the tag affinity column was applied to an immunoaffinity column specific for the -subunit ectodomain (TIC antibody(12) ). SDS-polyacrylamide gel electrophoresis analysis of the fraction bound to this second column confirmed enrichment of -subunit fusion protein when compared with the starting mixture or column flow-through (Fig. 2).


Figure 2: Gel electrophoresis. SDS-polyacrylamide gel electrophoresis analysis of immunoaffinity purification of the cc complex (Coomassie Blue-stained). Lane 1, the fraction of co-infection cell supernatant bound to the ectodomain specific tag epitope affinity column; lane 2, the flow-through fraction obtained when the tag epitope bound sample (lane1) was applied to a ectodomain-specific immunoaffinity column; lane 3, the fraction bound to the ectodomain specific immunoaffinity column, the cc complex. Acc and Bcc mark the positions of the - and -subunit coiled-coil fusion proteins, respectively.



Automated N-terminal Edman sequencing of this product matched the published amino acid sequence of both and receptor subunits. The results of the first seven cycles for the -subunit were Glu, 62 pM; Leu, 51 pM; Xaa, 0.7 pM; Asp, 41 pM, Xaa, not quantifiable; Asp, 0.4 pM; Pro, 43 pM, and the results -subunit were Ala, 39 pM; Val, 27 pM; Xaa, 0.4 pM; Gly, 20 pM; Thr, 15 pM; Xaa, not quantifiable; Gln, 20 pM. The / ratio of cycles 1, 2, 4, and 7 are 1.6, 1.9, 2.1, and 2.2, respectively (cycles 3, 5, and 6 occur at unstable or glycosylated positions in one or both subunits). Therefore, N-terminal sequence analysis of the cc complex revealed a 2:1 stoichiometry of -subunit to -subunit ectodomain. This stoichiometry was consistent with subsequent characterization of ligand binding via surface plasmon resonance(35) .

Studies of competitive neutralization of bioactivity (not shown) determined on Kit 225 cells (13) revealed that the fraction that bound to both columns was 2-fold more potent (IC = 0.2 µg/ml) than the initial tag column-bound fraction (IC = 0.4 µg/ml) and 3-fold more potent than the TIC column flow-through fraction (IC = 0.6 µg/ml). This heteromeric complex was the most potent ectodomain-related inhibitor ever tested in the competitive bioassay. Therefore, we carried out a quantitative ligand binding study to determine the solution affinity. In order to exclude any potential interference by the tag epitope sequence, we prepared another -ectodomain/coiled-coil fusion protein lacking this tag sequence and isolated the heteromeric complex by sequential affinity columns employing monoclonal antibodies specific for each ectodomain (, TAC(25) ; , TIC(12) ).

We compared the ability of this cc heterocomplex, a complex composed of the -subunit alone (cc) and a mixture of uncomplexed - and -ectodomains, to compete with the low affinity cell surface receptor for IL-2 binding on the MT-1 cell line (2) (Fig. 3). The results of the competitive binding assay clearly demonstrate that the cc complex is a much more efficient competitor than the other two preparations. Since this complex is a multimer of subunits, each of which is potentially capable of binding ligand independently, it was important to distinguish cooperative binding from the potential effects of avidity due to simple multivalency. To quantify the competition assays and determine if binding was cooperative, we calculated an effective dissociation constant for the complex (K) from the equilibrium binding constants (K = 1/K) listed in . If the three subunits of the complex act independently, then K = 3/(2K + K), where K and K are the equilibrium constants for the binding of labeled IL-2 to the and ectodomains, respectively. If the binding is positively cooperative, then the K will be smaller than this value, while if the binding is negatively cooperative, it will be greater than this value. We estimate () that for noncooperative, independent binding, K 76 nM. Nonlinear least square analysis of the competitive binding data (Fig. 3) yields a K = 0.32 nM for the heteromeric cc complex, a K = 15 nM for the cc complex, and a K = 42 nM for the mixture of individual ectodomains. Previously, we determined that for the cc trimeric complex, K = 300 nM per -ectodomain or K 100 nM for the complex(19) . Therefore, the results in indicate that ligand binding for the cc complex is highly cooperative. In addition, this complex is an efficient competitor at concentrations below 1 nM, where occupancy of the third -ectodomain is insignificant(35) . The data also suggest that binding of the homomeric cc complex is consistent with the three -ectodomains binding ligand independently and noncooperatively (as was previously observed for the cc complex). For the mixture of and ectodomains, there is no detectable ectodomain association in the absence of the recognition sequences. This is unlike the ligand-induced association of the and ectodomains in solution(13) . Since the full-length / pseudo high affinity site exists preformed on the surface of cells, it is likely that transmembrane or cytoplasmic contact contributes to the stability of the complex.


Figure 3: Radioligand binding. Competitive displacement of I-IL-2 on MT-1 cells. Total bound radioactivity is plotted versus concentration of soluble receptor for the cc complex (), the cc complex (), and an equimolar mixture of and ectodomains (). Maximum cpm bound in the absence of competitor (2085 ± 47 cpm) was obtained from the average of six determinations. The assay was performed at 37 °C under conditions where the depletion of free I-IL-2 is negligible. Thus, the fraction of maximum bound I-IL-2, b = c (1 + K*C*)/(1 + cK*C*), where C* is the total concentration of I-IL-2 (500 pM), C* is the concentration of I-IL-2 in the presence of soluble receptor, c = C*/C*, and K* is the equilibrium constant for I-IL-2 binding to the cell surface -subunit on MT-1 cells. In the nonlinear least-squares fits to the data (solidcurves), we took the concentration of I-IL-2 bound to soluble receptor to be KC* R/(1 + KC*), where R is the total concentration of soluble receptor (x axis) and K is an equilibrium binding constant for the binding of I-IL-2 to the soluble receptor. Since the total concentration of I-IL-2 remains unchanged, C* = C* +KC*R/(1 + KC*). The following equation for total radioactivity bound, cpm, was used to fit the data: cpm = ns + (cpm (0) - ns)b, where ns is the amount of nonspecifically bound I-IL-2 (410 cpm, visible as noncompetable cpm in figure). In control experiments where unlabeled IL-2 was used to compete I-IL-2 (not shown), we determined that the K for unlabeled binding to the -subunit on MT-1 cells was 2.0 10M (three separate experiments). When fitting the soluble receptor data, we took K* equal to this value.



The efficiencies of these complexes to inhibit IL-2 bioactivity in a human peripheral blood lymphocyte bioassay (8) were also compared (Fig. 4). The IC value obtained for the cc complex (0.9 nM) was slightly greater than its K, (0.32 nM) while the IC value obtained for the cc trimer (100 nM) was significantly greater than its K (15 nM). The cc homomeric complex did not compete in this assay at concentrations in excess of 1 µM (data not shown). Similar assays (not shown) were performed on the murine CTLL-2 bioassay (24) where the IC values were 2 and 500 nM for the cc and cc complexes, respectively. The rapid dissociation rate from the - and -subunits (2, 26) compared with the slower dissociation rate from the /- pseudo high affinity site (26, 35) may account for the poor efficiency of competition for the homomeric complexes during the 24-h bioassay. The 100-fold increase in potency for the heteromeric complex compared with the homomeric cc complex provides further support for the cooperative nature of ligand binding.


Figure 4: Competitive bioassays. Neutralization of IL-2 bioactivity by the cc complex () and the cc complex () in the human peripheral blood lymphocyte bioassay. The assay was performed as described (8) in the presence of an EC of IL-2 (10 pM). Each data point is the mean of triplicate determinations with standard deviation indicated. Maximum response to 10 pM IL-2 was 6055 cpm with a background response of 88 cpm in wells where no IL-2 was added.



In the previous study of the IL-2Rcc complex, the molecular weight of the trimer complex was determined by analytical ultracentrifugation(19) . Therefore, we analyzed the cc complex using both equilibrium sedimentation and sedimentation velocity techniques. Equilibrium sedimentation studies were consistent with a trimeric complex () with no significant evidence of monomer at any of the concentrations studied. Comparison with the cc complex previously examined (19) indicates that the heteromeric complex is at least as stable as the IL-2R-homotrimer. Sedimentation velocity experiments were consistent with sedimentation equilibrium results (). At concentrations below 0.25 mg/ml, the major component migrated at s of 4.6-4.8. At 0.52 mg/ml, the major peak was at 4.3 s, with a shoulder extending past 5 s (Fig. 5). All curves provide some evidence for small amounts of both smaller and larger species, but there is no evidence for either mass action association or dissociation. The decrease in s with increasing concentration is expected for a noninteracting asymmetric molecule(27) . The heterocomplex migrates as an extremely asymmetric molecule; assuming the complex behaves as a hydrated (0.4 HO/g of protein) prolate ellipsoid, the axial ratio is 15. This is consistent with the expected structural nature of the complex being globular ectodomains fused to an extended coiled-coil stalk. The observed formation of trimers using the designs depicted in Fig. 1is in accord with recent studies confirming that heptads containing Leu residues at both the a and d positions favor the formation of trimeric complexes(28, 29, 30) .


Figure 5: Sedimentation velocity analysis. Sedimentation coefficient distribution (19) of the cc complex at a loading concentration of 0.52 mg/ml (--), 0.25 mg/ml (- - -) and 0.08 mg/ml (). Aggregated material is noted from 8-12 s at 0.52 mg/ml. Trailing edge from 1-2 s for all loading concentrations is indicative of some smaller material. Integration of the major peak at each loading concentration indicates that the trimeric complex represents 80-95% of migrating species.



In this study, we have employed coiled-coil heptad repeats as recognition elements to mediate the solution assembly of receptor ectodomains that normally function cooperatively in cell surface ligand capture(8) . We employed seven coiled-coil heptads in order to achieve stability at submicromolar monomer concentrations. The results of the competitive ligand binding assays confirm that the stability of the cc complex extends into the subnanomolar range since significant competition is observable at these concentrations. The effectiveness of the use of like-charged electrostatic residues at positions e and g of the heptad repeat to favor heteromeric over homomeric association is uncertain. The cc trimeric complex in which each monomer carried like-charged residues at these positions was also stable. Although electrostatic interactions can direct heteromeric coiled-coil association(21, 31) , the contribution of electrostatic interactions is less significant than hydrophobic interactions to the overall stability of coiled-coil complexes. High resolution studies of triple stranded coiled-coil peptides suggest that like-charged side chains have sufficient flexibility to avoid close approach(28) .

Coiled-coil sequences from transcription factors have been employed previously to help mediate the formation of antibody complexes (for example, see Ref. 32). Unlike these studies, we have chosen multiple copies of idealized coiled-coil sequences due to their inherently greater stability. In addition, our goal was to assemble a cooperative system using noncovalent complexation. Cooperativity in ligand binding by the - and -IL-2R subunits in the cc complex is evident from the 45- and 300-fold increases in affinity when compared with the homomeric complexes of - and -subunits, respectively. How closely the cc complex mimics ligand binding by the cell surface -pseudo high affinity site is difficult to determine. Values ranging between 100 and 600 pM are often reported for the -receptor when the individual subunits are transfected into nonlymphoid cell lines(26, 33, 34) . The reason for this variation may be due to cell type or the ratio of subunits expressed, nevertheless, the K value for our cc complex is within the range of these reported values. Although the stoichiometry of the soluble heterocomplex is not 1:1, neither is the ratio of to IL-2 receptor subunits on the surface of most cells (2, 10, 33). It is highly likely that ligand binding by the soluble cc complex resembles, at least in part, the functional form of the cell surface pseudo high affinity site. Subsequent study of both equilibrium and kinetic ligand binding characteristics of this complex employing surface plasmon resonance support this conclusion (35). ()

Ligand-induced receptor subunit cross-linking to form both homomeric and heteromeric complexes is a hallmark of the hematopoietin receptor family(1) . Refinements in the designs of coiled-coil peptide complexes now make it possible to prepare stable assemblies containing two, three, or four subunits (29, 30) as well as to direct the formation of heteromeric complexes(21, 31) . This is the first study to demonstrate the feasibility of employing coiled-coil designs as recognition units to mediate the solution assembly of heteromeric cytokine receptor complexes. This approach can easily be extended to other receptor complexes composed of two to four similar or different subunits and may provide reagents for detailed receptor subunit and ligand binding characterization, crystallographic analyses, selection of novel ligands from random libraries, or in vivo therapeutic evaluation.

  
Table: Equilibrium binding constants

Equilibrium solution binding constants (K) determined from the competitive radioligand binding data shown in Fig. 3 as described in ``Materials and Methods.''


  
Table: Analytical ultracentrifugation cc IL-2R complex

Analytical ultracentrifugation was carried out as described under ``Materials and Methods.'' Concentration lists cell loading concentrations used in combined analysis at each rotor speed indicated. M is the apparent z-averaged molecular weight (19). Values in parentheses indicate the 95% confidence interval. r.m.s. is root mean square of variance of fit to a model consisting of a single ideal thermodynamic component. s is the sedimentation coefficient determined from the time derivative of the concentration profile as previously described (19).



FOOTNOTES

*
This work was supported by grants from the Hitchcock Foundation, the National Institutes of Allergy and Infectious Diseases (AI34331), General Medical Sciences (GM35556 to B. G.), the American Cancer Society (FRA-385), The National Science Foundation (DIR-9002027, to T. M. L.) and also by the Norris Cotton Cancer Center, the United States Department of Energy, and Chiron Corporation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 603-650-1474; Fax: 603-650-1129.

The abbreviations used are: IL-2R, interleukin-2 receptor; IL, interleukin; cc, coiled-coil receptor complex.


ACKNOWLEDGEMENTS

We thank Kendall Smith, Irwin Chaiken, and Randy Noelle for helpful comments; Abla Creasy for direction and support; and Renee Risingsong for technical expertise. We also thank Gwynn Pardee for help with recombinant baculovirus insect cell expression.


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