Proc. Natl. Acad. Sci. USA 85 (1988)

Correction. In the article "Structure-function relationshipsfor the interleukin 2 receptor: Location of ligand and anti-body binding sites on the Tac receptor chain by mutationalanalysis" by Richard J. Robb, Cynthia M. Rusk, and MichaelP. Neeper, which appeared in number 15, August 1988, ofProc. Natl. Acad. Sci. USA (85, 5654-5658), the authorsrequest that the following correction be noted. Mutant 49 ofthe Tac receptor subunit in which Thr-85, Thr-86 werereplaced by Ala-85, Gly-86 was also modified at the Cterminus of the polypeptide chain. The oligonucleotide usedin the mutation reaction apparently hybridized with thesingle-stranded template at the correct location surroundingThr-85, Thr-86 and also at a site after the codon for Gln-202.As a consequence, the C-terminal region of the mutant 49protein differed markedly from that of the native molecule.The mutant protein bound to affinity supports coupled withinterleukin 2 and the anti-Tac monoclonal antibody, but wasdefective for expression on the cell surface. Examination ofa new mutant of the Tac protein in which Thr-85 and Thr-86were replaced by Ala and Gly, respectively, but in which theremaining sequence was identical to that of the nativeprotein, indicates that this substitution has no effect oncell-surface expression. Moreover, the mutant receptor ex-pressed on the surface of transfected murine L cells boundinterleukin 2 and each of the eight monoclonal antibodiestested in the original paper with an affinity indistinguishablefrom that of native Tac protein. The effect seen on intracel-lular transport for the original mutant 49 was thus attributableto the aberration in the protein's C terminus and not thesubstitution for Thr-85, Thr-86. Kozarsky et al. (14) demon-strated that cells defective for O-linked glycosylation wereincapable of expressing native Tac protein on their surface.Our suggestion that Thr-85, Thr-86 might be the critical sitefor such glycosylation was, therefore, erroneous. This cor-rection does not otherwise affect the conclusions of thepaper.

Correction. In the article "Helper T-cell antigenic site iden-tification in the acquired immunodeficiency syndrome virusgp120 envelope protein and induction of immunity in mice tothe native protein using a 16-residue synthetic peptide" byKemp B. Cease, Hanah Margalit, James L. Cornette, ScottD. Putney, W. Gerard Robey, Cecilia Ouyang, Howard Z.Streicher, Peter J. Fischinger, Robert C. Gallo, CharlesDeLisi, and Jay A. Berzofsky, which appeared in number 12,June 1987, of Proc. Natl. Acad. Sci. USA (84, 4249-4253),the authors request that the following correction be noted. Inthe legend to Fig. 1 (page 4250), residue 9 of the sequence ofenv T2 (residues 112-124) should be aspartate. Thus, thesequence should read "His-Glu-Asp-Ile-Ile-Ser-Leu-Trp-Asp-Gln-Ser-Leu-Lys." This sequence is shown correctly inFig. 4.

14. Kozarsky, K. F., Call, S. M., Dower, S. K. & Krieger, M.(1988) Mol. Cell. Biol. 8, 3357-3363 (cited as "in press" in thepublished paper on p. 5658).

8226 Immunology: Corrections

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Proc. Natl. Acad. Sci. USAVol. 85, pp. 5654-5658, August 1988Immunology

Structure-function relationships for the interleukin 2 receptor:Location of ligand and antibody binding sites on the Tacreceptor chain by mutational analysis*

(lymphokines/site-specific mutagenesis)

RICHARD J. ROBBt, CYNTHIA M. RUSK, AND MICHAEL P. NEEPERMedical Products Department, Glenolden Laboratory, E. I. du Pont de Nemours & Company, Glenolden, PA 19036

Communicated by K. Frank Austen, April 15, 1988

ABSTRACT The Tac protein plays a role in high- andlow-affinity interleukin 2 (IL-2) receptors. A mutational surveyof this molecule identified several small segments in which thebinding of IL-2 was particularly sensitive to amino acid substi-tutions. Two of the segments (residues 1-6 and 35-43) locatedin the exon 2-encoded region of the molecule overlapped theapparent binding sites of three monoclonal antibodies (anti-Tac,GL439, and H31) that block high- and low-affinity Tac-IL-2interactions, thus supporting the hypothesis that these segmentsof the protein are at or near sites of receptor-lgand contact. Incontrast, the apparent binding sites ofantibodies (Hiei and H47)that selectively inhibit high-affinity IL-2 binding were mapped toa distinct location (residues 158-160) within the region encodedby exon 4 of the Tac gene. Since high-affinity receptors consistof a heterodimer of Tac and a second lignd-binding protein(p70), this portion of the Tac molecule may be involved in theinteraction between the two receptor subunits. As expected, thebinding sites of noninhibitory antibodies (7G7/B6, residues140-144; H48, residues 170-211) did not overlap those segmentsin which IL-2-binding mutants were observed. These resultsprovide a preliminary correlation of structure and function forthe Tac protein that should prove useful in evaluating detailedmodels of the IL-2--receptor complex.

Cellular receptors for the growth and differentiation factorinterleukin 2 (IL-2) exist in at least three forms, which differin their ligand-binding affinity and subunit composition (forreview, see ref. 1). Low-affinity receptors consist of the55-kDa Tac glycoprotein, intermediate-affinity sites consistof the distinct 70-kDa molecule(s) (p70), and high-affinitysites consist of a noncovalent heterodimeric structure involv-ing both components. In a series of reports, we haveexamined structure-function relationships for the Tac recep-tor subunit (2-4). This protein is encoded by eight exonslocated on human chromosome 10 (5). Initial experimentsemploying chemical crosslinking of IL-2 to high- and low-affinity receptors demonstrated a close proximity betweenthe ligand and the segment of the Tac molecule encoded byexon 2 (amino acids 1-64 ofthe mature protein) (2). Althoughthis result suggested that the exon 2 region contained theligand binding site, examination ofa series oftruncated formsof the Tac protein indicated that the shortest fragment of themolecule capable of binding IL-2 was composed of aminoacids 1-163 (3, 4). At least part of the reason why smallerfragments failed to bind IL-2 was due to the critical role ofintramolecular disulfide bonds in maintaining an active con-formation of the molecule. Substitution of alanine for any ofthe 10 cysteine residues within the first 163 positions, includ-ing Cys-163, resulted in proteins that were defective for li-gand binding (4). As an extension ofthese studies, the present

report uses mutational analyses to identify key sites in theNH2-terminal 163 positions of the Tac protein that areinvolved in the binding of IL-2 and a panel of inhibitory andnoninhibitory monoclonal antibodies.

MATERIALS AND METHODSMutagenesis. Oligonucleotide-directed mutagenesis ofa Tac

cDNA-containing expression vector (PKZ) was performed asdescribed (4, 6). Each of the oligonucleotides incorporatedboth the desired amino acid substitution and the creation ordeletion of a restriction enzyme site. After transformation ofcompetent bacterial cells, colonies containing the mutatedvector were identified from a random selection by digestionof plasmid DNA with the appropriate enzyme. Candidatemutants were then confirmed by dideoxy sequencing (13).To focus on possible contact residues for ligand and

antibody binding, the mutations were generally confined tocharged and uncharged polar amino acids present in theNH2-terminal 163 positions of the mature receptor protein(for the Tac protein sequence, see ref. 5). Substitute aminoacids were selected that substantially altered the ability of theside chain to participate in intermolecular bonding (i.e., basicand acidic residues were interchanged, hydrogen-bondingresidues were replaced by alanine) while minimizing disrup-tion of local secondary structure. The choices were con-strained, however, by the decision to introduce or delete arestriction enzyme site.

In addition to the site-specific mutations in amino acidpositions 1-163, the contribution of extracellular amino acidsbeyond residue 163 was examined by deleting a portion of theTac cDNA. Unique Spe I restriction sites were introduced atsites corresponding to amino acid positions 169-170, and211-212 by oligonucleotide-directed mutagenesis. The mod-ified vector was then treated with Spe I and religated, re-sulting in the removal of the bases encoding amino acids 170-211.

Analysis of Mutant Tac Proteins. Murine L cells weretransformed with plasmid DNA encoding native and mutantTac proteins by a modification of the DEAE-dextran method(3). After 48 hr of culture, the number and affinity of thecellular binding sites for radioiodinated IL-2 were measuredas described (4, 7). The level of binding of various murinemonoclonal antibodies was also determined, by using a singleconcentration of antibody in combination with a radioiodi-nated goat anti-murine immunoglobulin reagent (DuPont-NEN, Boston). The anti-Tac antibody was kindly provided byT. Waldmann and W. Greene (National Institutes of Health,

Abbreviation: IL-2, interleukin 2.*This paper is no. 6 in a series. Paper no. 5 is ref. 4.tTo whom reprint requests should be addressed at: GlenoldenLaboratory, Rm. 205, E. I. du Pont de Nemours & Co., 500 SouthRidgeway Avenue, Glenolden, PA 19036.

5654

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 85 (1988) 5655

Bethesda, MD); the 7G7/B6 antibody was provided by D.Nelson and L. Rubin (National Institutes of Health), and theHiei, HA26, H31, H47, and H48 antibodies were provided byK. Sugamura (Tohoku University, Sendai, Japan). Monoclonalantibody GL439 (IgG1) was prepared in our laboratory frommice immunized with the Tac-positive HUT 102B2 cell line.The assay results for each of the Tac mutants were normalizedto those obtained with the native PKZ vector based on thebinding of rabbit polyclonal antibody R239ap169186. R239,which was made against a synthetic peptide consisting of Tacresidues 169-186(2), recognizes normal and denatured forms ofTac protein, making it ideal for detecting Tac chains that do notfold properly (3, 4).

In addition to the cell binding assays, native and mutantforms of the Tac protein were also compared by NaDodSO4/PAGE. Proteins present in transfected L cells were labeledbiosynthetically with [35S]methionine (du Pont-NEN), ex-

tracted with detergent, and incubated with Sepharose beadscoupled with IL-2 and certain of the antibody reagents (3, 4).Purified Tac proteins were eluted from the beads withelectrophoresis sample buffer and fractionated on 12% acryl-amide gels as described (3).

RESULTS

A Scatchard analysis of IL-2 binding to 89 mutant forms ofthe Tac protein identified two small segments of the moleculein which amino acid substitutions drastically affected thereceptor-ligand interaction (Table 1). Virtually all IL-2 bind-ing was eliminated by certain mutations in positions 35-43,whereas a drop of 3-15 times in the binding constantaccompanied a variety of changes in the NH2-terminal sixpositions. The enormous decrease in affinity associated withreplacement of Arg-35 and Arg-36 was particularly notewor-thy since it was not limited to a single choice for the alternateamino acids (see mutations 22-24). The IL-2 binding affinitywas also markedly affected by substitution of His-120 and theresidues adjacent to Cys-28, -30, and -59, and modestlydecreased by mutations at positions 110, 154, and 157. Sincemutational analyses do not readily distinguish between directeffects on ligand-contact residues and indirect effects causedby changes in protein folding, these results must be inter-preted with caution. Some of the mutations, particularlythose occurring near cysteine residues involved in intramo-lecular disulfide bonds (Cys-3 to Cys-147; Cys-28 and -30 toCys-59 and -61, respectively; and Cys-131 to Cys-163; ref. 4),may have indirectly affected ligand binding by distorting thetertiary structure of the molecule. Nevertheless, the findingthat most of the substitutions that affected IL-2 binding werein the portion of the molecule encoded by exon 2 (residues 1-64) suggests that key elements of the binding site are locatedin this segment.The mutant Tac proteins were also tested for their reac-

tivity with a panel of monoclonal antibodies (Table 1). Threeof the antibodies, anti-Tac, GL439, and H31, block IL-2binding to the Tac protein present in high- and low-affinityreceptor configurations (refs. 7 and 8 and unpublished ob-servations). In a reciprocal fashion, IL-2 blocks the bindingof each of these antibodies, suggesting that the antibodyepitopes overlap the IL-2 binding site. Consistent with thisinterpretation, the binding of anti-Tac antibody was signifi-cantly affected only by mutations of Asn-4, placing itsapparent epitope within one of the segments (residues 1-6) inwhich pronounced IL-2-binding mutants were located. Bind-ing of the other two inhibitory antibodies was stronglyaffected by mutations of residues 42 and 43, placing theirapparent epitopes adjacent to another segment (residues 35-43) containing IL-2-binding mutants. These antibodies, there-fore, may interfere with IL-2 binding by sterically blocking

ligand access to one or the other of two potential contact siteson the receptor chain.Two of the other antibodies used to assess the mutant Tac

proteins (Hiei and H47) selectively reduce the binding con-stant of high-affinity receptors without affecting the bindingconstant or number of low-affinity sites (8). Since high-affinity binding presumably reflects the simultaneous inter-action of IL-2 with both the Tac and p70 receptor chains (1),these antibodies may exert their selective effect by interferingwith the association of the two receptor subunits (8). Thebinding of both antibodies to the Tac protein was eliminatedby modification of residues 158 and 160 (Table 1), placingtheir apparent epitopes within the segment of the moleculeencoded by exon 4 (residues 102-173). H47 binding, how-ever, was also weakly affected by certain mutations inresidues 135-156 and by changes in residues 9 and 12 (Table1), raising the possibility that these segments and residues158-160 are close to one another in the three-dimensionalstructure. None of these mutations significantly affected ligandbinding by the transfected L cells, consistent with the lack ofeffect of Hiei and H47 on low-affinity Tac-IL-2 interactions.Of the remaining antibodies tested, one (HA26) slightly

reduces the binding constants of high- and low-affinity recep-tors, whereas the others (7G7/B6 and H48) have no appre-ciable effect on either type of IL-2 binding (8, 9). The bindingof antibody HA26 was affected by the mutation of residues 158and 160, as well as by several substitutions at or near position140 (Table 1). The latter modifications also affected the bindingof 7G7/B6. Although the apparent binding sites for HA26 and7G7/B6 thus appeared to be in the general vicinity of those ofthe Hiei and H47 antibodies, subtle variations must occur toexplain the different effects these antibodies have on IL-2binding. The slight reduction in the IL-2 binding constantcaused by HA26 (8) may be related to the effect on IL-2binding that was observed when Thr-154 and Thr-157 weremodified. This segment of the Tac molecule might contain asecondary contact site for IL-2 or it might influence IL-2binding indirectly by affecting the folding of the critical exon2 segments. In contrast to the other antibodies, the binding ofH48 was affected only by the deletion of residues 170-211(mutation 89, Table 1). Consistent with the location of itsbinding site in this region, H48 was the only antibody of theeight tested that inhibited the binding of R239apl69186 (unpub-lished observation). Furthermore, while the other antibodieswere capable of binding a truncated form of Tac proteinconsisting of residues 1-163, H48 was totally ineffective(unpublished observation). The deletion of residues 170-211also reduced the binding of the Hiei, HA26, H47, and 7G7/B6antibodies, although the effect was less pronounced than thatfor antibody H48. By bringing the exon 4-encoded region closeto the cell membrane, the deletion of these residues may havesterically hindered access ofthe Hiei, HA26, H47, and 7G7/B6antibodies to their binding sites and/or altered the conforma-tion of the exon 4 segment of the molecule.

In several instances, the reagents employed in the bindingassays, including R239apl69i186, failed to detect a mutant Tacprotein on the surface of the transfected L cells (denoted byan X in Table 1). To evaluate such mutations, proteins werelabeled biosynthetically with [35S]methionine and tested fortheir reactivity with certain affinity supports. In contrast toPKZ-transfected control cells that synthesized a 55-kDaprotein that reacted with IL-2, anti-Tac, and R239ap169il86,cells transformed with mutant vectors 13, 32, 61, 65, 66, 72,73, 81, and 82 synthesized a 41-kDa molecule that bound onlyto R239-coupled beads (Fig. 1). The simultaneous defects inligand binding and intracellular transport seen in thesemutations exactly mimicked those obtained when intrachaindisulfide bonds were disrupted by the direct substitution ofkey cysteine residues (4). Cells transfected with mutantvector 49, on the other hand, synthesized a 39- to 41-kDa

Immunology: Robb et al.

5656 Immunology: Robb et al. Proc. Nati. Acad. Sci. USA 85 (1988)

Table 1. Relative level and affinity of binding of IL-2 and antibodies to mutant Tac proteins

Mutation*1. E'D4/KK2. E1/K3. E'/Q4. L2/A5. D4/K6. D4/N7. D5D6/KV8. E9/K9. H12/A

10. T14/A11. F15/L12. K16/E13. Y20/A14. Y20/F15. K21/E16. E22T24/KS17. N27/A18. E29/R19. E29/A20. K3IR32/EE21. F34/Y22. K31R35R36/NEE23. R35R36/TS24. R35R36/KK25. K38S39/ED26. K38/T27. S39S41/AA28. L42Y43/SL29. N49 N68/SS30. H52S54/QT31. S53S54/AG32. W55/Y33. W55/F34. D56N57/TS35. N57Q58/AG36. D56N57Q58/KST37. Q60/A38. T62S63S64T66/AGAA39. R67/A40. T70K71/AD41. K71/D42. T74Q76/AG43. E78E79/RS44. Q80/A45. K81/E46. E82/K47. R83T86/SS48. K84/S49. T85T86/AG50. T85E87/QS51. T86E87/QS52. E87/R53. Q89S90/AG54. Q93Q97/SS55. D96/K56. S99/A57. H103/Q58. R105/E59. E06/K60. W110/F61. E111E'13/KK62. E11El13/AA63. N"12T115/AG64. T'15El16/SQ65. T 15R 17/SE66. T" 5R'17/ST67. T115Rl17/SK68. Y'19/F69. H120V123/QL70. H120/A71. F'21/y

IL-2

Relative Relativelevel affinity(A) (B)0.9 0.081.2 0.241.0 0.371.1 0.140.8 0.201.0 0.551.0 0.061.1 0.830.9 1.151.0 1.000.8 1.001.0 0.910 _1.0 0.780.9 0.960.9 1.151.0 0.480.9 0.101.1 0.281.1 0.451.0 0.77

<0.1 <0.01<0.1 <0.01-0.1 -0.02-0.1 -0.01

1.0 0.531.1 0.201.0 0.330.9 1.030.9 0.911.2 0.960 _0.8 0.851.0 0.940.9 0.261.0 1.111.1 0.941.1 0.970.9 0.921.0 0.971.0 0.831.2 0.871.1 1.001.1 0.961.0 0.831.2 0.771.1 1.070.9 1.110 _0.8 0.790.9 1.011.0 1.111.1 0.910.8 0.841.2 0.781.0 1.111.2 0.831.1 0.910.9 0.910.9 0.590 -1.0 1.011.0 0.991.0 1.110 -0 -1.0 1.010.9 0.990.9 0.081.0 0.261.0 0.77

Relative level of antibody reaction

Anti-Tac(C)0.00.90.80.80.00.00.81.01.11.01.10.901.01.01.11.01.01.00.91.01.11.01.01.01.10.90.91.01.01.001.11.01.01.01.01.01.11.01.01.01.01.01.11.01.01.001.01.11.01.01.01.01.11.11.01.11.101.11.11.0001.01.00.91.01.0

GL439(D)0.20.90.80.80.91.00.91.01.01.00.90.9

0.91.00.90.81.00.91.01.20.80.90.90.80.91.00.01.11.00.9

1.00.90.90.91.00.91.00.81.00.91.01.01.11.01.01.0

1.11.00.90.81.01.01.01.01.11.11.1

1.01.00.8

01.01.10.90.81.0

H31 Hiei H47(E) (F) (G)0.7 1.0 1.00.9 1.1 1.01.1 1.0 1.01.0 0.9 1.01.0 0.8 1.01.2 1.1 1.01.2 1.0 1.10.8 1.0 0.51.0 1.0 0.40.9 0.9 0.91.0 1.0 1.01.2 0.9 1.1

1.2 1.0 1.01.2 1.0 1.01.0 0.8 1.01.0 1.0 1.01.0 1.0 1.01.2 0.9 1.21.2 1.0 1.01.1 0.8 1.20.9 1.0 1.11.1 1.0 1.01.1 0.9 1.10.9 1.0 1.00.9 1.0 0.91.1 1.0 0.90.3 1.1 1.01.0 1.0 1.00.9 1.0 1.01.1 0.9 1.1

0.9 0.9 1.01.2 1.1 1.01.1 0.9 1.01.0 0.9 1.10.9 0.9 0.91.2 1.2 1.01.1 0.9 1.01.2 0.9 1.00.9 0.9 1.21.0 1.1 1.01.2 0.9 1.20.8 1.0 1.11.1 1.1 1.00.9 1.0 0.90.9 0.9 1.01.2 1.0 0.8

1.0 0.9 0.91.0 0.9 1.01.2 1.2 1.01.1 1.0 0.91.0 1.0 1.01.1 1.2 1.01.2 1.2 1.01.0 1.1 1.01.1 1.2 1.00.8 0.9 0.91.1 1.0 1.0

1.1 1.1 1.01.0 1.2 1.01.1 1.0 1.0

0 0 00.9 0.8 1.01.1 0.8 1.00.9 1.1 1.11.0 1.0 1.01.2 1.0 1.0

HA26(H)1.01.01.01.00.80.81.00.90.91.01.00.9

0.91.01.01.01.01.01.01.01.01.01.10.91.10.90.91.01.00.8

1.01.11.01.00.91.01.00.90.90.90.81.01.01.01.00.9

1.01.01.00.91.01.11.20.80.91.01.0

0.91.11.0

00.91.01.10.91.0

7G7 H48(I) (J)1.0 0.91.0 0.91.0 1.11.1 1.01.0 1.00.9 1.01.0 1.01.0 1.01.0 0.91.0 1.01.0 1.01.0 1.00 -1.0 1.01.0 1.00.9 1.01.0 0.90.9 1.01.0 1.21.0 1.01.1 1.00.9 1.10.9 1.11.0 1.01.0 1.11.0 1.01.0 1.11.0 1.11.0 1.00.9 0.91.0 1.00 _0.9 1.11.0 1.01.0 1.11.0 1.11.0 1.11.0 1.00.9 1.11.0 0.80.9 1.21.0 1.01.0 0.91.0 1.21.1 1.01.1 1.11.0 1.01.0 1.00 _0.9 1.21.0 0.91.0 0.91.0 0.81.1 1.21.0 1.01.0 0.91.0 0.91.0 1.11.0 1.11.1 1.20 -1.0 1.01.0 1.01.0 0.90 -0 01.0 1.11.0 1.00.9 1.11.0 1.01.1 1.0

Comment(s)tB,C,D,eBbBB,Cb,CBgg

x

bBbb

A,BA,BA,BA,BbBb,D,e

xx

b

xx

x

bx

xx

Bb

Proc. Natl. Acad. Sci. USA 85 (1988) 5657

Table 1. ContinuedIL-2 Relative level of antibody reaction

Relative Relative Anti-level affinity Tac GL439 H31 Hiei H47 HA26 7G7 H48

Mutation* (A) (B) (C) (D) (E) (F) (G) (H) (I) (J) Comment(s)t72. Q125/H 0 - 0 - - - - - 0 - X73. Q125/A 0 - 0 - - - - - 0 - X74. Y128Y129/FF 0.8 0.83 0.9 1.1 0.8 1.1 1.0 1.1 0.9 1.175. Q130Q'33/AA 0.9 0.83 0.9 1.0 1.2 1.1 1.1 1.0 0.9 0.976. y'3-/A 1.0 1.00 1.0 1.0 1.1 1.1 1.0 0.9 1.0 1.177. Y'35R136/SE 1.2 1.11 1.0 0.9 1.1 1.0 0.5 0.5 0.7 1.0 gh,i78. H139/Q 1.2 0.84 1.1 0.9 1.1 0.8 1.1 0.5 0.9 1.2 h79. R140/E 1.0 0.83 1.0 1.0 1.2 0.9 1.1 0.2 0.0 1.0 H,!80. G41E"44/SG 1.1 0.77 1.0 1.0 0.8 0.9 1.0 0.8 0.0 0.9 181. S145V146/KI 0 - 0 - - - - - 0 - X82. S145/L 0 - 0 - - - - - 0 - X83. K'48T'50/TA 1.1 0.99 1.0 0.9 1.0 1.0 0.4 0.9 1.1 1.0 g84. T150H'5/SA 1.1 0.91 0.9 0.8 1.0 1.0 0.9 0.8 1.0 1.085. K153R'55/ES 1.1 0.91 1.0 0.9 1.1 1.0 0.5 1.1 1.0 1.0 g86. T'54lT'57/AA 1.1 0.40 1.0 1.0 0.8 0.9 0.5 1.0 1.0 1.0 b,g87. W'56/F 0.9 1.11 1.0 1.0 1.1 0.9 0.4 1.2 1.0 1.0 g88. Q158Q160/AG 1.1 0.91 1.0 1.0 1.1 0.0 0.0 0.1 1.0 1.1 F,G,H89. De1(170-211) 1.0 0.96 1.0 1.0 1.0 0.7 0.5 0.3 0.3 0.0 fgh,i,J

The level and affinity of 125I-labeled IL-2 binding to L cells transfected with vectors containing mutant Tac cDNA was derived by Scatchardanalysis (7). The association constant of each mutant was divided by that of control PKZ-transfected cells (1.32 x 108 M - 1) to obtain a valuereflecting relative changes in the ligand-binding affinity (column B). The number of binding sites for IL-2 (column A) and the level of bindingof eight monoclonal antibodies (columns C-J) were also normalized to that of PKZ-transfected cells (which was assigned a value of 1.0 for eachassay), by using the binding of rabbit polyclonal antibody R239 to adjust for variations in the extent of cellular transformation. As such, theseresults represent the relative binding efficiencies between the different assays and do not reflect variations in the absolute level of cell-surfaceexpression. The data for mutation 89, which involved deletion of the R239 binding site, were normalized to the PKZ control by using the bindingof the anti-Tac antibody. The recorded values are the means of at least two determinations. In instances where mutational effects were observed(see below), the results were confirmed two or more times on freshly transfected cells. Standard deviations were consistently <10% of the mean.*In the notation identifying each mutation, the amino acids in the native sequence precede the slash mark, and the substitutions follow the slash markin the same order. The superscript numbers refer to the location of the amino acid (abbreviated with the single-letter code) in the sequence of themature Tac protein (5). Unlike the site-specific modifications, mutation 89 involved the deletion ofcDNA encoding amino acids 170-211.

tMutations that affected the reactivity of the Tac protein are highlighted by the letter of the column of the reaction in which the effect wasobserved. Capital letters denote particularly strong effects (>75% decrease), and lowercase letters denote weaker effects (25-75% decrease).Mutations for which no Tac protein was detected on the cell surface are designated with the letter X, and mutations that consistently yieldedlow surface expression (5-15% of the PKZ control) are designated by the letter x.

protein that bound IL-2 and anti-Tac, as well as R239 (Fig. 1).A defect in surface expression was thus not invariablyaccompanied by a defect in the ligand binding site. Severalother mutations, 33, 50, and 59, resulted in the expression ofmodified proteins that reacted normally with IL-2 and theantibodies in the cell binding assays but were present atunusually low levels on the cell surface (denoted by an x inTable 1). Analysis of mutant 33 demonstrated that most ofthe35S-labeled protein migrated at 41 kDa on NaDodSO4/polyacrylamide gels and reacted equally well with anti-Tac-,IL-2-, and R239-coupled beads (data not shown). A smallfraction of the mutant 33 protein, however, migrated at 55kDa, possibly corresponding to the portion of the moleculesexpressed on the cell surface. Mutant 33 thus appeared to bea "leaky" version of the type of defect seen with mutant 49.A small fraction of the 35S-labeled Tac proteins made inresponse to vectors 50 and 59 also migrated at 55 kDa andreacted with anti-Tac antibody, but the vast majority mi-grated at 41 kDa and only reacted with R239-coupled beads(data not shown). The defects in expression and ligandbinding seen with these mutations thus resembled a "leaky"version of those illustrated by vector 13 (Fig. 1).

DISCUSSIONWhen IL-2 was chemically crosslinked to the Tac proteinpresent in high- and low-affinity receptor configurations, itbecame attached to an amino group located within theNH2-terminal 83 positions of the polypeptide chain (2). Themutational analysis presented in this report supports theconclusion that this portion of the molecule is in directcontact with IL-2 by identifying two short segments withinthis region (residues 1-6 and 35-43) in which substitutions

severely affect the binding ofboth the ligand and each ofthreeinhibitory monoclonal antibodies (anti-Tac, GL439, andH31). Both of these segments were near amino groups towhich IL-2 could have been chemically crosslinked (the NH2terminus in segment 1-6 and Lys-31 or Lys-38 in segment 35-43). The reciprocal fashion in which IL-2-and these particularantibodies block each other's access to the Tac chain pro-vides strong support for the correlation of the ligand binding

PKZ

200 -92.5 -

69 -

46 -

13 49

a b c d a b c d a b c d

so --55

*-39

30 -

14.3 -

FIG. 1. NaDodSO4/PAGE analysis of biosynthetically labelednative (PKZ) and mutant (13 and 49) Tac proteins isolated by affinitychromatography on Sepharose beads coupled with various reagents.Lanes: a, control murine plus rabbit antibody; b, anti-Tac antibody;c, IL-2; d, R239etp169-186. Mutant proteins 32, 61, 65, 66, 72, 73, 81,and 82, similar to mutant protein 13 shown here, were bound only bythe R239-coupled beads and migrated as a 41-kDa protein. Positionsof molecular mass markers in kDa are shown to the left and positionsof Tac protein are to the right.

Immunology: Robb et al.

Proc. Natl. Acad. Sci. USA 85 (1988)

site with these segments of the molecule. This view is alsobolstered by the consistent mapping of noninhibitory (forlow-affinity Tac-IL-2 binding) monoclonal (Hiei, H47, H48,and 7G7/B6) and polyclonal (R239ap169186 and R234ap8O-%,ref. 2) antibodies to binding sites outside the exon 2 region.Based on computer modeling (10), both of the exon

2-encoded segments (residues 1-6 and 35-43) identified withIL-2 binding and inhibitory monoclonal antibodies have ahigh probability for expression on the protein surface. Fur-thermore, each is predicted to form a p-sheet conformation(residues 1-3 and 33-37) followed by a p-turn (residuses 4-6and 38-40). As such, they could be adjacent in the three-di-mensional structure in a parallel or antiparallel configuration.In fact, two pieces of information indicate that the segmentsmay be part of a single ligand-binding site. The simultaneousmodification of Glu-1 and Asp-4 (mutation 1) affected thebinding of GL439 and H31, as well as that of the anti-Tacantibody. Furthermore, GL439 competitively inhibited thebinding of anti-Tac to the receptor chain but did not interferewith the binding of a control antibody (7G7/B6) (unpublishedobservation). The apparent spatial overlap of the anti-Tac,GL439, and H31 binding sites favors a similar proximity forthe two segments (residues 1-6 and 35-43) implicated in IL-2binding. The hypothetical P-turns present in the two seg-ments may play a particularly significant role in the ligandbinding site since computer predictions indicate that they arealtered by many of the mutations (1, 5, 7, 22, 24, 25, and 27)in which the affinity for IL-2 was drastically reduced.The protein segments encoded by exons 2 and 4 of the Tac

gene share considerable sequence homology (5). Neverthe-less, the data do not support a duplication in the exon 4 regionof the potential contact sites (residues 1-6 and 35-43)identified in the exon 2 segment (compare mutations 1-7 and22-28 with mutations 57-59 and 77-80). Within the exon 4region only substitution of His-120 markedly reduced theligand binding affinity, whereas mutations of Trp-110, Thr-154, and Thr-157 caused a modest decrease. Since Cys-3 andCys-147 (and perhaps Cys-46 and Cys-104) form a disulfidebond linking structural elements of exons 2 and 4 (4), theseresidues may be in proximity to the critical exon 2 segments.As such, one or more of them could play a direct role in theligand binding site. Alternatively, they might simply influ-ence the folding of the exon 2-encoded region. The criticalcontribution of the Cys-3 to Cys-147 disulfide bond (4) andsuch residues as Trp-55, Arg-117, and Gln-125 to an activeconformation of the molecule demonstrates the interdepen-dence of the exon 2 and 4 regions for proper folding.The binding sites of antibodies that selectively affect the

formation of the high-affinity ternary complex of IL-2, Tac,and p70 (Hiei and H47) were tentatively mapped by mutation88 to a site within the exon 4-encoded region. Thus, whileexon 2 may provide the Tac protein with key contact sites forIL-2, part of exon 4 may provide a contact surface for anydirect interaction that might occur with the p70 subunit. Thelatter hypothesis will be directly tested with the Tac mutantsdescribed in this report by transfection of cells that selec-tively express the p70 chains.Two types of expression mutants were observed in the

present study. One type illustrated by vector 13 was accompa-nied by a defect in IL-2 and antibody binding (Fig. 1). Sincethese dual defects also occurred when intramolecular disulfidebonds were disrupted by mutation of cysteine residues (4), thisclass of expression mutant may have indirectly had the sameeffect. Such denatured forms of the Tac protein are apparentlyblocked in their transport to the Golgi apparatus in a manneranalogous to folding mutants of the influenza virus hemagglu-

tinin (4, 11). A second type of expression defect illustrated byvector 49 was not accompanied by a defect in ligand andantibody binding (Fig. 1). Kozarsky etal. (14) showed deficientcell-surface expression of the Tac protein synthesized in cellswith a reversible defect in O-glycosylation (12). The O-glycan-deficient molecules were abnormally sorted after addition ofneuraminic acid in the trans-Golgi compartment (14). Of themodifications of threonine and serine residues made in thisstudy, only the simultaneous substitution of Thr-85 and Thr-86in mutation 49 affected expression in a manner analogous to thatobserved by Kozarsky et al. (14). Ifa particular O-glycan chainis critical to sorting of the Tac protein, Thr-85/Thr-86 is a likelysite of attachment. N-Glycosylation, in contrast, appearedunimportant for transport, since substitution ofthe two relevantasparagine sites (Asn49 and -68, mutation 29) failed to affectcell surface expression. Expression defects of the type illus-trated by mutant 49, however, were not confined to directsubstitution ofThr-85 and Thr-86. Conversion ofTrp-55 to Phe(mutation 33) had a similar effect. Paradoxically, conversion ofTrp-55 to Tyr (mutant 32) resulted in the type of defectivefolding and transport illustrated by mutant 13. Likewise, theindividual replacements of Thr-85 and Thr-86 (mutations 50and 51) had distinctly different effects on expression than thedual substitution in mutation 49. Intracellular transport of theTac protein was clearly sensitive to a wide variety of muta-tions, with the choice of substitute amino acid affecting theoutcome just as critically as its position in the polypeptidechain.By using an oligonucleotide-directed approach, modifica-

tions were made in virtually all of the NH2-terminal 163 aminoacids likely to be exposed on the Tac protein surface. Althoughthe relative potency of mutations at different positions in theprimary sequence may have been biased by the choice ofsubstitution, the extensive nature of this survey makes itunlikely that important elements of the ligand and antibodybinding sites were overlooked. This preliminary correlation ofligand and antibody binding with various portions of the Tacmolecule should thus be instrumental in evaluating the resultsof yet more sophisticated approaches (i.e., x-ray crystallogra-phy) for analysis of the ligand-receptor complex.We thank S. Wurm, D. Kenney, T. Costello, M. Moran, and A.

Cavanaugh for their excellent technical support; M. Kelley forassistance in preparing the GL439 antibody; and W. Ripka for helpfulsuggestions on the choice of mutations.1. Robb, R. J. (1988) in Molecular and Cellular Aspects of Inflammation,

eds. Poste, G. & Cooke, S. T. (Plenum, New York), pp. 97-122.2. Kuo, L.-M., Rusk, C. M. & Robb, R. J. (1986) J. Immunol. 137,1544-

1551.3. Neeper, M. P., Kuo, L.-M., Kiefer, M. C. & Robb, R. J. (1987) J.

Immunol. 138, 3532-3538.4. Rusk, C. M., Neeper, M. P., Kuo, L.-M., Kutny, R. M. & Robb, R. J.

(1988) J. Immunol. 140, 2249-2259.5. Leonard, W. J., Deeper, J. M., Kanehisa, M., Kronke, M., Peffer, N. J.,

Svetlik, P. B., Sullivan, M. & Greene, W. C. (1985) Science 230, 633-639.

6. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82, 488-492.7. Robb, R. J., Greene, W. C. & Rusk, C. M. (1984) J. Exp. Med. 160,

1126-1146.8. Fujii, M., Sugamura, K., Nakai, S.-I., Tanaka, Y., Tozawa, H. &

Hinuma, Y. (1986) J. Immunol. 137, 1552-1556.9. Rubin, L. A., Kurman, C. C., Biddison, W. E., Goldman, N. D. &

Nelson, D. L. (1985) Hybridoma 4, 91-102.10. Wolf, H., Modrow, S., Motz, M., Jameson, B., Hermann, G. & Fortsch,

B. (1988) Comput. Appl. Biosci. 4, 187-191.11. Gething, M.-J., McCammon, K. & Sambrook, J. (1986) Cell 46, 939-950.12. Kingsley, D. M., Kozarsky, K. F., Hobbie, L. & Krieger, M. (1986) Cell

44, 749-759.13. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci.

USA 74, 5463-5467.14. Kozarsky, K. F., Call, S. M., Dower, S. K. & Krieger, M. (1988) Mol.

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5658 Immunology: Robb et al.