Download IL-4 is a T cell product originally described as B cell growth factor by

HUMAN INTERLEUKIN 4 RECEPTOR CONFERS
BIOLOGICAL RESPONSIVENESS AND DEFINES A NOVEL
RECEPTOR SUPERFAMILY
BY REJEAN L. IDZERDA, CARL J . MARCH, BRUCE MOSLEY,
STEWART D. LYMAN, TIM VANDEN BOS, STEVEN D. GIMPEL,
WENIE S. DIN, KENNETH H . GRABSTEIN, MICHAEL B. WIDMER,
LINDA S. PARK, DAVID COSMAN, AND M . PATRICIA BECKMANN
From the Immunex Corporation, Seattle, Washington 98101
IL-4 is a T cell product originally described as B cell growth factor by virtue of
its ability to co-stimulate B lymphocyte proliferation (1). It has broad effects on B
cells, including the promotion of Ig class switching to IgE and IgGl isotypes in activated B cells (2-7). In addition to regulating B lymphocyte growth and differentiation, IL-4 modulates the survival, proliferation, and differentiation of T lymphocytes, as well as a wide range of hematopoietic cell lineages, thereby contributing
to allergic and other immune processes (8-10) . It has recently been reported that
murine IL-4 also exhibits antitumor effects in vivo (11). IL-4 mediates its effects through
cell surface receptors; a single class of high affinity receptors for IL-4 has been detected on nearly all hematopoietic cells examined (12-17), and on many nonhematopoietic cell types as well . These include human fibroblasts, epithelial cells,
and hepatic, bladder, and pancreatic tumor lines (13), as well as murine fibroblast,
muscle, neuroblast, epithelial, and a variety of stromal cell lines (12, 16). The biological effect of IL-4 on most of these cells is unknown, but it has been reported that
IL-4 can stimulate fibroblast proliferation, and as such may play a role in inflammation (18).
We recently reported the isolation and characterization of a murine IL-4 receptor
(IL-4-R) cDNA (19), but the mechanism(s) by which IL-4-R mediate the pleiotropic
effects of IL-4 remains largely unknown. In this report we describe the cloning of
a human IL-4-R cDNA . The predicted amino acid sequence shows homology with
four other recently cloned receptors, and thus defines a novel receptor superfamily.
When transfected into a murine T cell line, the human IL-4-R cDNA confers responsiveness to human IL-4, demonstrating that the encoded protein contains sufficient
information to transmit the IL-4 signal across the cell membrane . This expression
system may prove useful for identifying components of the IL-4 signal transduction
machinery.
Cells and Culture Conditions.
Materials and Methods
Human peripheral blood T cells (PBT)' were purified from
Address correspondence to Dr. M. Patricia Beckmann, Immunex Corp ., 51 University Street, Seattle,
WA 98101.
' Abbreviation used in this paper. PBT, peripheral blood T cells.
J . Exp. MED. C The Rockefeller University Press - 0022-1007/90/03/0861/13 $2 .00
Volume 171
March 1990
861-873
861
862 MOLECULAR CLONING OF THE HUMAN INTERLEUKIN 4 RECEPTOR
three donors as described (20), and cultured in 1 Fig/ml PHA-P (Sigma Chemical Co., St.
Louis, MO) for 44 h before RNA isolation . COS-7 cells were maintained in DMEM with
10% FCS and antibiotics . CTLL-2 cells were grown as described (21). CTLLT22-8 and CTLLneo lines were grown in the same medium with the addition of 2 .0 mg/ml G418.
RNA Isolation and cDNA Library Construction and Screening. Pooled human PBLs were obtained by standard Ficoll purification, and were cultured in IL-2 for 6 d followed by stimulation with PMA and Con A for 8 h. Polyadenylated RNA was isolated and used to construct
an oligo(dT)-primed cDNA library in Xgt10 using a cDNA synthesis kit (Amersham Corp.,
Arlington Heights, IL). A hybridization probe was produced by synthesizing an unlabeled
RNA transcript of the entire murine IL-4 receptor cDNA insert from clone 7119-4 (19), followed by synthesis of a 32 P-labeled single-stranded cDNA with reverse transcriptase using
random primers (Boehringer Mannheim Biochemicals, Indianapolis, IN) . Hybridization to
plaque filters was carried out in 50% formamide, 0.4 M NaCl, 10 mM Pipes, pH 6.4, 1 mM
EDTA, 0.2% SDS, 0.1 mg/ml salmon sperm DNA at 42°C, followed by washing in 2 x SSC,
0.2% SDS at 55°C . The T22-8 cDNA clone was isolated from a library similarly prepared
from T22 cell polyadenylated RNA (22, 23) and screened with a 32P-labeled random-primed
probe made from clone PBL-1 .
Subcloning DNA Sequencing and RNA Blot Hybridization . These methods were carried out
as previously described (19). RNA blot hybridization used antisense riboprobes derived from
human IL-4-R clone PBL-1 and murine IL-4-R clone 7139-2 (19) in 50% formamide, 5 x
SSC, 0.5o7o SDS, 5 x Denhardt's, 50 mM Na(H)P04, pH 7.0, 0.1 mg/ml salmon sperm DNA
at 63°C followed by washing at 68'C in 0.1 x SSC, 0.2% SDS.
Transfection, Metabolic Labeling and Affinity Purification . COS-7 cells were transfected (24)
with 5 hg of the mammalian expression vector pDC302 (19) or pDC302 containing the human
IL-4-R cDNA clone T22-8. 3 d after transfection, cells were metabolically labeled with 100
pCi of [s sS]methionine and cysteine (Trans 35S- label; ICN Biomedicals, Inc., Irvine, CA)
for 1 h at 37°C, and then lysed (19). Lysates were incubated with a slurry of IL-4 Affigel
as previously described for murine IL-4 receptor (19), except that recombinant human IL-4
synthesized in yeast (13) was coupled to the beads instead of recombinant murine IL-4. Incubation with the beads was with or without 10 NAg ofuncoupled human IL-4. IL-4 Affigel beads
were then washed (19), and eluted proteins were analyzed by SDS-PAGE under reducing
conditions. CTLL-2 cells were electroporated (25) with 1 Fcg pSV2neo (26) and 10 FZg of either pDC302 control plasmid or pDC302 containing the human IL-4-R cDNA T22-8. Cells
were cultured as previously described (21), with the addition of 2.0 mg/ml of G418.
Binding Assays.
Equilibrium binding studies were performed on CTLL-neo and CTLLT22-8 . Murine and human ' 251-IL-4 were prepared as described (13, 16) . Specific activities
of 1 x 10' 6 cpm/mmol were routinely achieved for both preparations . Briefly, equilibrium
binding analysis was done by incubating 2 x 106 CTLL-neo or CTLLT22-8 cells with various concentrations of murine '25 1-IL-4 or human '251-IL-4 for 1 h at 37°C in binding
medium (RPMI medium containing 2.5% bovine serum albumin, 0.2% sodium azide, and
0 .2 M Hepes, pH 7.4). Free and cell-bound '251-IL-4 were separated by the pthalate oil
method (16). Nonspecific binding of 125 1-IL-4 was measured in the presence of 200-fold
molar excess unlabeled IL-4. Binding data were analyzed with RStI software (Bolt, Beranek
and Newman, Boston, MA) .
Proliferation Assays.
[3H]TdR incorporation studies were done by incubating 2 x 103
CTLL-neo or CTLLT22-8 cells in microtiter plates at 37'C in DMEM, 10% FCS, with
various concentrations of recombinant murine IL-4 (16) or recombinant human IL-4 (13).
In some experiments, cells were also cultured in the presence of various concentrations of
anti-murine IL-4-R antibody (Beckmann, M. P, K. A. Schooley, B. Gallis, T. Vanden Bos,
D. Friend, A. R. Alpert, R. Raunio, K. S. Prickett, P E. Baker, and L. S. Park, manuscript
submitted for publication.). After 20 h of culture, cells were pulsed with 1 tuCi [3H]TdR
(Amersham Corp., Arlington Heights, IL) for 4 h. Cells were harvested onto glass strips and
[3H]TdR incorporation was determined on a Packard Tricarb 460 liquid scintillation counter.
Results shown are duplicate, independent experiments .
IDZERDA ET AL .
Results
Characterization ofHuman IL-4 Receptor cDNA Clones.
863
A probe from the murine
IL-4 receptor cDNA clone 7B9-4 (19) was used to isolate human IL-4-R cDNA clones
from two libraries, one derived from human PBL, and the second from a CD4''/
CD8- human T cell line, T22 (22, 23). A restriction map and two representative
clones are diagrammed in Fig. 1 A. A full-length 3.6-kb cDNA clone, T22-8, contains an open reading frame encoding 825 amino acids flanked by 175 by of 5' and
947 by of 3' noncoding sequence (shown in Fig. 1 B). The predicted protein sequence
exhibits 53% identity with the murine IL-4 receptor, and contains a predicted 25
amino acid signal peptide, 207 amino acid external domain, 24 amino acid transmembrane region, and 569 amino acid cytoplasmic domain . Three of six potential
N-linked glycosylation sites are conserved between the human and murine sequences.
Five of the seven cysteines present in the human IL-4-R extracellular domain are
also found in the murine IL-4-R . These presumably contribute to protein structure,
and take on added significance in view of their conservation in other related receptor
sequences (see below) .
The PBL-1 cDNA showed identical sequence to T22-8 except for a 68-bp insertion that terminates the open reading frame 45 amino acids downstream of the
predicted NH2 terminus of the mature protein. This insertion was found in other
cDNA clones and therefore is not a cloning artifact but could represent unspliced
intronic sequences.
IL-4 Receptor Is A Member of a New Superfamily. A search against the EMBL and
Genbank DNA and National Biomedical Research Foundation protein databases
(28) revealed that the human IL-4-R sequence is unique, as was found with the murine IL-4-R (19) . However, in comparing the predicted amino acid sequence of the
IL-4-R with the published sequences of several recently cloned receptors, statistically significant homology was found among the extracellular domains. These sequences are the human IL-6-R (29), the human IL-2-R ,8 subunit (p75 ; reference
30), the murine erythropoietin receptor (31), and the rat prolactin receptor (32) .
Like the IL-4-R, these receptor sequences are unique in the databases, and do not
contain tyrosine kinase domains or features that would classify them as members
of other gene super¬amilies (except IL-6-R, see below) . Pairwise alignments of the
predicted extracellular domains of the receptors were performed with the ALIGN
program (33, 34), generating scores ranging from 3.9 to 8.8 (given in Table 1) . Scores
>3 .0 are statistically significant and indicate common ancestry.
Shown in Fig. 2 is an alignment of the extracellular domains of these receptor
superfamily members. Invariant amino acids are designated in bold print, although
many more amino acids are conserved among the sequences. Distinguishing features of the extracellular domains include four conserved cysteines and a COOHterminal double tryptophan-serine motif (WSXWS; see asterisks) .
Expression ofHuman IL-4-R cDNA . Proof that cDNA clone T22-8 encodes a biologically active human IL-4-R was provided by biochemical and functional analyses
of the encoded protein in mammalian expression systems. COS-7 cells were transiently transfected with the mammalian expression vector pDC302 (19), or pDC302
containing cDNA clone T22-8, and were metabolically labeled with [35 S]methio-
86 4 MOLECULAR CLONING OF THE HUMAN INTERLEUKIN 4 RECEPTOR
A
a
T22-8
v
PBL-1
200 by
B
1
51 TCATPGAGRAGCCGrXCGrGGTGGCtrh=CTA
116 ATG GGG TIM CTT TOC TCT GGG CPC CIG TTC CCT GIG AM TOC CTG UK CTG CTC CAB GIG OG AGC TCT 00 AhC AN
Net AN ON TTS CAG
-25 Her Gly Tip Uu Cys set Gly LOU W "W Pro. Val sar CYS Lu Val W Seat Gin Val AW SetBas. GlyAND
14V Val Lwl Gln
266 GAG CCC AM AGTC TCC GC TAC IGG AOC ATC TCT ACT ?QC GAG TOO AN MG An GOT CDC ACC 1115 TOC 11GC WC GAG CPC COC CTS
6 Glu Pro Sht©Val Sac Amp Tyr "wt, Par lie set the©Glu T.P Lys Not Am Gly Pro Th. A:n®sat Tar al. LY AM I356 TTG TAC CAG CTG GTI TIT CSG CTC ICC GW GCC CAC ACG TGT ATC CCT GC A11C AW GOA Ow GCr OGG~ ow
GC CTG CIC AM
36 1- Tyr GI. Lau Val Ph. Lmu Is . Set Glu Ale His Thr©Tls Pro Glu Am Nn OXY Gly Ale Gy t't'1 Vala
-WWHet
666 GO G1C GIG GTC ACT OW GT AAC TAT ACA CTG WC CTG TOG GCC GGO CAG CAG CM C'TG TGG AAG GOC TCC MAAG CDC AOC GAG CAT
66 Asp Asp Val Val Sar Ale Asp Asn TV. Th. Lu Asp Lo Up Al . Gly GI. 01. Lw W Try Lys Oly Be. Ms Lys Pro sat Gln His
536 GTG MA CM AM GCC CCA Gd ARC CIG ACA GM CAC ACC N11 OTC TCC GC ALT C10 CTG CIO ACC TOG AM AAC CCG TAT GC OCT GAC
96 Val Lys Pro Aeq Ala Pro Gly As Iwu Thr Val His Thr han Val Box Asp ?hr Lw Lu Lao 21m TIP Par AM Pro Tyr Pro Pro App
626 MT TK CM TIT W CAT CTC WC TAT GG GC WASS TOG WT GIA MC GC CCG WA GAS TTC AM ATC 9T AAC Ol0 ACC TAC CU
126 AM Tyr Lu Tyr Aso His Lu Thr Tyr Ala Val Asn Ile Trp Set Glu Asn Asp Pro Ala Asp Phe Arq Ila Wt AID Val MTyr Lw
716 GW CCC IOC CTC COC ATC MA OCC AGC ACC CIO AM TCT MG ATT ?CC TAC AGO WA COG GTG AM CCC 100 OCT CAG
156 Glu Pro Sat lem Arq Ile Ala Ala ear Thr Lw Lys Sax Gly lie Sac Tyr Arq Ala Arq Val Arq Ala Tep AL GI.
PAT AAC ACC
ff. AM Thr
606 ACC TOG ACT GAG TOG AGC CCC AGC AM AAG TGG CAC AAC TCC TM AGO GAG CCC TIC GAG CG CAC CTC CTG C20 GOC ON AOC OT! IOC
166 Tht Sap Set Glu Ttp Set Pro Set Thr Lys Trp His ADD Set Tyr axq Glu Pro Phe Glu Oln His 4u WLGly Val Set VAl 36t
696 IGC ATT GTC ATC CTG QCC GTC TGC CTG TTG TGC TAT GTC AGC ATC ACC N1G ATT AM AIW CIA TGG TOG GT ChG ATT CCC AAC CrL OCC
216 Cys Ile Val Ile Lou Ala Val Cys 1w Lu Cys Tyr Val See Ile Thr Lys Ile Lys Lys Glu Trp Up Amp Gin IL Pro AYE Pro AIA
966 COC AOC CGC CTC GIG GCT An ATA ATC CJYG GAT GCT CNI GGG TCA CAG TOG GAG "G COG TCC CG GGC CAG WCC21 GCC AAG TOC CCA
266 Arq Set Atq Lou Val ALA 110 116 114 Gln ASP 1114 Gin Gly Set Gin Ttp Gln Lys Mq Sag Mg Gly Gin Glu Pro AL Lys Cys Pro
1016 CAC TOG AIG AAT TGT CST ACC AIG CTC TPG CCC TGT ITT CIO GAG CAC 1WC ATG AM AGG GT CPA GT CCT CAC AAG OCT OCC she GAIT
276 His Ttp Lys Asn Cys Lu Thr Lys Lu Lou Pro Cys PM Lw Glu His A4n Hot Lys Arq Asp Wu AV Pro HIS Lys Ala AL Lys Glu
1166 ATG CCT TIC CAG COC TCT GG ARA TG GG TGG TGC CG GIG GAG ATC AM AM I1CA GTC CTC TGG CG GO ADC ATC 1GC GIG MG CG
306 Her Pro PM Gin Gly Bar Gly Lys Set Ala Try Cy4 Pro Val GI. IL Ser Lys Thr Val Iwu Try Pro 01. Be. II* Set VU Vat ACV
1256 TLT MG GAG TTG TTT GAG WC COG GIG GM TOT GG GAG GG G1G G1q GTA GAG WA GA AM GOG AGC TTC TOP GG T'CC CCT GG AOC
336 Cys Val Glu Lou Phe Glu Ala Pro Vat Glu CPS Glu Glu Glu Glu Glu Val Glu Clu Glu Lys Gly Set Ma Cys AL Set Pro Clu Ser
1366 ACC AGG GT WE TIC CAC. GG GW AOG GAG GGC Art GIG GCC COG CU ACA GAG AM M TIC CIO GIC CTG CIC CA GAG WD AV GOG
366 8er Arq !up Am Plr Gn Glu Gly Arq Glu Gly LL Val Ala Atq Lu Thr Glu low Lw Phs Lu Asp ban Lu GAP Glu Olu Am Gly
1636 GGC TTr 'MC CM CAG GC ATG GGG GW TG TGC CTT CST CG OCT TOO GM AM ACG AUT GCT CAC AIG CCC TGG GT GAS TIC CG1 AOr
396 Gly Me Cys Gin Gin Asp Her Gly Glu Set Cys Lu Lu Pro Pro Set Gly Bar Thr Set Ala His )Or Pro Try Aup Glu Phe Pro Bar
1526 GfA GGG CCC AIG GG GG OCT CCC '1GG WC AM GAG CAG CCT CTC CJJC CTO GM CG AGr OCT OCT OCC AGC CCG 1100 CIA Box
Am CCw G&C
426 Ala Giy Pro Lys Glu Ala Pro Pro Trp Gly Lys Glu Gin Pro Lau His Lu Glu Pro Set Pro Pro AL Sax Pro The Gin
Pro AV
1616 AM CIO ACT TGC ACA GAG ACG CCC CTC GTC ASC GG GGC AAC CCT GCT the CGC AGC TIC AGC ARC T'CC CTC AGC CAO TCA CCG Tor CCC
656 A=n W Tht Cys Thr Glu Ths Pro tan, Val Ila Ala Gly hen Pro Ala tyr Arq Sac Ph* Sar Am Set Lu Set Gn Sec Pro Lys Pro
1706 AG GAL CTG GOT CG GAC CCA CIO CTC OCC AG CAC CG GAS
Gin GM OTA WCCC GAG MG OCC SOT GTC CCC CAW CTC TCT GG CG1 ACC
486 Arq Olu Lao Gly Pro Asp Pro LAU Lu Ala ASq His Lu
Glu Val Glu Pro Glu Hat Pro Cys Val Pta ®1n Lu Bet Glu Pro "s
1796 ACt GG CCC CAA CLI' GAG OCA GIA ACC TGG GG CAG ATC C'rC OW OW AM GIC CIC CAG CRT 000 GM OCT OCA OCC CDC O'IC TOG GOC
516 TIa Val Pro Gin Pro Glu Pro Glu lht Trp Glu Gln Ile Lu Arq Axq Am Val Lw Gin His Gly Ala AL Ala hla Pro Vat Ser Ala
1666 COC ACC AGr GGC TAT C1G GAG TIT Gth CAT GOO GIG GAG CAG OOl' OW ACC CAG OCC AM GN. No GIG GOC TTG GN OOC CG OG G10
54, Pro The Set Gly Tyx Gin Wu Phe Val His Ala val Glu GL Gly Gly TAr GL Ala Set Ala Val Val Gly I.w Gly Pro Pre, UP Glu
1976 OCT GOT TAC AJ1G GOC TIC TG 11GC CTG CTT GCC ACC Mr GC'T OTG TCC CCA GAG AM TOT 000 TTt G00 OCT AOC AM 000 GA GIG 000
576 AIa Gly Tyr Lys Ala PM Set Set W Lu Ala Set Her Ala Val Set Pro Glu Lys Cys Gly PM Gly AL $or Set Gly Oln Um Oly
2066 TM WA CCI TIC CM GC CTC ATT OCT GOC TGC CCT GGG G1C OCT GCC CG VIC CCT OPC CCC TTG TTC ACC ITT GG CTO OW AM GG
506 Tyr Lys Pro Phs Gin Asp Lu Ile Pro Gly Cys Pro Gly Aop Pro l11a Pro Val. Pro Val Pro Lw Ph. Thr PM Gly Lau Atp Mg Olu
2156 CG CCT CGC AGt COG CAC AGC '1'CA CAT CM CG MC AGC TCC CG GM CAC CM GOT CTG GM COG OGG GM AM GM GAG G1C ASG CG
636 Pro Pro Arq Set Pro Gin Set Set His law Pro Sat Set Set Pro Glu His Iwu Gly Lu Glu Pro Gly Glu Lys Vat CID lup Her Pro
2246 AM CCC CG CT CCC CAG GAG CAG GCC AG G1C CDC CTT 0% GC AGC CM OW AM GCC ATT GTC SAC TG GCC CTT ACC TOC CAC CTG
666 Lys Pro Pro 1wu Pro Gin Glu Gin Ala Thr Asp Pro 7wu Val Asp $or law Gly Ser Gly Ile Val Tyr Set Al. I= Th. Cys His Law,
2336 T'GC GGC CAC CTG AM CAG TGT CAT GGC CAG GAS G1T GOT GGC CM ACC CCT GTC ATC WC ACT CCT Toe TOT GOC TW TOC TOr GO GAC
696 Cys Gly His Lw Lys Gln Cys His Gly GL Glu Asp Gly Gly Gln Thr Pro Val Met Al. Set Pro Cys Cys Gy Cys Cys Cys Gly Asp
2426 AGG TCC TCG CCC CCr ACA WC CCC CTG AGG GCC WA GAC CCC WT CP21 GOT GGG Gyt CCA MGG Ga ACT CTG TOP CCG GCC T'CC CIO
726 Arq Ser Her Pro Pro Thr Thr Pro Lu Arq Ala Pro Asp Pro Set Pro Gly Gly Val Pro Lau Glu Ala Sex Lu Cya Pro AL Sor Lao
2516 GG COC TCG GGC ATC TCA GAG AG AGT 1VW ?CC 1TA TG TCC TIC GT CCT GCC CCT GGC AAT OCT CAG AM ICA ADC CAG ACC CCC AM
756 Ala Pro Sax Gly IL Set Glu Lys Set Lys Set Set Set Set Me His Pro Ala Pro Gly Aan Ala Gin Sat ear Ser Gin T11[ Pro Lys
2606 ATC GIG AX TIT GTC TCC 0% GG CCC ACA TAC ATG AGO GTC TCT x
186 110 Val Asn PM Val Set Val Gly Pro Thr Tyr Hot Arq Val Set
2710 GGGAAATCAOG(.'1'eGCAGl1
2629
2948
3061
3305
311,
3424 AAGOCACCROTTACCCTWN
3543
ATA
A~IIOTTTC'TiGRT'C
TGAOacTAGGOLTIaTarTxcT
ATOAAOf1G
86 5
IDZERDA ET AL .
TABLE I
Alignment Scores of the Predicted Extracellular Domains
mIL-4-R
hIL-6-R
hIL-2-RO
mEPO-R
rPRL-R
hIL-4-R
mIL-4-R
hIL-6-R
hIL-2-R13
mEPO-R
30 .4
6.5
6.1
6.7
3.9
6 .3
4.7
4.8
4.5
4.8
8 .1
8 .3
7.7
4.6
8 .8
Each receptor was compared using the ALIGN program, which computes the
optimal alignment of two sequences and generates an alignment score (33, 34).
A score > 3 .0 SD is statistically significant . Amino acids 1-207 of the human
IL-4-R (ML-4-R; Fig. 1 B), 1-208 of murine IL-4-R (mIL-4-R ; reference 19),
106-329 of human IL-6-R (hIL-6-R ; reference 29), 1-214 of human IL-2-RQ
subunit (ML-2-RS ; reference 30), 37-249 of murine erythropoietin receptor
(mEPO-R; reference 31), and 20-229 of rat prolactin receptor (rPRL-R; reference 32) were compared .
nine and [31 S]cysteine. Radiolabeled receptors were purified from cell lysates by
affinity chromatography on human IL-4 coupled to Affigel, and subjected to SDS
polyacrylamide gel electrophoresis. As shown in Fig. 3 A, a major IL-4 binding protein of 140 kD was produced in cells transfected with the T22-8 expression construct
(lane 3) but not in cells transfected with vector DNA (lane 1). Addition of excess
human IL-4 to the affinity purification (lanes 2 and 4) blocked the reactivity of the
IL-4 Afflgel with the expressed IL-4-R protein (lane 4). This size correlates well
with the observed Mr (139,000) of the native human IL-4 receptor on both gingival
fibroblasts and the Raji B cell line (13). Similarly, the recently isolated full length
murine IL-4 receptor cDNA encodes a 139 kD protein in transfected COS-7 cells (19).
Human IL-4-R cDNA Confers Human IL-4 Responsiveness to Murine CTLL-2 Cells. We
next sought to determine whether the cloned human IL-4-R could mediate a biological response to IL-4 . The cloned murine T cell line, CTLL-2 (35), typically exhibits 1,000-4,000 IL-4-Rs per cell (16) and shows short-term proliferation in response to murine IL-4 (10) . Because murine and human IL-4 are species-specific
with respect to receptor binding (13-16), CTLL-2 cells do not bind or respond to
human IL-4 . Thus, this cell line provides an ideal host for examining biological activity of the cloned human IL-4-R cDNA. A stable cell population (CTLLT22-8)
expressing the human IL-4-R was derived by cotransfection of CTLL-2 cells with
the pDC302/T22-8 expression construct and the plasmid pSV2neo (which encodes
resistance to neomycin; reference 26), followed by selection for growth in the presence of the antibiotic G418. A control line (CTLL-neo) was established by cotransfection of CTLL-2 cells with pDC302 vector and pSV2neo plasmid DNAs, followed by selection in G418.
FIGURE 1. Human IL-4-R cDNAs. (A) cDNA clones T22-8 and PBL-1 are diagrammed along
with a restriction map. Bar denotes coding region. Location of 68-bp insertion in clone PBL-1
is shown (0). (B) Nucleotide and predicted amino acid sequence of clone T22-8. The predicted
signal peptide and transmembrane region are marked by light and heavy underlining, respectively. Potential N-linked glycosylation sites and extracellular cysteines have asterisks and boxes,
respectively. The dotted line marks the putative polyadenylation signal .
866 MOLECULAR CLONING OF THE HUMAN INTERLEUKIN 4 RECEPTOR
huIL-4R
muIL-4R
huIL-2V
hUIL-6R
MURPOR
raPRLR
MKVL-----QEP---TCVSDY-MSISTCENKMNGPTNCSTELRLLYQLVFL-LSEAH
IKVL-----GEP---TCFSDY-IRTSTCENFLDSAVDCSSQLCLHYRLMFFEFSENL
AVN-----GTSQF-TCFYNS-RANISCVWSQDGALQ-DTSCQVHAWPDR--RRWNQ
VHLLVDVPPEEPQL-SCFRKSPLSNVVCENGPRSTPSLTTKAVLLVRKFQ--NSPAE
SKAALLASRGSEELL-CFTQR-LEDLVCFWEEAASSGMDFNYSFSYQLEGE--SRKQSPPGKPEIHKCRSPD-KETFTCWWVPGTDGGLPTNYSLTYSKEGE-KTTYE
----TCIPENNGGAGCV----CHLLMDDVVSADNYTLD---L----WAGQ--QLLWKGSFKPSEHVKP
----TCIPRNSASTVCV----CHMEMNRPVQSDRYQME---L----WAEH--RQLWQGSFSPSGNVKP
----TCELLPVSQASWA----CNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRL
DFQEPC-QYSQESQKFS----CQLAVPEGDSS-FYIVS---MCVASSVGS--KFSKTQTFQGCGILQP
----SCSLHQAPTVRGSVRFWCSL--PTADTSSFVPLE---LQVTEASGS---PRYHRIIHINEVVLL
-----C-PDYKTSGPNS----CFFSKQYTSIWKIYIIT---VNATNQMGS--SSSDPLYVDVTYIVEP
RAPGNL--TVHTNVSD-TLL-LTWSNPYPPD-NYLYNHLTYAVNIWSENDPADFRIYNVTYLEPSLRI
LAPDNL--TLHTNVSD-EWL-LTNNNLYPSN-NLLYKDLISMVNISREDNPAEFIVYNVTYKEPRLSF
MAPISL----QVVHVETHRCNISN-EISQAS-HYFERHLEFEARTLSPGHTWEEAPL-LTLKQKQEWI
DPPANIT--VTAVARNPRWLSVTWQDPHSWN--SSFYRLRFELRYRA-ERSKTFTT--WMVKDLQHHC
DAPAGL---LARRAEEGSHVVLRNLPPPGAPMTT---HIRYEVDVSAGNRAGGTQR--VEVLEGRTEC
EPPRNLTLEVKQLKDKKTYLWVKNSPPTITDVKTGWFTMEYEIRLKP-EEAEEWEI---HFTGHQTQF
AASTLKSGISYRARVRAW-A-QCYNTTWSENS-P---STKWHNSYREPFEQH
PINILMSGVYYTARVRVR-S-QILTGTWSEWS-P---SITWYNHFQLPLIQR
CLETLTPDTQYEFQVRVK-PLQGEFTTWSPNSQP------- LAFRTKPAALGKDT
VIHDAWSGLRHVVQLRAQ--EEFGQGENSEWS-PEAMGTPWTESRSPPAENEVST
VLSNLRGGTRYTFAVRARMAEPSFSGFWSAMSEP---ASLLTASDLDP
KVFDLYPGQKYLVQTRCK-PD---HGYNSRWSQES-------- SVEMPNDFTLKD
Sequence alignment of extracellular domains. Amino acidsconserved among all five
receptors are shown in bold print. Alignment of the sequences specified in Table I was performed
by computer using the algorithm of Feng and Doolittle (45), and then modified to more closely
reflect the pairwise alignments produced by the ALIGN program. The alignment shown is not
necessarily optimal for each individual pair of receptors.
FIGURE 2 .
Expression of murine and human IL-4-Rs in these lines was first examined by
radiolabeled-IL-4 binding and Scatchard analysis. Representative binding studies
(Fig. 3 B) indicate that both CTLL-neo cells (top paneo as well as CTLLT22-8 cells
(middle paneo have -1,400 murine IL-4-Rs on the cell surface with an affinity ofti4 x
109/M. As expected, no apparent human 125 1-IL-4 binding was detected on the
CTLL-neo cells (not shown) . However, >800 human IL-4-Rs could be detected on
the transfected CTLLT22-8 cells (Fig. 3 B, bottom paneo with an affinity for human
IL-4 of 1.1 x 10 1°/M. These binding affinities are consistent with the values previously reported for the native murine (12, 14-16) and human (13, 17) IL-4-Rs.
Expression of murine and human IL-4-R mRNA was examined in the transfected
lines by RNA gel blot analysis. A blot containing RNA from CTLLT22-8 and CTLLneo cells, and for comparison human peripheral blood T lymphocytes, was hybridized under stringent conditions with a human IL-4-R probe (Fig. 4 A) . Shown in
Fig. 4 B is a blot containing CTLLT22-8 and CTLL-neo RNAs that was hybridized with a probe specific for murine IL-4-R mRNA. The murine probe detected
only the endogenous 3.9-kb murine IL-4-R mRNA, which was expressed at similar
levels in the two CTLL sublines . The human IL-4-R probe hybridized to a 4.0-kb
endogenous IL-4-R mRNA in the human T cells, but did not detect the murine
form in the CTLL-neo control line . In the CTLLT22-8 transfectants the human
IDZERDA ET AL.
86 7
3. (A) Detection of the human IL-4-R expression product in transfected COS-7 cells.
Radiolabeled lysates from COS-7 cells transfected with pDC302 vector DNA (vector lanes) or
the pDC302 T22-8 expression construct (T22-8 lanes) were incubated with human IL-4 Affigel
in the presence (+) or absence (-) of an excess of human IL-4, and the bound proteins were
subjected to electrophoresis and autoradiography. The mobilities of molecular weight standards
are indicated. (B) IL-4 bindingcharacteristicsoftransfectedCTLL-2 cells. Forequilibrium binding
studiesCTLL-neo (top) or CTLLT22-8 (middleand bottom) cells were incubatedwith various concentrations of murine 125 1-IL-4 (top and middle) or human 125 ML-4 (bottom) for 1 h as previously described (13). Nonspecific binding of 125 1-11-4 was measured in the presence of 200-fold
molar excess of unlabeled IL-4. Binding data were analyzed with RS/l software (Bolt, Beranek
and Newman, Boston, MA).
FIGURE
probe hybridized to a 4.3-kb mRNA, consistent with the predicted size of the exogenous human IL-4-R mRNA, which should contain an additional 0.25 kb of 5' flanking
sequences transcribed from the expression vector. Surprisingly, a 3 .0-kb RNA species is also evident in the CTLLT22-8 lane . This could result from any of several
events such as aberrant splicing, gene rearrangement during integration into the
86 8 MOLECULAR CLONING OF THE HUMAN INTERLEUKIN 4 RECEPTOR
4. Expression of human IL4-R mRNA in transfected CTLL-2
cells. RNA gel blots containing 8 Wg
per lane total RNA from human peripheral bloodT cellsthat hadbeen cultured for 44 h in 1 Wg/ml of PHA(PBT,
A), CTLL-T22-8 umsformants (CTLLT22-8, A and B), and CTLL-neo transformants (CTLL-neo, A and B) were
probed for (A) human and (B) murine
IL-4-R mRNA . The positions of 28S
and 18S ribosomal RNAs are marked.
FIGURE
genome, or utilization of a cryptic polyadenylation signal within the cDNA. The
sequence AGTAAA, which occurs at position 2540 (Fig . 1 B) might function as such
a signal, although there is no evidence for its use in normal human cells.
The ability of the expressed human IL-4-Rs in CTLLT22-8 cells to convey a biological signal across the membrane was assessed by quantifying the proliferative response of these cells to human IL-4. The results, comparing [3H]thymidine incorporation in response to various concentrations of human and murine IL-4 in the
two CTLL sublines, are shown in Fig. 5 A. While the CTLL-neo control line proliferated only in the presence of murine IL-4, the CTLLT22-8 cells responded to both
murine and human IL-4. Moreover, addition of anti-murine IL-4-R mAb (Ml),
which blocks murine IL-4 binding (Beckmann, M. P, et al ., manuscript submitted
for publication), abrogated the response to murine IL-4 in the CTLLT22-8 cell
line, while having no effect on the biological response to human IL-4 (Fig. 5 B).
Similarly, the anti-murine IL-4-R antibody blocked the proliferative activity ofmurine
IL-4 on the CTLL-neo cells (data not shown) . These data demonstrate that the proliferative response of CTLLT22-8 cells to human IL-4 was mediated through the human
IL-4-R encoded by cDNA clone T22-8.
Discussion
We have isolated cDNA clones that hybridize with a murine IL-4 receptor probe.
The protein sequence encoded by these clones matches closely with that determined
for the murine IL-4-R (19), and its identity as the human IL-4-R is confirmed by
functional expression of the cDNA in COS-7 and CTLL-2 cells. In murine cells
IDZERDA ET AL.
A
869
s
X
5
4
3
d
1
0
rri
O_
Ea
0
a
o
0
2
5 CTLL-T22-8
4-
M
^ 15~ CTLL T22-8
+ Antibody
oM
a0 10
0
U X
c E
2
v u5
1
In
(r 3
H
o
p
4
0
1
2
3
IL-4 concentration (ng/ml)
I
C
0
50
100
Antibody concentration
(ng/ml)
FIGURE 5. (A) Proliferative responsiveness of transfected CTLL-neo and CTLLT22-8 cells (A
and B, respectively) to recombinant murine IL-4 (O) or recombinant human IL-4 (0). (B) Mito-
genic responsiveness of CTLLT22-8 in the presence of anti-human IL-4-R antibody. CTLLT22-8 cells were stimulated with 0.5 ng/ml of murine (O) or human (" ) IL-4 in the presence
of various concentrations of anti-murine IL-4-R antibody (1-140 ng/ml; reference 27). Anti-murine IL-4-R antibody alone (0) was used as a control.
we found an alternatively spliced mRNA that encoded a secreted form of the IL-4-R
(19), but an analogous cDNA has not yet been isolated in the human system . However, if a secreted form of the human IL-4 receptor is expressed at the same low
frequency as the murine secreted form (N1 in 15 cDNAs), then a large number of
human IL-4-R cDNAs would have to be examined to rule out its occurrence . Alternatively, expression of secreted IL-4-Rs may be restricted to the murine species.
The sequence comparisons shown in Table I and Fig. 2 define the IL-4-R as a
member of a novel superfamily of cytokine receptors. The ALIGN scores are in a
range similar to that found among the members of the Ig gene superfamily. Because
all of these receptors can be found on, and mediate effects on, cells of the hematopoietic lineage, we suggest the name, hematopoietin receptor superfamily. As additional
members of this family are discovered and as receptor structures are examined, the
significance of the conserved residues highlighted in Fig. 2 may become more evident.
It is interesting to note that these apparently structurally related receptors bind
molecules that also share similar structures . The x-ray crystallographic data of IL-2
shows that it has a four-helix bundle core structure (36) . IL-6 has also been modeled
as a helix bundle protein (37) . Although the structure of prolactin is not known,
x-ray crystallographic data on the closely related protein, growth hormone, reveal
that it has a four-helix bundle structure (38) . Therefore, conservation of the binding
region in this family of receptors may be due to the structural requirements necessary to bind a specific class of hormones, and would predict that erythropoietin and
IL-4 may also have four-helix bundle structures .
Interestingly, the first 86 amino acids of the mature IL-6-R extracellular domain
are unrelated in sequence to the hematopoietin receptor superfamily as determined
with the UWGCG GAP program (28), which uses the same algorithm as ALIGN,
870 MOLECULAR CLONING OF THE HUMAN INTERLEUKIN 4 RECEPTOR
but has a gap length penalty and no end gap penalty. However, the NH2-terminal
81 amino acids of the mature IL-6-R align with the Ig superfamily C2 domain (29,
39), indicating that the extracellular portion of the IL-6-R has an NH2-terminal
Ig-like domain, followed by a domain characteristic of the hematopoietin receptor
superfamily. Whether IL-6 binding and biological responsiveness require both domains is not yet known.
The demonstration of signal transduction by the human IL-4-R in murine CTLL-2
cells in response to human IL-4 indicates that homology between the murine and
human IL-4 receptor must be sufficient to allow interaction of the human IL-4-R
with the murine proteins involved in transmitting the IL-4 signal to the nucleus .
Presumably, the cytoplasmic domain functions in this capacity. The amino acid sequence identity between the predicted cytoplasmic domains of the human and murine IL-4-Rs is 54%, with the greatest conservation focused in two regions : amino
acids 232-259 and 685-727 (Fig. 1 B) . Additional evidence suggesting the importance of the cytoplamic domain comes from previous experiments with CTLL-2
cells . CTLL-2 cell line derivatives that express a high number of endogenous murine IL-4-Rs with a truncated cytoplasmic domain no longer respond to murine
IL-4 (19). This is in contrast to the IL-6 receptor, whose 86 amino acid cytoplasmic
domain is dispensable for signal transduction (40) .
Examination of the predicted cytoplasmic domain sequence of the human IL-4-R
revealed no clues about the signaling mechanism. No homologies with protein kinases or sequences found at phosphorylation acceptor sites for protein tyrosine kinases or protein kinase C (41, 42) were observed .
No significant homologies between the predicted cytoplasmic domains of the IL4-Rs and the other members of the hematopoietin receptor superfamily, which vary
greatly in length, were observed using the ALIGN program. D'Andrea et al . (43)
recently reported homology between the entire length of the ß subunit of the IL-2-R
and the erythropoietin receptor, which are roughly equivalent in size . In view of
the similarities between IL-4-R and these other receptors, it will be interesting to
determine whether components of the signaling machinery are shared among them .
In addition, analysis of the genomic structure of these receptor genes might shed
further light not only on their evolutionary relatedness, but on domain structural
and functional similarities as well .
During the preparation of this paper, we became aware that Bazan (44) had also
noticed the sequence similarities between the receptors described here (with the exception of IL-4-R) .
Summary
IL-4, a pleiotropic cytokine produced by T lymphocytes, plays an important role
in immune responsiveness by regulating proliferation and differentiation of a variety of lymphoid and myeloid cells via binding to high affinity receptors. In this
report we describe the isolation and functional expression of a human IL-4-R cDNA.
When transfected into COS-7 cells, the cDNA encodes a 140-kD cell-surface protein . After transfection into a murine T cell line, the cDNA encodes a protein that
binds human IL-4 with high affinity and can confer responsiveness to human IL-4 .
The predicted extracellular domain of the IL-4-R exhibits significant amino acid
IDZERDA ET AL .
87 1
sequence homology with the S subunit of the IL-2-R (p75), and the receptors for
IL-6, erythropoietin, and prolactin . These receptors comprise a novel superfamily
with extracellular domains characterized by four conserved cysteine residues and
a double tryptophan-serine (WSXWS) motif located proximal to the transmembrane
region.
We thank J. Sims and C. McMahan for the T22 cDNA library ; D. Anderson for the PBL
cDNA library, R. Armitage for preparing human T cells ; J. Wignall and C. McNutt for
transfecting COS cells ; K. Schooley for monoclonal antibody; T Farrah for assistance with
software; S. Lupton for assistance with electroporation ; W. Fanslow for helpful discussions ;
and S. Gillis, S. Dower, and D. Urdal for critical reading of the manuscript .
Received for publication 17 November 1989.
1.
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11 .
12.
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