.=) 1990 Oxford University Press

Nucleic Acids Research, Vol. 18, No. 13 3697

The human interleukin-2 receptor f-chain gene: genomic organization, promoter analysis and chromosomal assignment Hiroshi Shibuya*, Mitsutoshi Yoneyama, Yusuke Nakamural"+, Hisashi Harada, Masanori Hatakeyama, Seijiro Minamoto, Takeshi Kono, Takeshi Doi, Raymond White1 and Tadatsugu Taniguchi Institute for Molecular and Cellular Biology, Osaka University, 1 -3 Yamadaoka, Suita-shi, Osaka 565, Japan and 'Howard Hughes Medical Institute, Research Laboratories, University of Utah, 701 Wintrobe Building, Room 631, Salt Lake City, UT 84132, USA Received May 18, 1990; Accepted June 6, 1990

ABSTRACT The chromosomal gene for the human interleukin-2 receptor (3-chain (IL-2RjB) was isolated and characterized. The entire IL-2Rfl gene is composed of ten exons spanning about 24.3 kilobases, in which the protein is encoded by the exons 2-10. The cysteine rich extracellular region which displays- a significant evolutionary resemblance to other cytokine receptors, as well as growth hormone and prolactin receptors, is encoded primarily by exons 3 and 4, whereas the membrane proximal, cysteine poor domain showing a homology with type Ill modules of fibronectin is encoded by exon 7. Sequence analysis of the 5'-flanking region revealed the presence of potential binding sites for transcription factors such as Octamer binding factors, AP-1, AP-2 as well as the 'GC-clusters'. At least five potential cap sites were identified by SI mapping analysis. The 850 bp DNA sequence of the 5'-flanking region exhibited constitutive promoter activity when it was linked upstream of the HSV-tk reporter gene and then transfected into YT cells, a human leukemic cell line. By applying the RFLP linkage analysis, the IL-2R13 gene has been assigned to chromosome 22q12-13. INTRODUCTION The clonal proliferation of antigen-specific T lymphocytes (Tcells) is stimulated by interleukin-2 (IL-2) through its interaction with the homologous high-affinity receptor (IL-2R). Resting Tcells do not generate IL-2 and lack the expression of high-affinity IL-2R, unless they are activated by antigens or mitogens (1-4). At present, two distinct IL-2-binding membrane receptor components have been identified and characterized; IL-2Rc (p55), and IL-2Rj3 (p70-75) chains. The heterodimer, IL-2Rc *

EMBL accession

no.

X53093

and IL-2R3, constitute the functional high-affinity receptor capable of transducing the proliferative signal (3, 4). Studies on the structure and function of IL-2Rat have revealed that this receptor component solely constitutes the 'low-affinity' IL-2R (Kd: = lOnM) and it does not possess a sufficiently long cytoplasmic structure(s) required for IL-2 signaling (5-8). Subsequently, cDNAs encoding human IL-2R,3 were isolated and characterized (9). Unlike IL-2Rc, IL-2R(3 has a large cytoplasmic region consisting of 286 amino acids, but lacks an identifiable tyrosine kinase domain (9). The IL-2R,B cDNA gave rise to the expression of 'intermediate-affinity' IL-2R (Kd: 1 -2nM) in IL-2R-negative lymphocytic cell lines. Furthermore, functional, high-affinity IL-2R was reconstituted by co-expressing IL-2Ra and IL-2R13 cDNAs in the human leukemic T cell line, Jurkat (9). Subsequent studies have confirmed the essential role of IL-2R,3 in IL-2-mediated mitogenic signal transduction and have identified the critical cytoplasmic region for IL-2 signaling (10, 11). The expression of the genes for IL-2 and IL-2Ra is tightly regulated. In fact, the expression of both genes is controlled at the transcriptional level and it is induced upon T cell activation (1-4, 12). We, as well as others, have identified the cis-acting regulatory DNA sequences of the human IL-2 and IL-2Ra genes (13-16). Moreover, nuclear factors which bind to IL-2 and IL-2Ra gene sequences have been identified (17, 18). Those include the NF-x-like factor which regulates both genes (19, 20). On the other hand, little is known at present about the regulation of the gene for IL-2R(. Recent studies shows that IL-2R,B is constitutively expressed in a certain population of resting T-cells (9, 21), suggesting that the regulation of IL-2Ri3 gene expression may be distinct from that for the IL-2 and IL-2Ra genes. In order to study the structural organization as well as the mechanism of expression of human IL-2R,3 gene, we have cloned the chromosomal gene. We relate the genomic structure and the

To whom correspondence should be addressed

+ Present address: Department of Biochemistry, Cancer Institute Japanese Foundation for Cancer Research, Toshima-ku,

Tokyo 170, Japan

3698 Nucleic Acids Research, Vol. 18, No. 13

I~

chromosomal location of IL-2R,3 gene, 22q12 -13. Our results suggest that certain module structures within the IL-2Rf3 are encoded by distinct exons. Moreover we have characterized the 5' flanking region which functions as a promoter for IL-2R,B gene expression.

(Takara). The reaction was carried out for 25 cycles, with the use of DNA Thermal Cycler (Perkin Elmer Cetus). The cycle times were as follows: denaturation, 94°C 1 min; annealing, 55°C 2 min; polymerization, 72°C 3 min. The length of the amplified DNA fragments was determined by electrophoresis in 0.6% agarose gel.

MATERIALS AND METHODS Isolation of human IL-2R,B gene The Charon 4A library containing human genomic DNA which was digested partially with AluI/HaellI and ligated with EcoRIcleaved Charon 4A arms was kindly provided by Dr. Lawn (22). The Charon 4A library was screened by the in situ procedures using the two Sacd fragments (a; 10-1009, b; 1009-2387) and Sau3AI fragment (d; 3872-4034) of human IL-2R,B cDNA (9) as probes. These probes were labeled by the multiprime DNA labeling systems (Amersham) (specific activities, 6 x 108 cpm/Ag). Essentially, probe c (22-61) was chemically synthesized and labeled by T4 kinase at the 5' termini of both strands.

RFLP, genotyping and linkage analysis For screening RFLPs, DNAs from six unrelated individuals were digested with MspI, TaqI, RsaI, BglII, PvuH or PstI. After blotting, DNAs were hybridized with 32P-labeled IL-2Rf genomic DNAs. The marker, called IL-2R,B, was genotyped by using the panel of 60 three-generation families (822 individuals) at the Howard Hughes Medical Institute, University of Utah (Salt Lake City, UT). Linkage analysis was performed with the LINKAGE program package (23). The genomic map was constructed from the reference families by using the GMS (Gene Mapping System) alogorithm, as described by Lathrop et al. (24).

Construction of plasmids The pSVOOtk was constructed by replacing the region of CAT gene in the pSVOOcat (25) (HindIll-BamHI fragment) with the HSV-tk gene in the pRSVtk (19). To obtain the pSO,B854tk, the 5'-flanking region of IL-2RF3 gene was excised out of the fragment 8b (Figure 1) cloned into pUC 18 by EcoRI and ApaI digestion. After converting the 5' and 3' ends into the blunt end and Hindl site respectively, the fragment was inserted into the SmaI/HindI digested pSVOOtk.

DNA sequence analysis The nucleotide sequence was determined by the dideoxy method (SEQUENASE, United States Biochemical Corporation) using the synthetic DNA primers which were complimentary to human IL-2R,( cDNA, or by the Maxam-Gilbert procedure.

Polymerase chain reaction The PCR mixture contained 1 ng of template DNA, 100 pM of two oligonucleotide primers, 200,M of each dNTP, lO x PCR buffer (500 mM KCl, lOOmM Tris-HCl pH 8.3, 15 mM MgCl2, 0.1% gelatin), and 2.5 units of Taq DNA polymerase

DNA transfection and RNA extraction DNA transfection was performed by the DEAE dextran method for YT cells as described previously (13). Cells were harvested

b

probe

d

Ic i

500bp4 -

3,

2kb

I_MH , i

P .

E HE HPlH t

P H t

E- EcoR! H- HindM M- Smal

P- Sphl

IE

PH

eMM

E

M RPP

6a F

M P

E

leHP.M

I

M.p P

fi

M,

'1

Figure 1. The organization of human IL-2R,3 gene. Upper rectangle indicates cDNA, and the positions interrupted by introns are shown as vertical lines. The bold horizontal line indicates the genomic DNA. The positions of the exons are shown by the filled box. The four horizontal lines at bottom indicate the isolated genomic inserts, and the restriction sites within these DNA fragments are denoted by vertical lines: E, EcoRI; H, HindIII; M, SmaI; P, SphI. probe a and b, Sacl fragments (10-1009, 1009-2387 respectively), probe c, 40 mer oligonucleotides (22-61), and probe d, Sau3AI fragment (3872-4034) were used for screening.

Nucleic Acids Research, Vol. 18, No. 13 3699 30 hr after transfection, and subjected to RNA extraction. Total cellular RNA was prepared by the guanidine thiocyanate method. Polyadenylated RNA was isolated by binding to oligo(dT)cellulose (Pharmacia).

to the total cellular RNA (40 jig), and digested with 50 units of SI nuclease (Pharmacia). Protected DNA fragments were analyzed by electrophoresis in 6% polyacrylamide-urea gel. For the detection of transcripts from transfected plasmids, 10 Ag of poly(A)+RNA was analyzed similarly.

Si nuclease mapping analysis Si analysis was performed essentially as described previously (19). For detection of endogenous transcripts from IL-2Rj3 gene, the 1.1 kb genomic fragment containing the exon 1 of IL-2R,B gene was subcloned into the EcoRI site of M 13mp19. For an analysis of mRNA transcribed from the transfected plasmids, the DNA fragment excised from pSO,854tk (from the Stul site at -71 to NaeI site within the tk structural gene) was subcloned into Hincd site of M13mpl9. In addition, two oligonucleotides which were complementary to these templates were synthesized (5'-CAGGGGCCGGGAGGGAG-3', 5'-GGTACGAAGCCATACGCGCT-3'). In order to synthesize the radioactive probes, the oligonucleotide primers were labelled with [Fy-32P]ATP, and annealed with each single-strand template DNA, and polymerized by Klenow fragment. After digestion with BglIl and EcoRI, respectively, the labeled single strand probes were prepared. To detect the endogenous transcripts, the probe DNA was hybridized

RESULTS Isolation of the Recombinant Phage Containing the Human IL-2R,B Gene Sequence We screened a Charon 4A phage library from AluI-HaeII partial digests of human DNA using human IL-2R(3 cDNA fragments as probe. Approximately 1.5 x 106 phages in the library were first screened using probes a and b (Figure 1). Positive phages were further selected by Southern blot hybridization using (i) probe c, a 40 nucleotide particular synthetic oligonucleotide corresponding to the 5' end region of the human IL-2R(3 cDNA and (ii) probe d, the 179 bp Sau3AI fragment closest to the 3' end (9). The inserts of several selected clones were separately subcloned into pUC18 plasmid vector and the subsequent analysis was carried out using these recombinant plasmids. Four positive clones (8b, Sa, 6a and 14b) corresponding to insert DNAs of

Table 1. Exon-intron organization of IL-2RP gene Intron Size 5' Splice Donor Size [bp] 3' Splice Acceptor [kb] Size [bp][]

Exon

No.No.

1 2 3 4 5 6 7 8 9 10

*92 121 115 79 106 149 166 115 85 >3000

----GCCAG --G AAT G ---AGA CG ---CA GAT --G AAC C ---GG GAG --T GCA G ---CCA TG ---TC CAG

GGCTT---GC ACT--G CGG T-TCT CA--TT CGC--GAG GC--CC CTT--gtaagaaggt-----ctctgtccag G CTG A-gtaggaggcc-----ctcccaccag AAG TG---

gtgatgtccc-----tctccaccag gtgaggaccc-----tgcccggcag gtgagtccac-----gccatgaaag gtgagtagcc-----ttccttgcag gtgagtgagg-----ttgtctccag gtgagaatgt-----tccgacccag gtactggtgg-----ccctgggcag

5.5 0.5 1.0 3.2 1.4 1.3 1.0 2.9 3.5

Gly-4 Arg-42 Asp-68/Ser-69 Leu-104 Glu-153/Glu-154 Ala-209 Trp-247 Gln-275/Lys-276

capital letters, intron sequences are in lowercase letters. interrupted by each introns are denoted with numbers in IL-2Rfl cDNA. *Determined from position +1, shown in Figure 4.

Exon seauences are in

1

exon

2

3

I

4

5

Extracellular Region

\~~Signal Sequence

I

7

6

8

The amino acids

10

9

I

Amino Acid Interrupted

Cytoplasmic Region

\,-- Transmembrane Region

I

5'-Untranslated Region

Coding Region

3'-Untranslated Region

Figure 2. The relationship between exons and functional domains of IL-2R,3. Each rectangle indicates an exon. Dotted, horizontal, closed, and hatched regions in exons indicate a signal sequence, extracellular region, transmembrane region, and cytoplasmic region, respectively. Numbers immediately below each rectangles denote the nucleotide positions at which the introns interrupt the cDNA (number 1 indicates the major cap site determined in this study). The cysteine residues are indicated by the arrowheads. Especially, large arrowheads indicate the conserved cysteine residues in cytokine receptors (see DISCUSSION).

3700 Nucleic Acids Research, Vol. 18, No. 13

T CG A 1 2 3

7.8-16.7 kb in length were finally isolated. Restriction endonuclease analysis indicated that these clones overlapped with each other, as depicted in Figure 1.

-undigested probe

-."

p

.me _

gm

U. alk

3. Determination of the cap sites for human LL-2Rj3 mRNA by SI nuclease analysis. The total cellular RNA (40 pg) were subjected to SI mapping analysis. The arrowheads indicate the positions of potential cap sites, and their locations are denoted in the nucleotide sequences shown in Figure 4. The sequence ladder (lanes T, C, G, and A) was obtained using the same template DNA and primer used in synthesis of the probe. Lane 1, YT cells; lane 2, MT-4 cells; lane 3, C91/PL cells. Fue

mapping

GAATTCATGAAATGGGAAGGGCAGTGATGGGATGGAGGGG AMG

Nucleotide Sequence Analysis of IL-2R$ Gene DNA sequences were determined for selected regions of these inserts using synthetic DNA primers which encode 1L-2R,B cDNA. Comparison of the human genomic IL-2Rfl sequence with that of human IL-2Rf cDNA revealed that the gene is divided into ten exons and nine introns. The exon-intron organization of IL-2Rfl gene was determined according to a newly applied strategy using polymerase chain reaction (PCR) method. We carried out PCR with these insert DNAs as template and synthetic DNA primers consisting in the sequences of IL-2Rf8 cDNA, used in the sequence analysis. The length of the resulting fragments was determined, and the locations of certain restriction enzyme cleavage sites were compared with those of the cloned DNA. The overall distribution of the exons is identified (Figure 1). The approximate size of the introns was also confirmed by the restriction enzyme cleavage analysis (data not shown). This strategy might be available for the determination of rapid and exact exon-intron organization without complete sequencing. The IL-2Rfl gene spans about 24.3 kb. The nucleotide sequences of the exons were in agreement with that of IL-2Rfl cDNA reported previously. The consensus sequences for splicing junctions, GT and AG, were found at the boundaries of all nine introns (Table 1). As shown schematically in Figure 2, three of the nine introns interrupted the reading frame precisely between codons. The first exon contains the 5' untranslated region. The second exon contains the DNA sequence corresponding to the signal peptide and the NH2-terminal amino acid of the mature protein. Exons 2-8 encode the extracellular region. Exon 8 also encodes the transmembrane region and a part of the cytoplasmic region, and exon 9 and 10 encode the rest of the cytoplasmic region. Exon 10 also includes the entire 3' untranslated region with TGGAI GG -800

TG*G-TTGGGT

GAkAAACAGAATTAGATCGA

ATGAATAAGATCTAGTATTTGATAGCATAACAGGGTGACTTTAGTCAACAATAATTTATTGTACATTTAAAAATAACTAAAAGAGTATACTTGGATTTTA -700 Oct Oct ACACAAAGAAAGGATAAATACTTGAGGTGATGGATACCCCATTTACCCTGATGTGATTATTATACATTGTATGCCTGTATCAAAATAGCTCATGTGCCTC -600 Oct API ATGAATATAGACACCTACCACATGCCCACAAAATTAAAAACTAAAAAAAACAGTCATCTCTGAATGCTAAACGGAGTAAGGGGCTTCCT

GCT -500 ~~ ~~~~~~~3 37 T -AATGGGA-aCTCGGAAAGATGGTGTGTTGCA= T GGGTGAGAICTGgG 1 ACCTAGAGGGACCTGCTTGTGTGAAGCCTACGTAuA -400 1

GTGGGTATGTGTGTGACC>GGGGGTCAGAGFGTTGGTgCTGTGTGAGTT jGTGorGAGTAGGAGGGGAGAGAGGAGGGCCTGCG -300 TTCCCTTGGCTCCTGTGTGCAGCTAGGCCCCTATTTGACAATGTGTGTCTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCCGCCCCCAM -200 Spi

~M3

AGATCTTTATCTGGCCCTGGTaTTGAGGAGTTTCAGGCTTTCTCATAAGCCTCGTCTCCCCGCCTCTCCACCCCAGGCCTTGCCCCTCTATCC -100

AP2

r- rll TCTGCACAGGAAGTGGGCTGGCTCTGGGCTTTTAGTCTTTGCGGCCCCAGCAGCCAGAGCTCAGCAGGGCCCTGGAGAGATGGCCACGGTCCCAGCACCG r-

r

r

+1

GGGAGGACTGGAGAGCGCGCGCTGCCACCGCCCCATGTCTCAGCCAG GTGATGTCCCCCTGCCTCCCTCCCGGCCCCTGTGGACCArrrA& Istexon

I'

atItrTnn

intron

t

GAGTGAAAGTCACAGAGAAGACTTTCAGCTCTGACTCAGTTCCCCCAGCAGTTTCTGCCTGAACTCCCATCCCCCAACTTTGTCTTAGAATTC

Figure 4. Nucleotide

sequence of the 5'-flanking region of IL-2R(3 gene. The nucleotide sequences of 1.1 kb EcoRI fragment containing the 5'-flanking region and the 1st exon were determined. The minor cap sites and the major cap site are indicated by thin and bold arrows, respectively. The nucleotide corresponding to the major cap site is numbered as + 1. The direct repeats are shown by horizontal arrows, and the long GT clusters are underlined. The potential binding sequences for the transcription factors (Oct, AP-1, SpI, and AP-2) are doubly underlined. The purine-rich sequences (see RESULTS) are indicated by a dotted line. The nonamer sequences (see DISCUSSION) are boxed, and the numbers above these boxes (1, 2, and 3) indicate the group numbers classified in Table 3.

Nucleic Acids Research, Vol. 18, No. 13 3701 Table 2. Pairwise lod score of IL-2R3 with chromosome 22 markers

5'

T C G A 1 2 W

undigested probe

Marker

Recombination fraction (0)

Zmax

D22S11 H61 PDGFB EW7.20

0.28 0.11 0.09 0.25

2.3 6.8 1.7 2.5

The recombination fraction with the highest log of odds (lod) score (Zmax) between IL-2RO3 and four chromosome 22 markers is indicated.

_.K

....

-K

/ ft

P 1

12

-

* ,

_ _~~a I 'a 2~~~~~~~~~, am _

FH +1

/

_

0

Im 0. / ~ ~ ~

--

4

/.

q

IL-2RR

3, 10 cM

Figure 5. The promoter activity of 5'-flanking region of IL-2Rf3 gene. The 10 ,tg of poly(A)+RNA were prepared from the transfected YT cells, and subjected to SI mapping. The sequence ladder (T, G, C, and A) were obtained using the same template and primer used in synthesis of probe. Lane 1; pSVOOtk. Lane 2; pSO,B854tk.

polyadenylation signal sequences. It is worth noting that some of the characteristic motifs of the IL-2Rfl such as type HI modules of fibronectin and cysteine rich domain homologous to other cytokine receptors (26) are encoded by dlstinct exons (see DISCUSSION). Determination of the cap Sites for IL-2R,B mRNA In order to identify the 5' end (i.e. cap site) of IL-2R,B mRNA, we performed SI mapping analysis. As shown in Figure 3, at least five cap sites were detected in mRNA from YT cells (a natural killer like human leukemic cell line). The major cap site is designated with an bold arrowhead and is labeled '+ 1' (see Figure 4). Essentially the same results were obtained with the mRNA from IL-2R,8-positive MT-4 and C91/PL cells (HTLV-l transformed cell lines), whereas no SI-protected DNA band was seen when SI reactions were performed with the mRNA from IL-2R,B-negative Jurkat or FL (human amnion derived cell line) cells RNA or yeast tRNA templates (data not shown). The presence of the multiple cap sites was also confirmed by primer extension analysis (data not shown).

Sequence and Characterization of the Promoter Region We determined the sequence of about 850 bp of 5 '-flanking region for the human IL-2Rf gene. As shown in Figure 4, the promoter region of IL-2Rf lacks a conventional TATA or CAAT sequences. Upstream from the multiple cap sites, however, several characteristic sequences can be detected. Direct repeat sequences (21 bp) are found about 800 bp upstream of the major cap site. A purine rich region which shows sequence homology to the 5'-flanking region of IL-2 gene is present about 650 bp upstream. Several potential factor binding sites, such as those

Chromosome 22 Figure 6. Location of IL-2R13 gene on chromosome 22. A genetic map with 7 RFLP markers and the location of IL-2R,B gene on chromosome 22 is shown. The map is scaled in centimorgans (cM), and the distance is calculated as no sex difference. The arrow indicates the most likely position of IL-2R3 gene locus.

for AP-1, AP-2 and SpI are detectable within the sequenced region (Figure 4). In order to study the function of the 5'-flanking DNA sequences for the IL-2R,B gene, we constructed a chimeric gene with the 5'-flanking region, extending from + 12 to -854, of IL-2Rj3 gene fused to Herpes simplex virus thymidine kinase (HSV-tk) gene (pSO03854tk) (Figure 5). The pS0,B854tk was transfected into YT cells, and mRNA transcripts were analyzed by SI mapping analysis. As shown in Figure 5, the mRNA with the multiple cap sites of IL-2Rj3 gene was detectable in the poly (A)+ RNA from transfected YT cells. The detected cap sites were located by one and the same to the mRNA derived from endogenous IL-2R,B. Thus, it appears that the constitutive expression of the IL-2R13 gene in YT cells is mediated at least in part by the 5'-flanking DNA sequences. Chromosomal Assignment of IL-2Rf Gene In order to determine the chromosomal location of IL-2R3 gene,

restriction-fragment-length polymorphism (RFLP) linkage analysis was carried out following the established procedures (23, 24). Hybridization of human DNA from clone 8b to a panel of Southern blots with DNAs from six unrelated individuals revealed a common two-allele RFLP with TaqI (data not shown). The size of the polymorphic bands is 2.0 kb (allele 1) or 1.8 kb (allele 2), and the frequency of allele 1 has been observed as 0.35 among 120 unrelated Caucasians. In pairwise linkage analyses, there was a significant linkage of IL-2RO gene with chromosome 22 markers shown in Table 2. The most likely location for the

3702 Nucleic Acids Research, Vol. 18, No. 13 Table 3. The repetitive unit sequences in the 5' flanking region of IL-2R3 gene group group

1

-845 -824

AAATGGGAA

-837

AGATGGGAA

-816

-453 AAATGGGAG -445 group

2

group

3

-784 -357

TGTTGGGTA -776 TAGTGGGTA 149 -319 TGTTGGGTA -311 -812

CAGTGGGGG

-804

-797 GGGTGGGGA -789 -462 -421 -402 -337

-296 -161 -136

GGCTGGGTG GGCTGGGAG CGCTGGGGT

_454 413 -394

GGATGGAGG -329 GCGTGGGGG

-288

GCGTAGGAG

-153

CCCTGGGTG

-128

IL-2Rj3 gene has been obtained by multi-point analysis and the result is shown in Figure 6. As the platelet-derived growth factor F3-chain (PDGF B, c-sis) was mapped to q12-13 (27), the IL-2Rfl gene is also considered to be on chromosome 22q12 -13.

DISCUSSION In the present study, we have isolated and analyzed the genomic for the human IL-2RM. It has been found that IL-2R3 gene contains ten exons and nine introns spanning about 24.3 kb. The extracellular region of IL-2R43 is encoded by seven exons (exons 2-8). The exon 7 encodes the extracellular cysteine poor domain which appears to show significant homology with the type III modules of fibronectin (26). This domain also includes the 'WS motif (W-S-X-W-S; X is a nonconserved amino acid) which is strictly conserved in the corresponding region of the receptors belonging to the new receptor superfamily (see below). As predicted by Patthy (26), phase 1 introns which fall between the first and second base of a codon at the boundaries of most type HI domains of fibronectin can be found at the boundary of the exon 7 of the IL-2Rf, supporting the view that intronic recombination played a role in the generation of the IL-2R13 gene. In addition, the IL-2R,B possesses a cysteine rich domain at the N-terminal proximal region which seems to be evolutionally related to the receptors for many cytokines such as interleukin 3 (IL-3), 4 (IL-4), 6 (IL-6), 7 (IL-7), erythropoietin (EPO), granulocyte-macrophage colony stimulatry factor (GM-CSF), as well as for growth hormone and prolactin (28-30). This domain appears to be encoded mostly, if not entirely, by exons 3 and 4. Since many of these receptors also possess the type HI modules (26), one can speculate that the primordial receptor gene was generated through the recombination of at least exons 3, 4 and 7 (and presumably other exons) and the gene went through duplications to generate the receptor superfamily. The cytoplasmic region is encoded by three exons. Interestingly, almost the entire cytoplasmic region and the 3' untranslated region are encoded by a single exon (exon 10). We have previously demonstrated that the cytoplasmic region contains several characteristic domains; serine-rich, acidic, and proline rich domains (9). Recent studies demonstrated that the restricted cytoplasmic domain including the serine-rich region appears to have a critical role for mitogenic signal transduction. This critical gene

region shows the highest degree of homology between IL-2R,B and murine EPO receptor (EPO-R) molecules (11, 29). As indicated most of the above mentioned cytoplasmic regions except for a small portion of the serine-rich region are encoded by exon 10. Furthermore, the exon-intron organization of the human EPOR gene revealed that the cytoplasmic region is dissected by three exons in the same manner as for the IL-2R3 gene (Dr. A. D'Andrea, Whitehead Inst., Cambridge, USA; personal communication), suggesting that the cytoplasmic region of EPO-R probably has similar functional importance. We have also found that a common two-allele RFLP was identified with TaqI, and that linkage of this RFLP to other polymorphic DNA markers localized the human IL-2R13 gene on chromosome 22q12 -13. The localization of IL-2R,B gene to human chromosome 22ql2-13, is very close to the PDGF B gene (22ql2-13) (27). S 1 mapping and primer extension analyses of the RNA from YT cells showed the existence of at least five cap sites for IL-2RF3 mRNA. Similarly IL-2Ra gene was also found to contain multiple cap sites and lacked a typical TATA box sequence (31-33). However, IL-2Rcx and IL-2R,B seem to be regulated differently in their expression. In fact, constitutive mRNA expression is observed for IL-2R,B gene albeit at low levels, but not at all for IL-2 and IL-2Ra genes in resting peripheral blood lymphocytes (9). We and others have identified DNA sequences in the 5'-flanking region responsible for the mitogen-induced expression of the human IL-2 and IL-2Ra genes (13-18). Moreover, recent studies indicate that the inducible NF-xB or NF-xB-like factor binds to the critical cis-elements for both IL-2 and IL-2Ra regulatory regions (19, 20). The NF-xB binding motif has not been identified in the promoter sequence(s) of the human IL-2Rf8 gene (Figure 4). On the other hand, it may be worth noting that the GT stretch sequences which potentially form Z-type DNA (34) are found in the promoter region of IL-2Rj3 gene. Furthermore, the promoter region contains repetitive unit sequences which are roughly classified to three types (Figure 4, Table 3). The role of such unique sequence motifs in the IL-2Rg gene expression needs to be further investigated. These views suggest that the mechanism for gene regulation of IL-2R,3 might be different from that of the IL-2 and IL-2Ra genes. Further studies will allow a possible clarification of these points.

ACKNOWLEDGMENTS We thank Drs. Y. Ebina and J. Yodoi for providing us with pSVOOcat and YT cells, respectively. We also thank Drs. E. Barsoumian and Y. Tokino for invaluable advice and discussion. This work is supported in part by a grant-in-aid for Special Project Research, Cancer Bioscience from the Ministry of Education, Science and Culture of Japan, and The Mitsubishi Foundation.

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