Characterization of the membrane-bound and a soluble form of human IL-4 receptor {alpha} produced by alternative splicing

Susanne Kruse, Johannes Forster, Joachim Kuehr and Klaus A. Deichmann

University Children's Hospital, University of Freiburg, Mathildenstrasse 1, 79106 Freiburg, Germany

Correspondence to: K. A. Deichmann


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IL-4 plays a major role in IgE production. Its signal is conferred to effector cells through binding to the {alpha} chain of the membrane-bound human IL-4 receptor (huIL-4R{alpha}). Here we present the genomic structure and organization of huIL-4R{alpha}. The promotor region shows binding sites for several transcription factors involved in inflammatory processes. HuIL-4R{alpha} has been shown to be organized differently to that of mouse IL-4R{alpha}. A soluble form of huIL-4R{alpha} is produced by alternative splicing of the huIL-4R{alpha} gene (shuIL-4R{alpha}/splice). Expression of the corresponding mRNA coding for the extracellular part of the receptor and an additional three amino acids is also shown. A second form of huIL-4R{alpha}, i.e. shuIL-4R{alpha}/prot, is produced by limited proteolysis of the receptor (shedding) and is already known. These results reveal a complex pattern for the regulation of the IL-4 pathway at the receptor level. The patterns of expression of all three receptor proteins as well as their individual meaning in the context of inflammation still have to be elucidated.

Keywords: genomic structure, IL-4 receptor, soluble, splicing


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IL-4 plays a major role in stimulating B cell proliferation, influencing B cell differentiation towards IgE production and participating in the Th0/Th2 shift of the Th cells (13). The IL-4 signal is conferred to effector cells through binding to the IL-4 receptors (IL-4R). These represent transmembrane receptors composed of a 140 kDa high-affinity binding chain (IL-4R{alpha}) and optionally a further IL-4R{alpha} chain, the common {gamma} chain shared by several IL receptors (4) or the IL-13 receptor {alpha} chain (5). All chains are members of the hematopoietin receptor superfamily (6).

With respect to the numerous functions in IgE regulation, as well as B and T cell differentiation, the IL-4R{alpha} gene represents a candidate gene for atopy. Human IL-4R{alpha} exists in at least two forms: the membrane-bound form (huIL-4R{alpha}) and a soluble one (shuIL-4R{alpha}). In mice two distinct pathways lead to the soluble forms. These involve alternative mRNA-splicing and limited proteolysis (shedding) of IL-4R{alpha} (7). So far, only the genomic structure of the mouse IL-4R{alpha} gene has been described (8,9). The mRNA coding for the smaller soluble muIL-4R{alpha} contains a 114 bp insert that terminates the open reading frame upstream of the predicted transmembrane region. Both mRNAs of the muIL-4R{alpha} originate from the same gene and the 114 bp insert corresponds to an alternatively spliced exon (exon 8) (9). The production of the soluble receptor by proteolysis seems to be regulated in a different way from the mRNA form in mouse and can be stimulated by, for example, TCR (7). A recombinant soluble human IL-4R{alpha} receptor form was found to either enhance or inhibit IL-4 signaling in a dose-dependent manner in supernatants of activated T cells (10), similar to previous findings in mouse (11). In humans, it was postulated that the soluble receptor is only produced by proteolytic shedding (4). This is supposed to be accomplished under the control of metalloproteinases (12).

Our interest was to deduce the complete genomic structure of the huIL-4R{alpha}, including the promotor region. We investigated the existence of an additional form of soluble receptor in man, produced by mRNA splicing (shuIL-4R{alpha}/splice).


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Genomic structure
The genomic organization and sequence of huIL-4R{alpha} was derived from human chromosome 16 BAC clone CIT987SK-582J2 (NCBI HUAC004525).

Preparation of cDNA
mRNA was isolated from human whole blood by using the RNeasy blood mini-kit (Qiagen, Hilden, Germany). The cDNA was synthesized by the First-strand cDNA synthesis kit (Amersham/Pharmacia, Freiburg, Germany).

Exon 8
Exon 8 was detected by performing a nested PCR technique. First PCR: 5'-TCTGCTGCTGACCTGGAGCA-3' and oligo(dT) primer (resulting in a DNA fragment of 454 bp in length). Second PCR: 5'-GCACCCTGAAGTCTGGGATT-3' and oligo (dT) primer (resulting in a DNA fragment of 302 bp in length). PCR was carried out in a volume of 10 µl containing 30 ng cDNA, 5 pmol of each primer, 0.06 U Taq polymerase (Pharmacia) and 2 mmol dNTP mix with the buffer recommended by the supplier. Annealing temperatures were 35°C. The detection of exon 8 was confirmed by sequencing and comparison with the genomic sequence of huIL-4R{alpha}. PCR was performed with cDNA templates derived from whole blood of probands using the primers: exon 7: 5'-GCACCCTGAAGTCTGGGATT-3' and exon 8: 5'-TCTCCCTCCAGAATGTCAGC-3'.

Northern blot
Total RNA was isolated from human whole blood by using the RNeasy blood mini-kit (Qiagen, Hilden, Germany). Then 15 µg RNA was separated on a 1% agarose gel containing 2.2 M formaldehyde and afterwards transferred onto a Hybond N membrane (Amersham, Braunschweig, Germany). The blot was hybridized with a 32P-labeled 330 bp PCR fragment coding for an extracellular part of the IL-4 receptor. Primers: 5'-CTTGCGAGTGGAAGATGAAT-3' and 5'-GTAATTGTCAGGGGGATACG-3'.

Cell culture and IL-4 stimulation for characterization of a STAT6 binding site
Characterization of the STAT6 binding site followed the technique as described by Matsuno et al. (13). Peripheral blood mononuclear cells were derived from whole blood and were grown for 48–72 h at 37°C and 5% CO2 in the presence of 3 µg/ml phytohemagglutinin (Gibco/BRL, Eggenstein, Germany) in RPMI 1640 containing 2 mM L-glutamine, 20 mM HEPES, 100 U/ml penicillin, 50 mg/ml streptomycin and 10% FCS (PAN Systems, Aidenbach, Germany).

Cells were made quiescent for 5–16 h in RPMI 1640/glutamine/HEPES/penicillin/ streptomycin and 1% FCS. T cells (2x107) were stimulated with 100 nM human IL-4 (PAN Systems) for 5–15 min at 37°C. Cell pellets were then stored at –80°C.

After thawing, cells were suspended in lysis buffer (10 mM Tris, pH 7.8, 5 mM EDTA, 50 mM NaCl, 30 mM pyrophosphate, 50 mM sodium fluoride, 20 µM sodium orthovanadate, 1% Triton X-100, 1 mM PMSF, 5 µg/ml aprotinin, 1 µg/ml pepstatin A and 10 µg/ml leupeptin; 108 cells/ml buffer) and incubated for 60 min at 4°C. Insoluble material was removed by centrifugation. Part of the cell extract was separated on 12% native PAGE in several lanes and transferred onto PVDF filters (Millipore, Bedford, UK) (cell extract-blot). The residual binding sites on the filters were blocked overnight with TPBS (150 mM sodium chloride, 3 mM potassium chloride, 1 mM potassium dihydrogen phosphate, 7 mM disodium hydrogen phosphate and 0.05 % Tween 20) and 5% non-fat dried milk. One part of the filter was incubated with monoclonal anti-STAT6 (Transduction, Lexington, KY), the appropriate horseradish peroxidase-coupled secondary antibodies (Dako, Hamburg, Germany) and developed with ECL (Amersham). The other part of the filter was incubated with the rest of the cell extract and a 32P-radiolabeled PCR fragment bearing the potential STAT6 binding site (position intron 2: 4959; primers: 5'-CTGGCCCTTGGTGTACATTT-3' and 5'-GACACCACCTTCACCAAGTG-3'). As a negative control, the same experiment was done using a 32P-radiolabeled PCR fragment bearing the potential STAT but not STAT6 binding site from the promotor region of the gene (position –2328 of the promotor region, see Fig. 2Go, primers: 5'-GCCTGGGATGAAGCATCAAC-3' and 5'-GGCACTGACAAGTGAGCAGA-3').



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2. Collection of signal sequences in the promotor region and introns 1 and 2. Nucleotide positions are indicated below the boxes.

 

    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Genomic structure
The genomic structure of the huIL-4R{alpha} was found to consist of a total of 12 exons and 11 introns (Fig. 1Go). The membrane-bound form of huIL-4R{alpha} is thereby coded by exons 3–7 (extracellular domain), exon 9 (transmembrane domain) and exons 10–12 (intracellular domain). Exons 1 and 2 bear untranslated regions of the mRNA (4). Furthermore, the DNA sequence coding for a signal peptide consisting of 25 amino acids was located to exon 3. In comparison to the mouse genomic structure (9), the flanking base pair sequences of exon–intron boundaries, the exact sizes of introns and the amino acids found at the boundaries are shown in Table 1Go.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1. Exon–intron organization of the huIL-4R{alpha} gene and correlation to the protein structure. The 12 exons (112) are depicted by boxes and coding regions by solid boxes. P, promotor region; signal, signal protein; Sol, soluble receptor form (shuIL-4R{alpha}/splice); TM, transmembrane domain; aa, amino acids. *Additional amino acids in the soluble receptor form.

 

View this table:
[in this window]
[in a new window]
 
Table 1. Exon–intron junctions of the huIL-4R{alpha} gene compared to the mouse genome (9)
 
Promotor region and transcription factors
The promotor region bears several signal sequences for transcription factors. A selection is shown in Fig. 2Go. The existence of a STAT6 signal sequence could be verified by Southern/Western blotting analysis and was located to position intron 2: 4959 (Fig. 3Go). The sequence is: 5'-TTCTCAGGAA-3'. A similar potential STAT but not a STAT6 sequence from the promotor used as a negative control (5'-TTCCAGGAA-3') was shown not to bind to the STAT6 proteins.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 3. Characterization of a STAT6 binding site by Southern/Western blotting. Lane 1, size marker; lane 2, incubation of the cell extract-blot with anti STAT6; lane 3, autoradiography after incubation of the cell extract-blot with 32P-labeled PCR fragment including the STAT6 signal sequence and the rest of the cell extract.

 
Differential mRNA splicing; exon 8
By applying PCR on cDNA derived from human whole blood cells, a short mRNA form of huIL-4R{alpha} was detected. Sequencing and comparison to the genomic sequence revealed it to be located between exon 7 and 9 (start position in intron 7: 2323). In the exon 8 sequence (195 bp) the first 9 bp code for three additional amino acids (Asn, Ile and Cys) followed by a stop codon (Fig. 4Go). The existence of two distinct mRNAs for IL-4R{alpha} products was confirmed in Northern blotting (Fig. 5Go). The short mRNA form could be amplified from at least 10 cDNA probes derived from whole blood of different probands. The concentration of the transcripts was similar (data not shown).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4. Nucleotide sequence of exon 8 (position 2323 in intron 7). Additional amino acids and the `STOP' codon are shown in bold letters. The poly(A) tail is underlined.

 


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5. Northern blot showing two distinct RNAs hybridizing with a 330 bp DNA fragment from the extracellular part of the IL-4R{alpha} gene. The size of the RNAs corresponds to those for the membrane bound IL-4R{alpha} (the complete receptor) and the soluble IL-4R{alpha} as deduced from cDNA sequencing.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Association studies with common polymorphisms in the coding part of huIL-4R{alpha} gene demonstrated a possible involvement of this gene in atopy (1416). Functional studies revealed that amino acid variants of the huIL-4R{alpha} protein have a strong influence on the structure, and consequently on substrate binding and the signaling processes of the receptor (17,18).

Due to the increasing importance of huIL-4R{alpha} in the context of IgE regulation as well as its role in the Th1/Th2 shift and in order to allow further studies on variants in regulatory elements of the gene, the whole genomic structure has been characterized in this work.

Genomic structure
In general, the genomic structure of the human IL-4R{alpha} gene resembles that of the mouse gene (9). It consists of 12 exons being interrupted by 11 introns. As the size of the mouse and human proteins slightly differ, the number of amino acids coded by the individual exons is slightly different. For example, we detected a signal peptide of 25 amino acids in human, in contrast to the mouse showing a signal peptide of 23 amino acids. Furthermore, the intracellular part of the human protein is bigger in size (exon 12).

Introns
Regarding the intron sequences, they are on the whole larger than the mouse introns, so that the complete huIL-4R{alpha} gene spans ~51 kb on chromosome 16p11.2–12.1 (19), which is double in size compared to 26 kb on mouse chromosome 7.

Promotor analysis
Analyzing the promotor region, we could detect binding sites for those transcription factors having an influence on genes of the inflammatory processes (Fig. 2Go). These include NF{kappa}B, AP-1, NF-AT, CREB and STAT (20). It may well be that huIL-4R{alpha} is differentially controlled by these factors, which still has to be investigated.

Strikingly, we could detect several potential TATA boxes in the promotor region and intron 1. There is no TATA box directly preceding the proposed cap site (mRNA start) (4) in human, whereas there is one at position –29 in mouse (9). We therefore postulate multiple transcription initiation sites in human, which have already been postulated for the muIL-4R{alpha} gene (9).

As the mouse IL-4R{alpha} chain is known to be up-regulated by IL-4 through STAT6 binding (21), we were interested in a potentially similar function of STAT6 in humans. By using a combined Western/Southern blot technique we could detect a typical STAT6 binding site at position 4959 of intron 2. It is situated about 5 kb 5' of the start codon (ATG) of the huIL-4R{alpha} gene. The sequence TTCTCAGGAA and the position is completely different from the mouse IL-4R{alpha} gene, where the STAT6 binding site has been located to position –402 in the promotor and the sequence was shown to be TTCATCTGAA (21). STAT6 is an important factor for the activation of genes belonging to the IL-4 pathway (22). There is strong evidence for STAT6 not only being a direct effector substrate of the IL-4R{alpha} protein itself (23), but also influencing its transcription.

From our findings we conclude that due to strong variations in structure from human IL-4R{alpha} to that of the mouse, transcription is probably organized in a different way. Further studies are planned to verify this hypothesis.

Exon 8; shuIL-4R{alpha} produced by alternative splicing
Here we present for the first time evidence for the existence of an additional exon (exon 8) being located in intron 7/8 of huIL-4R{alpha}. Due to the observations in the mouse system (9), exon 8 is very likely to code for a soluble form of huIL-4R{alpha}. A corresponding mRNA coding for the soluble receptor form could be shown in Northern blotting (see Fig. 5Go).

Likewise in mice we found a stop codon in the sequence after additional amino acids. This soluble form is lacking transmembrane and intracellular domains. Therefore the shorter receptor form is likely to be excreted into the cytoplasm. Whereas in mice six amino acids are added to the cytoplasmic domain of IL-4R{alpha} (4), in human we find three (Asn, Ile and Cys).

As proposed in mice (9), the shorter form of IL-4R{alpha} mRNA is produced by alternative splicing mechanisms. We could detect shuIL-4R{alpha}/splice mRNA in several samples derived from human whole blood. As the samples were collected randomly (not only from atopics), we suspect that shuIL-4R{alpha}/splice is expressed in relatively equal amounts in mononuclear cells, regardless of the stimulation status. Our hypothesis is supported by similar findings in mice (24); however, it has to be further investigated.

shu IL-4R{alpha} produced by limited proteolysis (shedding)
A soluble receptor form produced by the process of proteolytic shedding has already been described (12). It remains to be shown if both forms of shuIL-4R{alpha} exhibit similar functions and if they are regulated by distinct processes.

It has been found previously that in atopics and asthmatics, as well as other inflammatory states of the respiratory epithelium, the concentration of shuIL-4R{alpha} is much lower compared to healthy subjects (25). If we assume that the mRNA concentration is the same in all cases, it may well be that either altered post-translational regulations of the mRNA spliced form occur or that the shedding is somehow reduced, probably due to different surface conditions of huIL-4R{alpha}, due to influences on the activity of metalloproteinases itself or due to increased degradation of shuIL-4R{alpha} forms. In order to deduce the exact regulatory mechanisms it will be also necessary to identify the specific metalloproteinase responsible for the huIL-4R{alpha} shedding.

The exact function of shuIL-4R{alpha} forms still has to be elucidated. Inhibitory effects and co-stimulatory effects on IL-4 signaling as well as depot effects for IL-4 have been discussed (10).

We suggest careful discrimination between the different shuIL-4R{alpha} forms regarding their two distinct origins. Furthermore, the meaning of the polymorphism Ile50Val (17), which appears in all three forms, has to be further evaluated.

Our findings indicate complex regulatory mechanisms for the expression of membrane bound as well as soluble IL-4R{alpha}. This opens a wide spectrum of further studies investigating the regulation either on the genomic, mRNA or protein level.


    Acknowledgments
 
This project is supported by a grant from the German Science Foundation (DFG-De386/2-2) and the Klinische Forschergruppe: Pathomechanismen der allergischen Entzündung (BMFT 01GC9701/5).


    Abbreviations
 
IL-4R{alpha} IL-4 receptor {alpha}

    Notes
 
Transmitting editor: A. Radbruch

Received 18 May 1999, accepted 24 August 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Howard, M., Farrar, J., Hilfiker, M., et al. 1982. Identification of a T cell-derived ß cell growth factor distinct from interleukin 2. J. Exp. Med. 155:914.[Abstract]
  2. Coffman, R. L., Lebman, D. A. and Rothman, P. 1993. Mechanism and regulation of immunoglobulin isotype switching. Adv. Immunol 54:229.[ISI][Medline]
  3. Hu-Li, J., Shevach, E. M., Mizuguchi J., et al. 1987. B cell stimulatory factor-1 (interleukin 4) is a potent costimulant for normal resting T lymphocytes. J. Exp. Med. 165:157.[Abstract]
  4. Idzerda, R. L., March, C. J., Mosley, B., et al. 1990. Human interleukin-4 receptor confers biological responsiveness and defines a novel receptor superfamily. J. Exp. Med. 171:861.[Abstract]
  5. Russell, S. M., Keegan, A. D., Harada, N., et al. 1993. The interleukin-2 receptor {gamma} chain is a functional component of the interleukin-4 receptor. Science 262:1180.
  6. Cosman, D. 1993. The hematopoietin receptor superfamily. Cytokine 5:95.[ISI][Medline]
  7. Blum, H., Wolf, M., Enssle, K., et al. 1996. Two distinct stimulus-dependent pathways lead to the production of soluble murine interleukin-4 receptor. J. Immunol. 157:1846.[Abstract]
  8. Mosley, B., Beckmann, M. P., March, C. J., et al. 1989. The murine interleukin-4 receptor: molecular cloning and characterization of secreted and membrane bound forms. Cell 59:335.[ISI][Medline]
  9. Wrighton, N., Campbell, L. A., Harada, N., et al. 1992. The murine interleukin-4 receptor gene: genomic structure, expression and potential for alternative splicing. Growth Factors 6:103.[Medline]
  10. Jung, T., Wagner, K., Neumann, C., et al. 1999. Enhancement of human IL-4 activity by soluble IL-4 receptors in vitro. Eur. J. Immunol. 29:864.[ISI][Medline]
  11. Sato, T. A., Widmer, M. B., Finkelmann, F. D., et al. 1993. Recombinant soluble murine IL-4 receptor can inhibit or enhance IgE responses in vivo. J. Immunol. 150:2717.[Abstract/Free Full Text]
  12. Jung, T., Schrader, N., Hellwig, M., et al. 1999. Soluble human interleukin-4 receptor is produced by activated T cells under the control of metalloproteinases. Int. Arch. Allergy Immunol. 119:23.[ISI][Medline]
  13. Matsuno, K., Suzuki, T., Takiya, S. and Suzuki, Y. 1989. Complex formation with the fibrin gene enhancer through a protein–protein interaction analyzed by a modified DNA-binding assay. J. Biol. Chem. 264:4599.[Abstract/Free Full Text]
  14. Deichmann, K. A., Bardutzky, J., Forster, J., et al. 1997. Common polymorphisms in the coding part of the IL-4-receptor gene. Biochem. Biophys. Res. Commun. 231:696.[ISI][Medline]
  15. Deichmann, K. A., Heinzmann, A., Forster, J., et al. 1998. Linkage and allelic association of atopy and markers flanking the IL-4-receptor gene. Clin. Exp. Allergy 28:151.[ISI][Medline]
  16. Hershey, G. K. K., Friedrich, M. F., Esswein, L. A., et al. 1997. The association of atopy with a gain-of-function mutation in the {alpha} subunit of the interleukin-4 receptor. N. Engl. J. Med. 337:1720.[Abstract/Free Full Text]
  17. Mitsuyasu, H., Izuhara, K., Mao, X. Q., et al. 1998. Ile50Val variant of IL-4R alpha upregulates IgE synthesis and associates with atopic asthma. Nat. Genet. 19:119.[ISI][Medline]
  18. Kruse, S., Japha, T., Tedner, M., Hauschildt Sparholt, S., Forster, J., Kuehr, J. and Deichmann, K. A. 1999. The polymorphisms S503P and Q576R in the IL-4a-receptor gene are associated with atopy and influence the signal transduction. Immunology 96:365.[ISI][Medline]
  19. Pritchard, M. A., Baker, E., Whitmore, S. A., et al. 1991. The interleukin-4 receptor gene (IL-4R{alpha} maps to 16p11.2.p12.1 in the human and to the distal region of mouse chromosome 7. Genomics 10:801.[ISI][Medline]
  20. Barnes, P. J. and Adcock, I. M. 1998. Transcription factors and asthma. Eur. Respir. J. 12:221.[Abstract/Free Full Text]
  21. Kontanides, H. and Reich, N. C. 1996. Interleukin-4-induced STAT6 recognizes and activates a target site in the promotor of the interleukin-4 receptor gene. J. Biol. Chem. 271:25555.[Abstract/Free Full Text]
  22. Mikita, T., Campell, D., Pengguang, W., et al. 1996. Requirements of interleukin-4 induced gene expression and functional characterization of STAT6. Mol. Cell. Biol. 10:5811.
  23. Wang, H., Zamorano, J. and Keegan, A. 1998. A role for the insulin-interleukin (IL)-4 receptor motif of the IL-4 receptor {alpha}-chain in regulating activation of the insulin receptor substrate 2 and signal transducer and activator of transcription 6 pathways. J. Biol. Chem. 273:9898.[Abstract/Free Full Text]
  24. Chilton, P. M. and Fernandez-Botran, R. 1997. Regulation of the expression of the soluble and membrane forms of the murine IL-4 receptor. Cell. Immunol. 180:104.[ISI][Medline]
  25. Schauer, U., Schmitt, M., Müller, S., et al. 1995. Soluble interleukin-4 receptor in atopic children. Int. Arch. Allergy Immunol. 108:175.[ISI][Medline]