Identification of Critical Residues in Bovine IFNAR-1 Responsible for Interferon Binding
2001; Elsevier BV; Volume: 276; Issue: 20 Linguagem: Inglês
10.1074/jbc.m009663200
ISSN1083-351X
AutoresElizabeth Cali Cutrone, Jerome A. Langer,
Tópico(s)Immune Cell Function and Interaction
ResumoInterferons have antiviral, antigrowth and immunomodulatory effects. The human type I interferons, IFN-α, IFN-β, and IFN-ω, induce somewhat different cellular effects but act through a common receptor complex, IFNAR, composed of subunits IFNAR-1 and IFNAR-2. Human IFNAR-2 binds all type I IFNs but with lower affinity and different specificity than the IFNAR complex. Human IFNAR-1 has low intrinsic binding of human IFNs but strongly affects the affinity and differential ligand specificity of the IFNAR complex. Understanding IFNAR-1 interactions with the interferons is critical to elucidating the differential ligand specificity and activation by type I IFNs. However, studies of ligand interactions with human IFNAR-1 are compromised by its low affinity. The homologous bovine IFNAR-1 serendipitously binds human IFN-αs with nanomolar affinity. Exploiting its strong binding of human IFN-α2, we have identified residues important for ligand binding. Mutagenesis of any of five aromatic residues of bovine IFNAR-1 caused strong decreases in ligand binding, whereas mutagenesis of proximal neutral or charged residues had smaller effects. These residues were mapped onto a homology model of IFNAR-1 to identify the ligand-binding face of IFNAR-1, which is consistent with previous structure/function studies of human IFNAR-1. The topology of IFNAR-1/IFN interactions appears novel when compared with previously studied cytokine receptors. Interferons have antiviral, antigrowth and immunomodulatory effects. The human type I interferons, IFN-α, IFN-β, and IFN-ω, induce somewhat different cellular effects but act through a common receptor complex, IFNAR, composed of subunits IFNAR-1 and IFNAR-2. Human IFNAR-2 binds all type I IFNs but with lower affinity and different specificity than the IFNAR complex. Human IFNAR-1 has low intrinsic binding of human IFNs but strongly affects the affinity and differential ligand specificity of the IFNAR complex. Understanding IFNAR-1 interactions with the interferons is critical to elucidating the differential ligand specificity and activation by type I IFNs. However, studies of ligand interactions with human IFNAR-1 are compromised by its low affinity. The homologous bovine IFNAR-1 serendipitously binds human IFN-αs with nanomolar affinity. Exploiting its strong binding of human IFN-α2, we have identified residues important for ligand binding. Mutagenesis of any of five aromatic residues of bovine IFNAR-1 caused strong decreases in ligand binding, whereas mutagenesis of proximal neutral or charged residues had smaller effects. These residues were mapped onto a homology model of IFNAR-1 to identify the ligand-binding face of IFNAR-1, which is consistent with previous structure/function studies of human IFNAR-1. The topology of IFNAR-1/IFN interactions appears novel when compared with previously studied cytokine receptors. type I interferon receptor interferon human IFN interferon-γ receptor subdomains 1 through 4 bovine IFNAR Dulbecco's modified Eagle's medium splice overlap extension hemagglutinin phosphate-buffered saline monoclonal antibody The type I interferon receptor, IFNAR,1 mediates the binding of all type I interferons (IFNs), which, in the human, derive from genes for 13 IFN-αs, one IFN-β, and one IFN-ω (1Merlin G. Falcoff E. Aguet M. J. Gen. Virol. 1985; 66: 1149-1152Crossref PubMed Scopus (100) Google Scholar, 2Flores I. Mariano T. Pestka S. J. Biol. Chem. 1991; 266: 19875-19877Abstract Full Text PDF PubMed Google Scholar, 3Pestka S. Langer J.A. Zoon K.C. Samuel C.E. Annu. Rev. Biochem. 1987; 56: 727-777Crossref PubMed Scopus (1605) Google Scholar, 4Allen G. Diaz M.O. J. Interferon Cytokine Res. 1996; 16: 181-184Crossref PubMed Scopus (36) Google Scholar, 5Branca A.A. 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Wilson I.A. Science. 1996; 273: 464-471Crossref PubMed Scopus (558) Google Scholar). In the cases of IFNGR-1, IFNGR-2, growth hormone receptor, tissue factor, and IFNAR-2 there are two FNIII domains, each containing ∼100 amino acids with seven β-strands and connecting loops. The extracellular domain of IFNAR-1 is atypical, consisting of a tandem array of four FNIII domains, here denoted subdomains 1 through 4 (SD1–4; beginning from the N terminus). The four-domain structure of IFNAR-1 appears to represent a tandem duplication of the more common two-domain structure (27Bazan J.F. Immunol. Today. 1990; 11: 350-354Abstract Full Text PDF PubMed Scopus (514) Google Scholar, 38Gaboriaud C. Uze G. Lutfalla G. Mogensen K. FEBS Lett. 1990; 269: 1-3Crossref PubMed Scopus (18) Google Scholar, 39Mogensen K.E. Lewerenz M. Reboul J. Lutfalla G. Uze G. J. Interferon Cytokine Res. 1999; 19: 1069-1098Crossref PubMed Scopus (224) Google Scholar). The low intrinsic affinity of HuIFNAR-1 for IFNs has hampered studies seeking to identify residues involved in ligand binding and specificity. Previously, the identity of elements of the ligand binding site of HuIFNAR-1 could only be deduced indirectly from studies involving antibody epitope mapping (40Lu J. Chuntharapai A. Beck J. Bass S. Ow A. De Vos A.M. Gibbs V. Kim K.J. J. Immunol. 1998; 160: 1782-1788PubMed Google Scholar, 41Eid P. Tovey M.G. J. Interferon Cytokine Res. 1995; 15: 205-211Crossref PubMed Scopus (23) Google Scholar, 42Eid P. Langer J.A. Bailly G. Lejealle R. Guymarho J. Tovey M.G. Eur. Cytokine Netw. 2000; 11: 560-573PubMed Google Scholar) or homology modeling based on other cytokine receptors (40Lu J. Chuntharapai A. Beck J. Bass S. Ow A. De Vos A.M. Gibbs V. Kim K.J. J. Immunol. 1998; 160: 1782-1788PubMed Google Scholar, 43Seto M.H. Harkins R.N. Adler M. Whitlow M. Church W.B. Croze E. Protein Sci. 1995; 4: 655-670Crossref PubMed Scopus (36) Google Scholar). Without large-scale mutagenesis and ligand binding analysis, key residues could not be identified in HuIFNAR-1. The bovine IFNAR-1 homologue is an attractive target for mutagenesis and analysis of the IFN binding site. Although the type I interferons are predominantly species-specific, human interferons display uniformly high binding and biological activity on bovine cells (3Pestka S. Langer J.A. Zoon K.C. Samuel C.E. Annu. Rev. Biochem. 1987; 56: 727-777Crossref PubMed Scopus (1605) Google Scholar, 44Rehberg E. Kelder B. Hoal E.G. Pestka S. J. Biol. Chem. 1982; 257: 11497-11502Abstract Full Text PDF PubMed Google Scholar, 45Zoon K. zur Nedden D. Arnheiter H. J. Biol. Chem. 1982; 257: 4695-4697Abstract Full Text PDF PubMed Google Scholar, 46Zoon K.C. Arnheiter H. Pharmacol. Ther. 1984; 24: 259-278Crossref PubMed Scopus (129) Google Scholar). This appears to reflect the ability of bovine IFNAR-1 (BoIFNAR-1) to bind human type I IFNs with moderately high affinity. Thus, human or murine cells expressing BoIFNAR-1 greatly increase their responsiveness to a variety of human type I IFNs (18Lim J.K. Xiong J. Carrasco N. Langer J.A. FEBS Lett. 1994; 350: 281-286Crossref PubMed Scopus (26) Google Scholar, 47Mouchel-Vielh E. Lutfalla G. Mogensen K.E. Uze G. FEBS Lett. 1992; 313: 255-259Crossref PubMed Scopus (37) Google Scholar, 48Lim J.K. Langer J.A. Biochim. Biophys. Acta. 1993; 1173: 314-319Crossref PubMed Scopus (25) Google Scholar). The nanomolar binding affinity of BoIFNAR-1 for HuIFN-αs provides an elegant way to circumvent difficulties in the studies of the human IFN type I receptor complex (47Mouchel-Vielh E. Lutfalla G. Mogensen K.E. Uze G. FEBS Lett. 1992; 313: 255-259Crossref PubMed Scopus (37) Google Scholar, 48Lim J.K. Langer J.A. Biochim. Biophys. Acta. 1993; 1173: 314-319Crossref PubMed Scopus (25) Google Scholar, 49Goldman L.A. Cutrone E.C. Dang A. Hao X.M. Lim J.K. Langer J.A. 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COS-1 cells transfected with BoIFNAR-1 cDNA express very high levels (0.5–1.0 × 106/cell) of nanomolar affinity binding sites for HuIFN-α2a, -α8b, -α1b, -β, and -ω (11Cutrone E.C. Langer J.A. FEBS Lett. 1997; 404: 197-202Crossref PubMed Scopus (70) Google Scholar, 48Lim J.K. Langer J.A. Biochim. Biophys. Acta. 1993; 1173: 314-319Crossref PubMed Scopus (25) Google Scholar, 49Goldman L.A. Cutrone E.C. Dang A. Hao X.M. Lim J.K. Langer J.A. Biochemistry. 1998; 37: 13003-13010Crossref PubMed Scopus (17) Google Scholar). Consistent with these results, a soluble BoIFNAR-1/Fc fusion protein bound HuIFN-α2a with an affinity of at most 10 nm (the true affinity is expected to be closer to a Ki of 0.14 nm (23Langer J.A. Yang J. Carmillo P. Ling L.E. FEBS Lett. 1998; 421: 131-135Crossref PubMed Scopus (14) Google Scholar)). All human type I IFNs tested were able to compete with HuIFN-α2a for binding to BoIFNAR-1/Fc, although they varied in affinity. Thus, the extracellular domain of BoIFNAR-1 by itself displays moderate affinity and differential binding of a broad range of human type I IFNs. Previously, we used BoIFNAR-1 to regionally localize the determinants that confer strong binding of IFN (49Goldman L.A. Cutrone E.C. Dang A. Hao X.M. Lim J.K. Langer J.A. Biochemistry. 1998; 37: 13003-13010Crossref PubMed Scopus (17) Google Scholar). A series of 14 HuIFNAR-1/BoIFNAR-1 chimeric receptors representing various human/bovine subdomain substitutions were assayed for their ligand binding properties. Only when the two central domains of BoIFNAR-1 (SD2 and SD3) were simultaneously substituted into the HuIFNAR-1 was a significant increase in the binding of HuIFN-α2a measured over the low affinity binding characteristic of HuIFNAR-1, indicating that ligand binding was focused in this region. The BoIFNAR-1 N-terminal (SD1) and membrane-proximal (SD4) domains each further enhanced binding when transferred with SD2 and SD3 into HuIFNAR-1 (49Goldman L.A. Cutrone E.C. Dang A. Hao X.M. Lim J.K. Langer J.A. Biochemistry. 1998; 37: 13003-13010Crossref PubMed Scopus (17) Google Scholar). Thus, the IFN binding site is not localized on one FNIII subdomain or even on one tandem pair of FNIII subdomains, as previously predicted from other cytokine family members (43Seto M.H. Harkins R.N. Adler M. Whitlow M. Church W.B. Croze E. Protein Sci. 1995; 4: 655-670Crossref PubMed Scopus (36) Google Scholar); instead the ligand-binding determinants of IFNAR-1 appear to be distributed in a more complex array centered on SD2 and SD3. Kumaran et al. (52Kumaran J. Colamonici O.R. Fish E.N. J. Interferon Cytokine Res. 2000; 20: 479-485Crossref PubMed Scopus (9) Google Scholar) used murine/human IFNAR-1 hybrids to confirm that SD1 plays at most a minor role in species-specific ligand binding. The current study of BoIFNAR-1 identifies amino acids that are critical for IFN binding, using a series of alanine substitutions throughout its extracellular domain. By presenting these results in the context of a new three-dimensional homology model of IFNAR-1, we shed light on the interaction of type I IFNs with IFNAR-1. IFN-α2a (1.56 × 108 IU/mg) was provided by Dr. Sidney Pestka. The M2 anti-FLAG antibody was purchased from Sigma Chemical Co.R-Phycoerythrin-conjugated F(ab′)2 goat anti-mouse IgG was purchased from Jackson ImmunoResearch. Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.) supplemented with 10% cosmic calf serum (HyClone), 2 mm glutamine (Sigma), and 10 units/ml penicillin with 10 μg/ml streptomycin (Life Technologies, Inc.), was used to maintain all cell lines. The HuIFN-α2a analogue IFN-α2a-P1 (53Li B.-L. Langer J.A. Schwartz B. Pestka S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 558-562Crossref PubMed Scopus (62) Google Scholar) was phosphorylated to a radiospecific activity of ∼1 × 103 Ci/mmol with [γ-32P]ATP (6000 Ci/mmol, PerkinElmer Life Sciences) and bovine heart cAMP-dependent protein kinase (Sigma). This corresponds to a fractional labeling of ∼0.15 mol of 32P/mol of protein. For simplicity, [32P]IFN-α2a-P1 is referred to as [32P]IFN-α2. Bovine and Human IFNAR-1 were transiently expressed from the EF-1α promoter in the vector pcDEF3 (54Goldman L.A. Cutrone E.C. Kotenko S.V. Krause C.D. Langer J.A. BioTechniques. 1996; 21: 1013-1015Crossref PubMed Scopus (152) Google Scholar). Unique restriction sites were engineered into the bovine IFNAR-1 cDNA by using oligonucleotides containing silent mutationsRsrII, BspEI, AgeI, andNheI. These restriction sites were placed between each respective subdomain and at the beginning of the transmembrane domain, resulting in a modified version of BoIFNAR-1. This version of BoIFNAR-1 was also tagged with the FLAG epitope (55Hopp T.P. Prickett K.S. Price V. Libby R.T. March C.J. Cerretti P. Urdal D.L. Conlon P.J. Bio/Technology. 1988; 6: 1205-1210Crossref Scopus (754) Google Scholar) between the signal peptide and the beginning of the first subdomain of the protein, allowing for its recognition on the cell surface. Mutations were engineered into the receptor using either a two-step splice overlap extension (SOE) polymerase chain reaction method (56Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene. 1989; 77: 51-59Crossref PubMed Scopus (6851) Google Scholar, 57Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene. 1989; 77: 61-68Crossref PubMed Scopus (2648) Google Scholar) or the QuikChange site-directed mutagenesis kit (Stratagene). All clones were screened by restriction digests and confirmed by sequencing. In most cases altered receptors were analyzed using two independent clones, to eliminate false decreases in binding due to reasons other than the engineered mutation. Mutants were made from the BoIFNAR-1 cDNA template in three different versions of pcDEF-3 at various stages in this project. The BoIFNAR-1 gene was the same for all three; however, the first had an HA tag at the N terminus (which was not effective for flow cytometric detection); in the second, the HA tag was replaced with a FLAG epitope to allow for cell surface confirmation by flow cytometry; the third version contained an internal ribosome entry site coupled to enhanced green fluorescent protein (IRES-EGFP) (CLONTECH) after the FLAG-bovine IFNAR-1, and a zeocin marker in place of the original neomycin marker. These plasmids, when transfected into COS cells, were indistinguishable in their transient BoIFNAR-1 expression and IFN binding. The two-step Splice Overlap Extension (SOE) polymerase chain reaction method (56Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene. 1989; 77: 51-59Crossref PubMed Scopus (6851) Google Scholar, 57Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene. 1989; 77: 61-68Crossref PubMed Scopus (2648) Google Scholar) was carried out using ELongase (Life Technologies, Inc.). SOE fragments were restriction-digested and then ligated into the proper vector. All reactions were carried out in a 50-μl volume in 0.5-ml thin-wall tubes in a PerkinElmer Life Sciences DNA thermal cycler. The QuikChange site-directed mutagenesis kit was used according to the manufacturer's directions (Stratagene), except that template and primer concentrations were doubled; extension time was increased to 3.5 min per kilobase; and DH10B Electromax competent cells (Life Technologies, Inc.) were used for transformations. COS-1 cells (58Gluzman Y. Cell. 1981; 23: 175-182Abstract Full Text PDF PubMed Scopus (1460) Google Scholar), derived from a simian kidney CV-1 line, were transfected with 5–10 μg of plasmid using the DEAE-dextran/Me2SO shock protocol (48Lim J.K. Langer J.A. Biochim. Biophys. Acta. 1993; 1173: 314-319Crossref PubMed Scopus (25) Google Scholar, 59Seed B. Aruffo A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3365-3369Crossref PubMed Scopus (789) Google Scholar, 60Sussman D.J. Milman G. Mol. Cell. Biol. 1984; 4: 1641-1643Crossref PubMed Scopus (124) Google Scholar). Briefly, tissue culture dishes (10-cm dish, Falcon) containing 10 ml of 10% cosmic calf (Hyclone) serum-supplemented DMEM were seeded with 1.75–2.0 × 106 cells. Cells were incubated overnight at 37 °C before transfecting. Cells were harvested for assays after 48–72 h. Under our transfection conditions, we generally see about 0.25–1 × 106 receptors per cell, averaged over the entire cell population, as measured by ligand binding to BoIFNAR-1. This arises from high level expression on 20–50% of the cell population, as demonstrated by flow cytometry detection of the common FLAG epitope at the N terminus. Much of this expression variability is between experiments; within any experiment, the variation of expression between constructs has a much smaller range. Each clone was transfected at least two times, and for most mutations binding was measured with transfections of two independent DNA clones. Saturation binding assays were done as previously described (48Lim J.K. Langer J.A. Biochim. Biophys. Acta. 1993; 1173: 314-319Crossref PubMed Scopus (25) Google Scholar). Briefly, COS cells were trypsinized and resuspended to 1 × 106 cells/ml. Aliquots of cells, with and without excess cold IFN (1–3 μg/ml), were combined with [32P]IFN-α2 (maximum concentration of 1.5–4 × 10−9 m), serially diluted, and then incubated while rocking for 1 h at room temperature. Cell-bound [32P]IFN-α2 was separated from unbound by brief centrifugation of 100 μl of sample through a cushion of 10% (w/v) sucrose in PBS. Tubes were then frozen and cut, and the tips and tops were counted separately. Suppliers' determinations of IFN concentration (in mg/ml) were used for all calculations. Data were analyzed by non-linear regression to one-site binding using the program Prism v.2.01 (GraphPad Software, Inc., San Diego, CA). A mutant receptor that lost 75% of the binding seen for unaltered BoIFNAR-1 was defined as having a significant loss of binding activity. Among the mutants, there is a clear demarcation between those mutants that lost 75–100% and a secondary group of mutants, which lost 0–56% of binding. This criterion is consistent with thresholds used in studies of other receptors (61Cunningham B.C. Wells J.A. J. Mol. Biol. 1993; 234: 554-563Crossref PubMed Scopus (487) Google Scholar, 62Wells J.A. Methods Enzymol. 1991; 202: 390-411Crossref PubMed Scopus (312) Google Scholar, 63Wells J.A. Bio/Technology. 1995; 13: 647-651PubMed Google Scholar) and with the accuracy and precision of our data, thereby guarding against over-interpretation of marginal effects. Transfected cells were resuspended in DMEM supplemented with 5% cosmic calf serum (Life Technologies, Inc.). Cells (7.5 × 105 to 1.25 × 106 in 25 μl) were incubated for 1 h at 4 °C with 25 μl (0.12–0.25 μg) of primary antibody (M2 anti-FLAG antibody, Sigma) or medium. The cells were washed with PBS and resuspended in 50 μl (0.125 μg) of secondary antibody (R-phycoerythrin-conjugated F(ab′)2 goat anti-mouse IgG, Jackson ImmunoResearch) and incubated for 1 h. Cells were again washed with PBS and then incubated in 100 μl of 3% paraformaldehyde at 4 °C for 1 h. The cells were washed once with 1 ml of PBS containing 50 mm Tris and then resuspended in 500 μl of PBS with 50 mm Tris and stored at 4 °C until analysis with a Coulter Epics Profile II cell sorter. Homology models were generated for the extracellular domains of bovine and human IFNAR-1, based on the coordinates of the extracellular domain of IFNGR-1 (30Walter M.R. Windsor W.T. Nagabhushan T.L. Lundell D.J. Lunn C.A. Zauodny P.J. Narula S.K. Nature. 1995; 376: 230-235Crossref PubMed Scopus (360) Google Scholar, 70Thiel D.J. le Du M.H. Walter R.L. D'Arcy A. Chene C. Fountoulakis M. Garotta G. Winkler F.K. Ealick S.E. Structure. 2000; 8: 927-936Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) graciously provided by Drs. Mark Walter (University of Alabama, Birmingham) and Steve Ealick (Cornell University). The homology model was created using GeneMine (LOOK v.3, Molecular Applications Group) and manipulated with GeneMine and Sybyl (Tripos, Inc.). The IFNAR-1 (4-domain receptor) was modeled against IFNGR-1 (2-domain receptor) in two steps. Because of the size disparity, it was necessary to separately model the N-terminal half (SD1 and SD2) and the C-terminal half (SD3 and SD4) of the receptor. This procedure is consistent with the finding that the four domains of IFNAR-1 likely evolved from a tandem duplication of a two-domain ancestor, which diverged to generate the cytokine receptor superfamily (27Bazan J.F. Immunol. Today. 1990; 11: 350-354Abstract Full Text PDF PubMed Scopus (514) Google Scholar, 38Gaboriaud C. Uze G. Lutfalla G. Mogensen K. FEBS Lett. 1990; 269: 1-3Crossref PubMed Scopus (18) Google Scholar,39Mogensen K.E. Lewerenz M. Reboul J. Lutfalla G. Uze G. J. Interferon Cytokine Res. 1999; 19: 1069-1098Crossref PubMed Scopus (224) Google Scholar). The sequence alignment of the N- and C-terminal halves of bovine and human IFNAR-1 with HuIFNGR-1 is shown in Fig.1, with β-strands from the crystal structure of IFNGR-1 indicated (underlined in the sequence) and sequentially labeled as B1–B7 within a FNIII domain. The alignments are facilitated by the similar sequence lengths and conserved characteristic residues of IFNGR-1 and the N- and C-terminal halves of IFNAR-1. Sequence alignments were similar with GCG version 9.0 and with ClustalW. (Expected loops are designated by the flanking β-strands: e.g. the loop connecting β-strands 4 and 5 is denoted “L4–5.”) As commonly observed within families of homologous proteins, the “core” β-sheet structure is well conserved, and much of the variability arises in the interstrand loops, particularly the loops L2–3 and L4–5 in SD2 and SD4 of IFNAR-1. In our homology model
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