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A Role for the Insulin-Interleukin (IL)-4 Receptor Motif of the IL-4 Receptor α-Chain in Regulating Activation of the Insulin Receptor Substrate 2 and Signal Transducer and Activator of Transcription 6 Pathways

1998; Elsevier BV; Volume: 273; Issue: 16 Linguagem: Inglês

10.1074/jbc.273.16.9898

ISSN

1083-351X

Autores

Helen Y. Wang, José Luís Zamorano, Achsah Keegan,

Tópico(s)

Immune Response and Inflammation

Resumo

The interleukin (IL)-4 receptor α-chain (IL-4Rα) contains a sequence motif (488PLVIAGNPAYRSFSD) termed the insulin IL-4 receptor motif (I4R motif). Mutation of the central Tyr497 to Phe blocks the tyrosine phosphorylation of the insulin receptor substrate 1 (IRS1) and diminishes proliferation in response to IL-4. Recent data suggest that the I4R motif encodes binding sites for several protein tyrosine binding (PTB) domain-containing proteins such as IRS1 and Shc and potentially for the Src homology 2 domain of signal transducer and activator of transcription 6 (STAT6). To analyze the function of the I4R motif in regulating IL-4 signaling, we changed conserved residues upstream and downstream of the central Tyr to Ala in the human IL-4Rα. We analyzed the ability of these constructs to signal the tyrosine phosphorylation of IRS2 and STAT6, the induction of DNA binding activity, and CD23 induction in response to human IL-4 (huIL-4) in transfected M12.4.1 cells. Mutagenesis of residues downstream of Tyr497, such as Arg498 or Phe500, to Ala had no effect on any of these responses, suggesting that the I4R motif may not be important for functional Src homology 2 domain interactions. However, mutagenesis of Pro488 to Ala (P488A) greatly diminished the tyrosine phosphorylation of IRS2 and abolished tyrosine phosphorylation of STAT6, induction of DNA binding activity, and CD23 induction in response to huIL-4. By contrast, a P488G mutant signaled these responses to huIL-4. Mutagenesis of hydrophobic amino acids previously shown to contact the PTB domain of IRS1, Leu489 or Ile491, to Ala had only minimal effects on responses to huIL-4. However, changing both Leu498 and Ile491 to Ala greatly diminished the tyrosine phosphorylation of IRS2 and abolished STAT6 activation. Taken together, these results indicate the important role of the I4R motif in regulating IRS docking and suggest that I4R docking to a PTB domain-containing protein regulates activation of the STAT6 pathway. The interleukin (IL)-4 receptor α-chain (IL-4Rα) contains a sequence motif (488PLVIAGNPAYRSFSD) termed the insulin IL-4 receptor motif (I4R motif). Mutation of the central Tyr497 to Phe blocks the tyrosine phosphorylation of the insulin receptor substrate 1 (IRS1) and diminishes proliferation in response to IL-4. Recent data suggest that the I4R motif encodes binding sites for several protein tyrosine binding (PTB) domain-containing proteins such as IRS1 and Shc and potentially for the Src homology 2 domain of signal transducer and activator of transcription 6 (STAT6). To analyze the function of the I4R motif in regulating IL-4 signaling, we changed conserved residues upstream and downstream of the central Tyr to Ala in the human IL-4Rα. We analyzed the ability of these constructs to signal the tyrosine phosphorylation of IRS2 and STAT6, the induction of DNA binding activity, and CD23 induction in response to human IL-4 (huIL-4) in transfected M12.4.1 cells. Mutagenesis of residues downstream of Tyr497, such as Arg498 or Phe500, to Ala had no effect on any of these responses, suggesting that the I4R motif may not be important for functional Src homology 2 domain interactions. However, mutagenesis of Pro488 to Ala (P488A) greatly diminished the tyrosine phosphorylation of IRS2 and abolished tyrosine phosphorylation of STAT6, induction of DNA binding activity, and CD23 induction in response to huIL-4. By contrast, a P488G mutant signaled these responses to huIL-4. Mutagenesis of hydrophobic amino acids previously shown to contact the PTB domain of IRS1, Leu489 or Ile491, to Ala had only minimal effects on responses to huIL-4. However, changing both Leu498 and Ile491 to Ala greatly diminished the tyrosine phosphorylation of IRS2 and abolished STAT6 activation. Taken together, these results indicate the important role of the I4R motif in regulating IRS docking and suggest that I4R docking to a PTB domain-containing protein regulates activation of the STAT6 pathway. Interleukin (IL)-4 1The abbreviations used are: IL, interleukin; IL-4Rα, IL-4 receptor α-chain; JAK, Janus family kinase; STAT, signal transducer and activator of transcription; IRS, insulin receptor substrate; I4R motif, insulin IL-4 receptor motif; PTB, protein tyrosine binding; γc, γ-chain; huIL-4, human IL-4; huIL-4Rα, human IL-4Rα; muIL-4, murine IL-4; SH2, Src homology 2; FACS, fluorescence-activated cell sorting; WT, wild type. evokes a wide variety of biological responses by binding to a high affinity receptor complex (1Paul W.E. Blood. 1991; 77: 1859-1870Crossref PubMed Google Scholar). In murine lymphoid cells, the receptor complex predominantly consists of a 140-kDa, high affinity binding chain (IL-4Rα) and the common γ-chain (γc) (2Keegan A.D. Ryan J.J. Paul W.E. Immunologist. 1996; 4: 194-198Google Scholar) that is also a component of the receptors for IL-2, IL-7, IL-9, and IL-15 (3Leonard W.J. Annu. Rev. Med. 1996; 47: 229-239Crossref PubMed Scopus (153) Google Scholar). Both chains of the IL-4 receptor complex are members of the hematopoietin receptor superfamily (4Cosman D. Cytokine. 1993; 5: 95-106Crossref PubMed Scopus (268) Google Scholar). These receptor subunits do not contain any consensus sequences encoding tyrosine or serine/threonine kinases. However, it has been shown that the IL-4Rα associates with the Janus family kinase JAK-1 (5Yin T. Tsang M.L. Yang Y-C. J. Biol. Chem. 1994; 269: 26614-26617Abstract Full Text PDF PubMed Google Scholar) and the γc associates with JAK-3 (6Miyazaki T. Kawahara A. Fujii H. Nakagawa Y. Minami Y. Lui Z-J. Oishi I. Silvennoinen O. Witthuhn B.A. Ihle J.N. Taniguchi T. Science. 1994; 266: 1045-1047Crossref PubMed Scopus (503) Google Scholar, 7Russell S.M. Johnston J.A. Noguchi M. Kawamura M. Bacon C.M. Friedmann M. Berg M. McVicar D.W. Witthuhn B.A. Silvennoinen O. Goldman A.S. Schmalstieg F.C. Ihler J.N. O'Shea J.J. Leonard W.J. Science. 1994; 266: 1042-1045Crossref PubMed Scopus (589) Google Scholar). Binding of IL-4 to its receptor results in the tyrosine phosphorylation of several molecules, including JAK1, JAK3, and the IL-4Rα (2Keegan A.D. Ryan J.J. Paul W.E. Immunologist. 1996; 4: 194-198Google Scholar). IL-4 treatment also results in the tyrosine phosphorylation of the insulin receptor substrate-1 (IRS1) and IRS2 (8Wang L-M. Keegan A.D. Li W. Lienhard G.E. Pacini S. Gutkind J.S. Myers Jr., M.G. Sun X.-J. White M.F. Aaronson S.A. Paul W.E. Pierce J.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4032-4036Crossref PubMed Scopus (168) Google Scholar, 9White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 10Sun X-J. Wang L-M. Zhang Y. Yanush L. Myers M.G. Glasheen E. Lane W.S. Pierce J.H. White M.F. Nature. 1995; 377: 173-177Crossref PubMed Scopus (764) Google Scholar). IRS1 and IRS2 are large cytoplasmic proteins (170–180 kDa) that contain numerous potential tyrosine and serine/threonine phosphorylation sites. Tyrosine-phosphorylated sites within IRS1 and IRS2 associate with SH2 domains found in cytoplasmic signaling molecules, including the p85 subunit of phosphatidylinositol 3-kinase and the growth factor receptor-bound protein 2. IRS1 and IRS2 have been shown to regulate both the proliferation and the protection from apoptosis of a factor-dependent myeloid cell line 32D in response to IL-4 (10Sun X-J. Wang L-M. Zhang Y. Yanush L. Myers M.G. Glasheen E. Lane W.S. Pierce J.H. White M.F. Nature. 1995; 377: 173-177Crossref PubMed Scopus (764) Google Scholar, 11Wang L-M. Myers Jr., M.G. Sun X-J. Aaronson S.A. White M. Pierce J.H. Science. 1993; 261: 1591-1594Crossref PubMed Scopus (370) Google Scholar, 12Zamorano J. Wang H.Y. Wang L-M. Pierce J.H. Keegan A.D. J. Immunol. 1996; 157: 4926-4934PubMed Google Scholar). Deletional studies of the IL-4Rα have led to the identification of a sequence motif (488PLX4NPXYRSFSD) termed the insulin IL-4 receptor motif (I4R motif) (13Keegan A.D. Nelms K. White M.F. Wang L-M. Pierce J.H. Paul W.E. Cell. 1994; 76: 811-820Abstract Full Text PDF PubMed Scopus (287) Google Scholar). Mutation of the central Tyr497 to Phe blocked the tyrosine phosphorylation of IRS1 and diminished proliferation in response to IL-4 in 32D cells. It has been shown that IRS1 contains a protein tyrosine binding (PTB) domain, also called a phosphotyrosine interaction domain (14Gustafson T.A. He W. Craparo A. Schaub C.D. O'Neill T.J. Mol. Cell. Biol. 1995; 15: 2500-2508Crossref PubMed Scopus (327) Google Scholar, 15Bork P. Margolis B. Cell. 1995; 80: 693-694Abstract Full Text PDF PubMed Scopus (172) Google Scholar, 16van der Geer P. Pawson T. Trends Biochem. Sci. 1995; 20: 277-280Abstract Full Text PDF PubMed Scopus (233) Google Scholar), which is important for the interaction of the I4R motif with IRS1. Several independent studies have demonstrated that the I4R motif encodes a binding site for the PTB domain of IRS1/2 and Shc (17Wolf G. Trub T Ottinger E. Groninga L. Lynch A. White M.F. Miyazaki M. Lee J. Shoelson S.E. J. Biol. Chem. 1995; 270: 27407-27410Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 18Nelms, K., Gustafson, T. A., and Paul, W. E. (1995) 9th International Congress of Immunology, San Francisco, CA, 1995,Abstr. 1837, American Association of Immunologists.Google Scholar). More recently, solution structure analyses of the binding of a phosphopeptide derived from the I4R motif of the huIL-4Rα with the PTB domain derived from IRS1 (19Zhou M.M. Huang B. Olejniczak E.T. Meadows R.P. Shuker S.B. Miyazaki M. Trub T. Shoelson S.E. Fesik S.W. Nat. Struct. Biol. 1996; 3: 388-393Crossref PubMed Scopus (107) Google Scholar) have indicated that amino acid residues at the −8 and −6-positions relative to Tyr497 in the I4R motif (Leu489 and Ile491) make contacts with residues in the PTB domain of IRS1. In addition to IRS family members, IL-4 also induces the tyrosine phosphorylation of a member of the signal transducer and activator of transcription (STAT) family, STAT6 (20Darnell J. Kerr I. Stark G. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5021) Google Scholar, 21Ihle J.N. Kerr I.M. Trends Genet. 1995; 11: 69-74Abstract Full Text PDF PubMed Scopus (821) Google Scholar, 22Hou J.U. Schindler U. Henzel W.J. Ho T-C. Brasseur M. McKnight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (726) Google Scholar, 23Quelle F.W. Shimoda K. Thierfelder W. Fischer C. Kim A. Ruben S.M. Cleveland J.L. Pierce J.H. Keegan A.D. Paul W.E. Ihle J.N. Mol. Cell. Biol. 1995; 15: 3336-3343Crossref PubMed Scopus (303) Google Scholar). The general model is that after tyrosine phosphorylation, STAT6 dimerizes in the cytoplasm, translocates to the nucleus, and subsequently binds to consensus sequences (termed γ-activated sequences) found within the promoter regions of IL-4-inducible genes. Recent studies of mice with a targeted disruption of the STAT6 gene clearly demonstrate that STAT6 is necessary for the induction of genes (CD23, major histocompatibility complexes II and Iε, and IL-4Rα genes) in response to IL-4 (24Kaplan M.H. Schindler U. Smiley S.T. Grusby M.J. Immunity. 1996; 4: 313-319Abstract Full Text Full Text PDF PubMed Scopus (1331) Google Scholar, 25Takeda K. Tanaka T. Shi W. Matsumoto M. Minami M. Kashiwamura S-I. Nakanii K. Yoshida N. Kishimoto T. Akira S. Nature. 1996; 380: 627-629Crossref PubMed Scopus (1267) Google Scholar, 26Shimoda K. Deursen J. Sangster M.Y. Sarawar S.R. Carson R.T. Tripp R.A. Chu C. Quelle F.W. Nosaka T. Vignali D.A.A. Doherty P.C. Grosveld G. Paul W.E. Ihle J.N. Nature. 1996; 380: 630-633Crossref PubMed Scopus (1106) Google Scholar). Several lines of evidence suggest that the IRS and STAT6 pathways are separate at the initiation phase of the signal. In 32D cells lacking IRS expression, IL-4 treatment was able to activate the DNA binding activity of STAT6 as well as in cells expressing IRS1 (27Pernis A. Witthuhn B. Keegan A.D. Nelms K. Garfein E. Ihle J.N. Paul W.E. Pierce J.H. Rothman P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7971-7975Crossref PubMed Scopus (82) Google Scholar). On the other hand, IL-4 was able to stimulate the tyrosine phosphorylation of IRS2 in lymphocytes derived from mice deficient in STAT6 expression as well as in lymphocytes derived from normal mice (28Wang H.Y. Zamorano J. Yoerkie J.L. Paul W, E. Keegan A.D. J. Immunol. 1997; 158: 1037-1040PubMed Google Scholar). A series of deletion, mutagenesis, and chimeric receptor studies of the huIL-4Rα (13Keegan A.D. Nelms K. White M.F. Wang L-M. Pierce J.H. Paul W.E. Cell. 1994; 76: 811-820Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 29Ryan J.J. McReynolds L.J. Wang L-H. Keegan A.D. Garfein E. Rothman P. Nelms K. Paul W.E. Immunity. 1996; 4: 123-132Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 30Wang H.Y. Paul W.E. Keegan A.D. Immunity. 1996; 4: 113-121Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) demonstrated that a region containing three tyrosine residues with a consensus sequence of GY(K/Q)XF 90 amino acids downstream of the I4R motif was necessary for maximal IL-4-induced activation of STAT6 DNA binding activity and CD23 induction in M12.4.1. They also indicated that STAT6 activation, in the absence of IRS activation, was sufficient to signal maximal CD23 induction in these cells. Furthermore, these same studies also suggested that Tyr497 in the I4R motif could signal partial STAT6 activation, implying that any one of the first four cytoplasmic tyrosine residues could potentially act as a docking site for the SH2 domain of STAT6. Interestingly, mutation of Tyr497 to Phe, known to consistently affect activation of the IRS pathway in response to IL-4 (13Keegan A.D. Nelms K. White M.F. Wang L-M. Pierce J.H. Paul W.E. Cell. 1994; 76: 811-820Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 29Ryan J.J. McReynolds L.J. Wang L-H. Keegan A.D. Garfein E. Rothman P. Nelms K. Paul W.E. Immunity. 1996; 4: 123-132Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), also affected activation of the STAT6 DNA binding activity in approximately half of expressing clones, while the others responded normally (27Pernis A. Witthuhn B. Keegan A.D. Nelms K. Garfein E. Ihle J.N. Paul W.E. Pierce J.H. Rothman P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7971-7975Crossref PubMed Scopus (82) Google Scholar, 31Ryan, J., Nelms, K., Keegan, A., McReynolds, L., Wang, L., and Paul, W. E. (1995) 9th International Congress of Immunology, San Francisco, CA, 1995, Abstr. 1838, American Association of Immunologists.Google Scholar). To analyze the function of the I4R motif in regulating IL-4 signaling in detail, we changed conserved residues upstream and downstream of the central Tyr to Ala. We analyzed the ability of these constructs to signal the tyrosine phosphorylation of IRS2 and STAT6, the induction of DNA binding activity, and CD23 induction in response to huIL-4 in transfected M12.4.1 cells. The results presented herein indicate the important role of the I4R motif in regulating IRS recruitment and suggest that I4R docking to a PTB domain-containing protein(s) regulates activation of the STAT6 pathway. The murine B cell lymphoma M12.4.1 (obtained from Dr. Richard Asofsky, NIAID, National Institutes of Health) was maintained in RPMI (BioWhittaker, Inc., Walkersville, MD) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm glutamine, and 1 × 10−7m 2-mercaptoethanol. Recombinant muIL-4 and recombinant huIL-4 were obtained from R & D Systems (Minneapolis, MN). For the mutagenesis, we cloned the huIL-4R cDNA (provided by Dr. Melanie Spriggs, Immunex, Seattle, WA) into the pAlter-1 vector obtained from Promega (Madison, WI). Amino acid numbering begins with +1 as the initiator methionine of the signal peptide of the huIL-4Rα cDNA. There are 25 amino acids in the signal peptide. Oligonucleotide-directed mutagenesis was performed according to the manufacturer's protocol using mutant oligonucleotides that would convert the wild type codons to an A codon (or as indicated a G). For example, the oligonucleotide used for the L489A mutation was designed as follows: 5′-ACA GAG ACG CCCGCC GTC ATC GCA GGC-3′ (L489A). We prepared single-stranded DNA containing the huIL-4Rα using helper phage R408. The mutant oligonucleotides were annealed with aliquots of the single-stranded DNA along with an oligonucleotide that repairs the mutation in the ampicillin resistance gene. Subsequently, synthesis and ligation were performed to link them. The DNA was transformed into a repair minus strain of Escherichia coli (BMH 71–18 mutS), and the cells were grown in the presence of ampicillin. A second round of transformation in JM109 and selection for ampicillin resistance was performed. Bacterial colonies containing the desired mutation were identified by sequence analysis of plasmid DNA. Mutant huIL-4Rα was then cloned into the EcoRI site of pME18s. M12.4.1 cells were washed and resuspended in phosphate-buffered saline. For each transfection, 2 × 107 cells were mixed with 2 μg of vector carrying neomycin resistance and 20 μg of huIL-4Rα-pME18s and subjected to electroporation using a Bio-Rad gene pulser set on 200 V and 960 microfarads. After transfection, the cells were cultured overnight before selection with 800 μg/ml neomycin (Life Technologies, Inc.). Neomycin-resistant cells were tested for the expression of huIL-4Rα by FACS analysis using monoclonal antibodies, M8 and M10 (a generous gift from Dr. Melanie Spriggs, Immunex, Seattle, WA) as described previously (13Keegan A.D. Nelms K. White M.F. Wang L-M. Pierce J.H. Paul W.E. Cell. 1994; 76: 811-820Abstract Full Text PDF PubMed Scopus (287) Google Scholar). Analysis of phosphotyrosine-containing proteins was performed as described previously (32Keegan A.D. Beckmann M.P. Park L.S. Paul W.E. J. Immunol. 1991; 146: 2272-2279PubMed Google Scholar). Briefly, cells were deprived of serum in RPMI for 2 h at 37 °C. After washing, 107 cells were resuspended in RPMI with 50 μmNa3VO4 and incubated in the presence or absence of murine or human IL-4 (10 ng/ml) for 10 min at room temperature. The reaction was terminated by 10-fold dilution in ice-cold phosphate-buffered saline containing 100 μmNa3VO4. Cell pellets were lysed in HEPES lysis buffer (50 mm HEPES, 50 mm NaCl, 0.5% Nonidet P-40, 1 mm Na3VO4, 50 mm NaF, 10 mm pyrophosphate, 1 mmphenylmethylsulfonyl fluoride, and protease inhibitor mixture) and clarified. The soluble fraction was immunoprecipitated with a polyclonal rabbit anti-IRS (a generous gift of Drs. Ling-Mei Wang and Jacalyn Pierce, NCI, National Institutes of Health), anti-STAT6 (Santa Cruz, CA), or anti-JAK3 (Upstate Biotechnology, Inc., Lake Placid, NY). The precipitates were washed in lysis buffer and solubilized in SDS sample buffer. The samples were separated on 7.5% SDS-polyacrylamide gels before transfer to a polyvinylidene difluoride membrane. The membranes were then probed with a monoclonal anti-phosphotyrosine antibody, RC20-H (Transduction Laboratories, Lexington, KY). The bound antibody was detected using enhanced chemiluminescence (Amersham Pharmacia Biotech). Where indicated, the blots were stripped and probed with control antibodies. Band intensities were analyzed using the public domain software NIH Image. M12.4.1 cells expressing huIL-4Rα constructs were incubated with media, 10 ng/ml muIL-4, or 10 ng/ml huIL-4 as indicated for 30 min and washed with phosphate-buffered saline. Total cell extracts were prepared exactly as described (29Ryan J.J. McReynolds L.J. Wang L-H. Keegan A.D. Garfein E. Rothman P. Nelms K. Paul W.E. Immunity. 1996; 4: 123-132Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) and stored at −70 until use. Extracts (4 μg) were incubated with 1 ng of labeled double-stranded oligonucleotide (5 × 105 cpm) corresponding to the N4 γ-activated sequence element found in the promoter of the Cε gene (5′-CAACTTCCCAAGAACAGA) at room temperature for 20 min as described previously (29Ryan J.J. McReynolds L.J. Wang L-H. Keegan A.D. Garfein E. Rothman P. Nelms K. Paul W.E. Immunity. 1996; 4: 123-132Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Where indicated, unlabeled Cε probe, anti-STAT3, or anti-STAT6 (1 μg, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were included in the binding reaction. Protein-DNA samples were analyzed by electrophoresis on 4.5% polyacrylamide gels in 0.22× TBE followed by autoradiography. M12.4.1 cells expressing the various constructs of the huIL-4Rα were incubated at 1 × 105/ml in media in the presence or absence of murine or human IL-4 (10 ng/ml) for 48 h at 37 °C. Expression of murine CD23 was tested by FACS analysis using fluorescein isothiocyanate-B3B4 (anti-murine CD23), a generous gift of Dr. Daniel H. Conrad (MCV, Richmond, VA), in the presence of the anti-Fc receptor antibody 2.4G2 to block Fc binding before analysis on a FACScan (Becton-Dickinson). For binding and cross-linking studies, 125I-huIL-4 was purchased from Amersham Pharmacia Biotech. Saturation binding analyses were done using 25 ng/ml 125I-huIL-4 essentially as described previously (13Keegan A.D. Nelms K. White M.F. Wang L-M. Pierce J.H. Paul W.E. Cell. 1994; 76: 811-820Abstract Full Text PDF PubMed Scopus (287) Google Scholar). 125I-huIL-4 was cross-linked to the IL-4 receptor complex using 3 mmbis(sulfosuccidinimidyl)suberate (Pierce) as described previously (32Keegan A.D. Beckmann M.P. Park L.S. Paul W.E. J. Immunol. 1991; 146: 2272-2279PubMed Google Scholar). Previous studies have shown that the central tyrosine in the I4R motif (Tyr497) of the huIL-4Rα is critical for the recruitment of IRS1 to the IL-4 receptor complex and the activation of the IRS pathway. These studies also suggested that Tyr497has some capacity to recruit STAT6 and to influence the activation of the STAT6 pathway. Residues downstream of phosphotyrosines participate in the binding to SH2 domains, especially the +1 and +3 residues (33Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2228) Google Scholar). It has recently become clear that residues upstream of phosphotyrosines participate in the binding to PTB domains (19Zhou M.M. Huang B. Olejniczak E.T. Meadows R.P. Shuker S.B. Miyazaki M. Trub T. Shoelson S.E. Fesik S.W. Nat. Struct. Biol. 1996; 3: 388-393Crossref PubMed Scopus (107) Google Scholar, 33Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2228) Google Scholar). Therefore, we changed conserved residues upstream and downstream of the central tyrosine, those likely to participate in PTB or SH2 domain binding, respectively, to alanine (Fig. 1) to further understand the role of the I4R motif of the huIL-4Rα. We transfected these huIL-4Rα constructs into the murine B lymphoma M12.4.1 and screened for stable expression by FACS using monoclonal anti-huIL-4Rα (data not shown). Receptor expression was verified using 125I-huIL-4 binding assays. All clones demonstrated similar levels of expression of the transfected huIL-4Rα ranging from 1000 to 4000 molecules/cell. We analyzed the ability of these mutant receptors to transmit signals in response to huIL-4. Since M12.4.1 cells express endogenous muIL-4 receptors and human and murine IL-4 do not cross-bind, we used muIL-4 as a positive control for each transfectant. For each of the responses assayed, we show representative results for each construct; at least three independent clones expressing the indicated constructs gave comparable results. We first analyzed the ability of these mutant receptors to signal the activation of the IRS pathway in response to huIL-4 by analyzing the induction of tyrosine phosphorylation of IRS2 (Fig. 2). All M12.4.1 cells demonstrated the tyrosine phsophorylation of IRS2 in response to muIL-4. M12.4.1 cells expressing the wild type (WT) huIL-4Rα demonstrated potent tyrosine phosphorylation of IRS2 in response to human IL-4, as expected (29Ryan J.J. McReynolds L.J. Wang L-H. Keegan A.D. Garfein E. Rothman P. Nelms K. Paul W.E. Immunity. 1996; 4: 123-132Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Cells expressing receptors with mutations downstream of Tyr497, such as R498A and F500A, also responded to huIL-4 treatment with potent tyrosine phosphorylation of IRS2 comparable with the levels seen in muIL-4-treated cells. However, cells expressing the P488A mutant showed greatly diminished tyrosine phosphorylation of IRS2 to levels that were <10% of the signal seen in WT-expressing cells in response to huIL-4 or of the signal elicited by the muIL-4 control. Proline residues can induce bends or kinks in protein structure, and Pro488 is just upstream to the hydrophobic residues shown to interact with residues found in a hydrophobic pocket of the PTB domain of IRS1 (19Zhou M.M. Huang B. Olejniczak E.T. Meadows R.P. Shuker S.B. Miyazaki M. Trub T. Shoelson S.E. Fesik S.W. Nat. Struct. Biol. 1996; 3: 388-393Crossref PubMed Scopus (107) Google Scholar) (Fig. 1). To determine whether Pro488might allow the downstream residues of the I4R motif to recruit and activate IRS2 in cells, we changed this residue to a glycine, an amino acid without a side chain, thus allowing protein flexibility. Cells expressing the P488G mutant demonstrated significant tyrosine phosphorylation of IRS2 in response to huIL-4, although slightly reduced compared with the response elicited by muIL-4, suggesting that Pro488 participates in IRS recruitment by regulating the availability of the downstream I4R motif residues to interact with the PTB domain. Interestingly, cells expressing huIL-4Rα with mutations in either of the hydrophobic residues shown to contact the PTB domain of IRS1, Leu498 or Ile491, demonstrated significant tyrosine phosphorylation of IRS2 in response to huIL-4, although the L498A-expressing cells showed a slightly diminished level (∼60% of signal elicited of endogenous muIL-4). However, cells expressing a construct with both Leu489 and Ile491 changed to Ala (L,I-A) showed greatly diminished tyrosine phosphorylation of IRS2 (<10% of WT) in response to huIL-4. These results suggest that these hydrophobic residues participate in the recruitment of IRS2, as shown in the solution structure analysis (19Zhou M.M. Huang B. Olejniczak E.T. Meadows R.P. Shuker S.B. Miyazaki M. Trub T. Shoelson S.E. Fesik S.W. Nat. Struct. Biol. 1996; 3: 388-393Crossref PubMed Scopus (107) Google Scholar), and that expression of either Leu489 or Ile491 alone is sufficient to activate the IRS2 pathway. We next examined the ability of huIL-4 to activate the STAT6 pathway and CD23 induction in M12.4.1 cells expressing the huIL-4Rα mutants (Figs. 3 and4). Treatment of cells expressing WT huIL-4Rα with human or mouse IL-4 induced a DNA binding activity specific for the Cε probe, indicating the activation of STAT6 (Fig. 3 A). The IL-4-induced complex can often be resolved into two bands, 2H. Wang, J. Zamorano, and A. D. Keegan, unpublished observations. where at other times the two bands are not distinguished. This DNA-binding complex was supershifted by anti-STAT6 antibody but not by anti-STAT3. Interestingly, when we analyzed the cells expressing the mutant constructs, we found that the pattern of responsiveness to huIL-4 was similar to the pattern we observed for IRS2 tyrosine phosphorylation (Fig. 3 B); i.e. cells expressing the P488A mutant or the L,I-A mutant did not demonstrate STAT6 DNA binding activity in response to huIL-4, while they clearly responded to muIL-4. Cells expressing the P488G, L489A, I491A, and F500A mutants responded to huIL-4 by inducing STAT6 DNA binding activity. In keeping with these results, the pattern of CD23 induction in response to huIL-4 also corresponded to the pattern we observed for STAT6 DNA binding activity (Fig. 4). Treatment of cell lines expressing WT, P488G, L489A, I491A, R498A, and F500A with huIL-4 resulted in CD23 induction to levels seen in cells treated with the muIL-4 control. However, treatment of cells expressing P488A or L,I-A with huIL-4 did not result in the induction of CD23, while treatment with muIL-4 was able to induce its expression.Figure 4CD23 induction. M12.4.1 cells expressing the indicated mutations of the I4R motif were incubated with media (dotted line), 10 ng/ml muIL-4 (dashed line), or 10 ng/ml huIL-4 (solid line) for 48 h. CD23 expression was analyzed by FACS using fluorescein isothiocyanate-anti-CD23.View Large Image Figure ViewerDownload (PPT) These results indicate that residues within the I4R motif that are likely to be important for the interaction with PTB domains also participate in the regulation of the STAT6/gene induction pathway. It is possible that the I4R motif serves to regulate the activation of an additional signaling pathway, such as a serine kinase (34Boulton T.G. Zhong Z. Wen Z. Darnell Jr., J.E. Stahl N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6915-6919Crossref PubMed Scopus (192) Google Scholar, 35Zhang X. Blenis J. Li H.-C. Schindler C. Chen-Kiang S. Science. 1995; 267: 1990-1994Crossref PubMed Scopus (523) Google Scholar), which would further modulate STAT6 DNA binding activity. On the other hand, the I4R motif could regulate the tyrosine phosphorylation of STAT6 itself. Therefore, we examined the ability of huIL-4 to induce the tyrosine phosphorylation of STAT6 (Figs.5 and 6). Again, the pattern of responsiveness to huIL-4 corresponded to the pattern we observed for STAT6 DNA binding activity. Treatment of cell lines expressing WT, P488G, L489A, I491A, R498A, and F500A with huIL-4 resulted in the tyrosine phosphorylation of STAT6 to levels seen in cells treated with the muIL-4 control. However, treatment of cells expressing P488A or L,I-A with huIL-4 did not result in the induction of STAT6 tyrosine phosphorylation, while treatment with muIL-4 was able to do so. To det

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