Importance of the Membrane-Proximal Extracellular Domains for Activation of the Signal Transducer Glycoprotein 130 This information is current as Ingo Kurth, Ursula Horsten, Stefan Pflanz, Andreas of September 28, 2021. Timmermann, Andrea Küster, Heike Dahmen, Ingrid Tacken, Peter C. Heinrich and Gerhard Müller-Newen J Immunol 2000; 164:273-282; ; doi: 10.4049/jimmunol.164.1.273 http://www.jimmunol.org/content/164/1/273 Downloaded from References This article cites 40 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/164/1/273.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 28, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Importance of the Membrane-Proximal Extracellular Domains for Activation of the Signal Transducer Glycoprotein 1301 Ingo Kurth, Ursula Horsten,2 Stefan Pflanz, Andreas Timmermann, Andrea Ku¨ster, Heike Dahmen, Ingrid Tacken, Peter C. Heinrich,3 and Gerhard Mu¨ller-Newen The transmembrane glycoprotein gp130 is the common signal transducing receptor subunit of the IL-6-type cytokines. The gp130 extracellular part is predicted to consist of six individual domains. Whereas the role of the three membrane-distal domains (D1–D3) in binding of IL-6 and IL-11 is well established, the function of the membrane-proximal domains (D4–D6) is unclear. Mapping of a neutralizing mAb to the membrane-proximal part of gp130 suggests a functional role of D4–D6 in receptor activation. Individual deletion of these three domains differentially interferes with ligand binding of the soluble and membrane- bound receptors. All deletion mutants do not signal in response to IL-6 and IL-11. The deletion mutants D4 and, to a lesser extent, D6 are still activated by agonistic monoclonal gp130 Abs, whereas the deletion mutant D5 does not respond. Because membrane- Downloaded from bound D5 binds IL-6/soluble IL-6R as does wild-type gp130, but does not transduce a signal in response to various stimuli, this domain plays a prominent role in coupling of ligand binding and signal transduction. Replacement of the fifth domain of gp130 by the corresponding domain of the homologous G-CSF receptor leads to constitutive activation of the chimera upon overex- pression in COS-7 cells. In HepG2 cells this mutant responds to IL-6 comparable to wild-type gp130. Our findings suggest a functional role of the membrane-proximal domains of gp130 in receptor activation. Thus, within the hematopoietic receptor family the mechanism of receptor activation critically depends on the architecture of the receptor ectodomain. The Journal of Immu- http://www.jimmunol.org/ nology, 2000, 164: 273–282. he coordination and regulation of immune responses is Subsequently, the activated Jaks phosphorylate tyrosine residues mainly mediated by cytokines that specifically bind to cell of the receptor. These phosphotyrosine residues are docking sites T surface receptors. Cytokine receptors are usually classi- for latent transcription factors of the STAT family, which also fied due to structural similarities. Class I cytokine receptors are become phosphorylated at the receptor. The phosphorylated characterized by the presence of at least one cytokine binding STATs dimerize and translocate into the nucleus to regulate target module (CBM)4 that consists of two fibronectin-type III-like gene expression (2). by guest on September 28, 2021 (FNIII) domains. The N-terminal domain contains a set of four Whereas the ectodomains of the receptors for growth hormone conserved cysteine residues, and the C-terminal domain contains a (GH), erythropoietin (Epo), or prolactin solely consist of a single WSXWS motif or a closely related sequence. Receptors belonging CBM, other members of the class I cytokine receptor family, such to this family are engaged by helical cytokines consisting of four as gp130, G-CSFR, or the leptin receptor show a more complex tightly packed a-helices (1). The cytoplasmic parts of cytokine architecture, which in respect to their biological functions is not receptors do not contain a kinase domain, but constitutively asso- understood (3). The glycoprotein gp130 is the common signal ciate with Janus tyrosine kinases (Jaks). Binding of the ligand transducing receptor subunit of the IL-6-type cytokines IL-6, IL- leads to receptor dimerization and juxtaposition of the associated 11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor Jaks, which results in their activation by tyrosine phosphorylation. (CNTF), oncostatin M (OSM), and cardiotrophin-1 (4). The extra- cellular part of gp130 contains an Ig-like domain (D1) followed by a single CBM (D2 and D3) and three FNIII domains (D4, D5, and Institut fu¨r Biochemie, Rheinisch-Westfa¨lische Technische Hochschule Aachen, Aachen, Germany D6) (5). Signal transduction is achieved by either homodimeriza- tion of gp130 in response to IL-6 (6) and IL-11 (7) or heterodimer- Received for publication April 15, 1999. Accepted for publication October 8, 1999. ization of gp130 with the LIFR in response to LIF (8), CNTF (9), The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance OSM (8), or CT1 (10). Alternatively, OSM induces the het- with 18 U.S.C. Section 1734 solely to indicate this fact. erodimerization of gp130 with the recently cloned OSMR (11). 1 This work was supported by grants from the Deutsche Forschungsgemeinschaft The cytokines IL-6, IL-11, and CNTF alone do not efficiently en- (Bonn, Germany) and the Fonds der Chemischen Industrie (Frankfurt am Main, gage the signal transducing receptor chains. They first bind to their Germany). specific a receptors that can functionally be replaced by the re- 2 Current address: Institut fu¨r Experimentelle und Klinische Pharmakologie und Tox- ikologie der Universita¨t Erlangen-Nu¨rnberg, Universitätsstrasse 22, D-91054 Erlan- spective soluble counterparts lacking the transmembrane and cy- gen, Germany. toplasmic regions (12). In addition to at least one CBM, all recep- 3 Address correspondence and reprint requests to Dr. Peter C. Heinrich, Institut fu¨r tors involved in IL-6-type cytokine signaling contain an Ig-like Biochemie, Rheinisch-Westfa¨lische Technische Hochschule Aachen, Pauwelsstrasse domain located N-terminally of the most membrane-proximal 30, D-52057 Aachen, Germany. E-mail address: [email protected] CBM. Besides the Ig-like domain and CBM(s), those receptors 4 Abbreviations used in this paper: CBM, cytokine binding module; FNIII, fibronectin type III-like; Jak, Janus kinase; GH, growth hormone; GHR, GH receptor; Epo, eryth- triggering the cytoplasmic signal transduction cascade (gp130, ropoietin; EpoR, Epo receptor; G-CSFR, G-CSF receptor; LIF, leukemia inhibitory LIFR, and OSMR) all contain three additional membrane-proximal factor; LIFR, LIF receptor; CNTF, ciliary neurotrophic factor; OSM, oncostatin M; OSMR, OSM receptor; SIE, sis-inducible element; sIL-6, soluble IL-6; sgp130, sol- FNIII domains whose function is still unknown. Mutagenesis stud- uble gp130. ies using deletion mutants of gp130 revealed that the CBM of Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 274 MEMBRANE-PROXIMAL DOMAINS OF gp130 gp130 as well as the Ig-like domain are required for the interaction plasmids were sequenced using an ABI Prism Automated sequencer (Per- with IL-6/IL-6R (13–15), IL-11/IL-11R (16), and OSM (17). A kin-Elmer, Norwalk, CT). For expression in HepG2 cells, the cDNAs were more detailed analysis of the binding epitope revealed that the subcloned into the expression vector pRcCMV. To express the deletion constructs and chimera in the context of CBM of gp130 binds the ligand in a way similar to the GH/GHR gp130g, respective cDNAs were subcloned into the vector pSVLEg-Yg440 interaction (15, 18). These findings were supported by the crystal (27) using the restriction endonucleases XhoI and BstEII. The resulting structure of the gp130 CBM (19) as well as the solution structure vector encodes the extracellular domain, the transmembrane, and a trun- of gp130-D3 (20). cated intracellular part of gp130 (containing boxes 1 and 2) fused to the IFN-g receptor motif YDKPH, which predominantly activates STAT1. For Ligand-induced dimerization of cytokine receptors is widely ac- construction of plasmids encoding the soluble gp130 proteins the deletion cepted as a prerequisite for receptor activation (21, 22). In this constructs were subcloned into a vector (pSVLsgp130-Flag) (18) encoding respect, the functional role of the membrane-proximal domains of the extracellular domain of gp130 fused to a Flag epitope at the C-terminus. gp130 (D4–D6) is not understood. Because these three domains represent a common structural feature of the signal transducing Transfection of cells IL-6-type cytokine receptors, D4–D6 of gp130 may be essential Plasmid DNA was transfected into Ba/F3 cells by electroporation. Twenty- for receptor activation. The studies presented here using deletion eight micrograms of the gp130 expression vector were coelectroporated m 3 6 mutants of gp130 reveal that the membrane-proximal domains with 2 g of pSV2neo into 3.5 10 cells in 0.8 ml of medium applying a single 70-ms pulse at 200 V.