Biochem. J. (2010) 426, 307–317 (Printed in Great Britain) doi:10.1042/BJ20091745 307 The Ig-like domain of human GM-CSF receptor α plays a critical role in cytokine binding and receptor activation Shamaruh MIRZA*, Andrew WALKER*, Jinglong CHEN*, James M. MURPHY† 1 and Ian G. YOUNG* 1 *Structural Biology Program, John Curtin School of Medical Research, Australian National University, Canberra, ACT 0200, Australia, and †Molecular Medicine Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia GM-CSF (granulocyte/macrophage colony-stimulating factor) is an important mediator of inducible haemopoiesis and inflammation, and has a critical role in the function of alveolar macrophages. Its clinical applications include the mobilization of haemopoietic progenitors, and a role as an immune stimulant and vaccine adjuvant in cancer patients. GM-CSF signals via a specific α receptor (GM-CSFRα) and the shared hβ c (human common β -subunit). The present study has investigated the role of the Ig-like domain of GM-CSFRα in GM-CSF binding and signalling. Deletion of the Ig-like domain abolished direct GM- CSF binding and decreased growth signalling in the presence of hβ c. To locate the specific residues in the Ig-like domain of GM- CSFRα involved in GM-CSF binding, a structural alignment was made with a related receptor, IL-13Rα1 (interleukin-13 receptor α1), whose structure and mode of interaction with its ligand has recently been elucidated. Mutagenesis of candidate residues in the predicted region of interaction identified Val 51 and Cys 60 as having critical roles in binding to the α receptor, with Arg 54 and Leu 55 also being important. High-affinity binding in the presence of hβ c was strongly affected by mutation of Cys 60 and was also reduced by mutation of Val 51 , Arg 54 and Leu 55 . Of the four key residues, growth signalling was most severely affected by mutation of Cys 60 . The results indicate a previously unrecognized role for the Ig-like domain, and in particular Cys 60 , of GM-CSFRα in the binding of GM-CSF and subsequent activation of cellular signalling. Key words: common β -subunit (β c), cytokine receptor, cytokine-receptor homology module (CRM), dissociation constant, granulocyte/macrophage colony-stimulating factor (GM-CSF), immunoglobulin-like domain, interleukin-3 (IL-3), interleukin-5 (IL-5). INTRODUCTION GM-CSF (granulocyte/macrophage colony-stimulating factor), interleukin (IL)-5 and IL-3 are three cytokines produced by activated T-cells during immune responses that are important mediators of inducible haemopoiesis and inflammation. They signal through a shared receptor {hβ c [human β c (common β -subunit)]} and play an important role in the pathogenesis of allergic disorders and inflammatory diseases of the lung, such as asthma. Eosinophilia is controlled primarily by IL-5 [1,2] and, to a lesser extent, by IL-3 and GM-CSF. IL-3 plays a critical role in basophil and mast cell responses in parasite infections [3]. GM- CSF is believed to be centrally involved in chronic inflammatory diseases, such as arthritis and multiple sclerosis [4]. Of the three cytokines, GM-CSF has received the most attention in terms of clinical applications. GM-CSF has been applied to the supportive care of cancer patients and, more recently, to the mobilization of progenitors. The ability of GM-CSF to stimulate cytotoxic immune responses gives it great potential as an immune stimulant and vaccine adjuvant in cancer patients [4]. GM-CSF is also an essential factor in maintaining alveolar homoeostasis through its action on alveolar macrophages, and mutations affecting GM-CSFRα (GM-CSF receptor α subunit) have recently been detected in patients with pulmonary alveolar proteinosis [5]. A critical first step in GM-CSF signalling involves its binding to the specific GM-CSFRα. In the present study, we have demonstrated an important role of the first domain of GM-CSFRα in binding GM-CSF, an aspect that has not been studied previously. The receptors for IL-5, IL-3 and GM-CSF consist of cytokine- specific α receptors essential to the activation of hβ c, which is believed to be the main signalling entity [6–9]. Human GM-CSF and IL-3 bind to their cognate α receptors with low affinities (1–50 nM) but, in the presence of hβ c, high-affinity complexes are formed (30–200 pM). By convention, the terms ‘low’ and ‘high’ affinity are used to distinguish the two receptor-binding modes in studies of cytokine–receptor interactions, even though in many areas of biochemistry the nanomolar affinity associated with ‘low’-affinity binding would be considered very high. The formation of a complex involving ligand, α and β c receptors is necessary for receptor activation and signalling. The cytoplasmic portions of α and β c subunits possess no intrinsic tyrosine kinase activity [10], but the activated receptor complexes formed with all three ligands interact with and activate JAK2 (Janus kinase 2) [11], leading to the phosphorylation of eight tyrosine residues located in the β c cytoplasmic domain [12,13]. Subsequently, several signalling pathways are induced [14]. Structurally, the α and β c subunits of hGM-CSFR (human GM- CSFR), hIL-3R (human IL-3 receptor) and hIL-5R (human IL- 5 receptor) belong to the cytokine class I receptor superfamily (or haemopoietin receptor family), which includes GHR [GH (growth hormone) receptor], the EPO (erythropoietin) receptor, gp130 and IL-4R (IL-4 receptor)/IL-13R (IL-13 receptor). The characteristic feature of this family is the extracellular cytokine- receptor homology module (CRM or CRH), composed of two fibronectin III domains that contain a number of conserved sequence elements (reviewed in [15]). Crystal structural analyses Abbreviations used: (h)βc, (human) common β-subunit; CRM, cytokine-receptor homology module; EPO, erythropoietin; FBS, fetal bovine serum; GH, growth hormone; GHR, GH receptor; (h)GM-CSF, (human) granulocyte/macrophage colony-stimulating factor; (h)GM-CSFR, (human) GM-CSF receptor; IL, interleukin; IL-3R, IL-3 receptor; (h)IL-5R, (human) IL-5 receptor; IL-6R, IL-6 receptor; (h)IL-13R, (human) IL-13 receptor; D1, the N-terminal Ig-like domain of IL-13Rα1, GM-CSFRα, IL-5Rα or IL-3Rα, α subunits of GM-CSFR, IL-5R or IL-3R respectively; K d , dissociation constant; mIL-2, murine IL-2. 1 Correspondence may be addressed to either of these authors (email jamesm@wehi.edu.au or Ian.Young@anu.edu.au). c  The Authors Journal compilation c  2010 Biochemical Society www.biochemj.org Biochemical Journal