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  • br Acknowledgements We thank Jeffrey D Konowalchuk and John


    Acknowledgements We thank Jeffrey D. Konowalchuk and John Sony Robbins for their technical assistance. This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) grants to DRB (RGPIN-2013-355303) and MB (RGPIN-2014-96395). AMR was supported by NSERC Vanier Doctoral Scholarship and a University of Alberta Dissertation Fellowship. JJH was supported by an NSERC PGS-M, a University of Alberta teaching assistantship, and an NSERC Vanier Doctoral Scholarship.
    Introduction The colony-stimulating factor-1 receptor (CSF-1R) is a cell-surface glycoprotein encoded by the c-fms proto-oncogene in humans [1], [2]. Its ligand CSF-1, also named macrophage-colony stimulating factor (M-CSF), is the primary regulator of the survival, proliferation and differentiation of mononuclear phagocytes, such as macrophages, osteoclasts, microglia, and cells of the female reproductive tract [3], [4], [5], [6]. Recently, interleukin-34 (IL-34) was identified as an alternative ligand of CSF-1R, although it shares no apparent sequence homology with CSF-1 [7]. IL-34 acts as a regulator of myeloid lineage differentiation, proliferation and survival, and triggers phosphorylation of CSF-1R and ERK1/2 that may also be induced by CSF-1, indicating that IL-34 and CSF-1 generate signals that at least partially overlap [7], [8], [9]. However, since different anti-CSF-1R monoclonal cox 2 inhibitors demonstrated different blocking effects on CSF-1R signaling, it was suggested that IL-34 and CSF-1 are not identical in biological activity and signal activation kinetics, and that macrophage phenotype and function are differentially regulated by the two ligands [10]. The different spatiotemporal expression patterns of IL-34 and CSF-1 also suggests that these ligands have complementary rather than identical functions [8]. CSF-1R is a member of the class III receptor tyrosine kinases (RTKs), which also include KIT (the receptor for Stem Cell Factor, or SCF), Flt3 (the receptor for Flt3 Ligand, or Flt3L), PDGFRα and PDGFRβ (the receptors for Platelet-Derived Growth Factors, or PDGFs) [11]. The class III RTKs are composed of a glycosylated extracellular region comprising five immunoglobulin(Ig)-like domains, a single transmembrane segment, and a split intracellular kinase domain. The structures of SCF:KIT, PDGF-B: PDGFRβ, CSF-1:CSF-1R, and Flt3L:Flt3 complexes have indicated that the N-terminal three Ig-like domains of these receptors are responsible for ligand recognition, and the membrane-proximal domains are responsible for complex stabilization and accurate positioning which are required for activation [12], [13], [14], [15], [16]. Both KIT and Flt3 have only one ligand, but akin to CSF-1R, each PDGFR can recognize multiple PDGF ligands [17]. Given the lack of homology between IL-34 and CSF-1, it has been puzzling how receptor sharing is achieved for these two CSF-1R ligands. Comparative co-evolution analysis with all vertebrate genes suggests that the two ligands interact with distinct regions of the CSF-1R [18], but it is unclear how this paradigm is compatible with overlapping but distinct functions of IL-34 and CSF-1. Here, we report the crystal structures of IL-34 alone and in complex with the ligand recognition domains of CSF-1R. Combined with thermodynamic and functional data, our studies reveal a basis for CSF-1R cross-reactivity as aided by flexibility in both conformation and recognition chemistry, which may explain the differentiated functional outcomes of the two ligands, IL-34 and CSF-1.
    Materials and methods
    Acknowledgements We thank P.J. Focia and Z. Wawrzak for support in data collection and S. Gomes for support in generating IL-34 mutant clones. X.H. is supported by the NIH grant 1R01GM078055. The Structural Biology Facility is supported by the R.H. Lurie Comprehensive Cancer Center of Northwestern University. Data were measured at the LS-CAT beamline 21-ID-D at the Advanced Photon Source (APS), Argonne, IL.