The b Common (bc) Family of Cytokines, Receptors and Ligands Prof. Angel Lopez and Dr. Timothy Hercus

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1 The b ommon () Family of ytokines, The b ommon () Family of ytokines, 1 Division of Human Immunology entre for ancer Biology S Pathology delaide, ustralia The βc cytokines 2 Beta ommon (βc) is the shared receptor subunit for a family of cytokines Granulocyte-Macrophage olony Stimulating Factor () Interleukin-3 (IL-3) Interleukin-5 (IL-5) Multiple, overlapping & redundant biological functions Survival, proliferation, differentiation, activation of myeloid cells Typically display considerable functional redundancy ytokines important for reactive hemopoiesis rather than steady-state hemopoiesis n exception is IL-5; Lack of it causes a decrease in eosinophils onserved receptor organization and signal transduction, IL-3 and IL-5 functions Mast cell Survival Stem ell Hemopoietic progenitor ommitted progenitors Basophil Eosinophil Neutrophil dhesion Migration Phagocytosis Degranulation Monocyte O 2 radicals Macrophage Dendritic cell Survival 3 Proliferation Differentiation Effector function 1

2 The b ommon () Family of ytokines,, IL-3 and IL-5 cell targets Basophil Monocyte Macrophage IL-3 Mast cell IL-5 Dendritic cell 4 Eosinophil Neutrophil, IL-3 and IL-5 in disease 5 ytokines important for "reactive hemopoiesis rather than "steady-state hemopoiesis IL-3 IL-5 Implicated in leukaemia; JMML, MML, ML, ML Pro-inflammatory cytokine; Rheumatoid arthritis, sthma, OPD, EE, rohn s disease Essential role in alveolar macrophage function, PP ML, autocrine production Receptor alpha chain over-expressed on leukaemic stem cells Hypereosinophilic syndrome llergic inflammation; Rhinitis, Polyposis, sthma, IL-3 and IL-5 expression is produced by many cell types: Macrophages, T-cells, endothelial cells, fibroblasts, mast cells IL-3 is usually expressed by T-cells Other sources include macrophages, mast cells, basophils IL-5 is primarily expressed by T-cells Other sources include mast cells and eosinophils 6 2

3 The b ommon () Family of ytokines,, IL-3 and IL-5 structure Type I cytokines, short chain a-helix bundle glycoproteins 127 aa IL aa IL aa, dimer D N' D B D B N' ' B ' N' ' D B 7 Human, 2.4Å crystal structure; Rozwarski D.. et al., 1996 Proteins 26, p304 Human IL-3, S variant, NMR structure; Feng. et al., 1996 J..Mol..Biol. 259, p524 Human IL-5, 2.4Å crystal structure; Milburn M.V. et al., 1993 Nature 363, p172 The, IL-3 and IL-5 receptors Heterodimeric receptors ytokine specific alpha chain, binds ligand with low affinity ommon b chain (), provides affinity conversion, signal transduction Non-tyrosine kinase receptor Low level of receptor expression, typically 200 1,000 receptors per cell IL-3 IL-5 IL3Ra IL5Ra Rα Kd nM 2-10nM 0.2-1nM Kd 100pM 8 Low affinity binding No signal No binding High affinity binding Biological signal Heterodimeric cytokine receptors ytokine families with shared receptor subunits family gp130 family gc family 9 For further information: Wang X. et al., 2009 nnu. Rev. Immunol. 27, p29 3

4 The b ommon () Family of ytokines,, IL-3 and IL-5 receptors, alpha subunit N-terminal immunoglobulin-domain Glycoproteins, 4-11 N-linked glycosylation sites Two extracellular fibronectin type III domains Short intracellular domain, essential for function RM N' I I I I I I I II I I N-terminal Domain 1 Domain SP 2 TM w yto ' ytokine Receptor Module (RM) N-linked glycosylation ( I ) WSXWS motif ( w ) 10 (SF2R, 16) 378aa eleven N-linked glycans 54aa cytoplasmic domain IL3Ra (IL3R, 23) 360aa six N-linked glycans 53aa cytoplasmic domain IL5Ra (IL5R, 25) 400aa four N-linked glycans 58aa cytoplasmic domain, IL-3 and IL-5 receptors, beta subunit Four extracellular fibronectin type III domains No direct cytokine binding Glycoprotein, 3 N-linked glycosylation sites Large cytoplasmic domain Principal signal transduction subunit ssociates with Multiple substrates for phosphorylation; Eight Tyr residues, one Ser residue Interacts with numerous signalling/adapter molecules N' RM 1 RM 2 I I I SP Domain 1 Domain 2 Domain 3 Domain 4 TM w S ytoplasmic domain ' 11 (SF2RB, 31) 881aa, three N-linked glycans ytokine Receptor Module (RM) N-linked glycosylation ( I ) WSXWS motif ( w ) Tyrosine ( ) Serine ( S), IL-3 and IL-5, beta subunit Intertwined, strand-swapped, homodimer : swap : swap Monomer Monomer B Human 2.7Å crystal structure arr P.D. et al., 2001 ell 104, p291; 12 arr P.D. et al., 2006 cta rystallogr Sect F Struct. Biol. ryst. ommun. 62, p509 4

5 The b ommon () Family of ytokines, Principal signalling pathways ytokine * * * JK/ MPK PI-3-K Extracellular ytoplasm Nucleus a S Shc Ras MEK Grb2 Raf-1 ERK p85 p110 c-fos, c-jun Bcl-2, Bcl-x L PIP 3 kt Bad S poptosis Survival Proliferation Differentiation 13 Gene transcription Guthridge M.. et al., 1998 Stem ells 16, p301; Martinez-Moczygemba M. et al., 2003 J. llergy lin. Immunol.112, p653; Hercus T.R. et al., 2009 Blood 114, p1289 ccessory signalling pathways ytokine * JK/ MPK PI-3-K ccessory Extracellular ytoplasm Nucleus a S Shc Ras MEK Grb2 Raf-1 ERK p85 p110 c-fos, c-jun Bcl-2, Bcl-x L PIP 3 kt Bad b1 integrin Vasculogenesis FcRg Syk S BP poptosis Survival Proliferation Differentiation poptosis IL-4 production 14 Gene transcription BP; Kao.J. et al., 2008 Oncogene27, p1397; FcRg; Hida S. et al., 2009 Nat. Immunol. 10, p214; b1 integrin; Uberti B. et al., 2010 Oncogene 29, p lpha subunits and signalling ytoplasmic sequences are essential for signalling ; Muto. et al., 1995 J. llergy lin. Immunol. 96, p1100 IL3Ra; Barry S.. et al., 1997 Blood 89, p842 IL5Ra; Takaki S. et al., 1994 Mol. ell Biol. 14, p7404 ssociate with specific signalling molecules Src family kinases PI-3-K p85 IkKb Perugini M. et al., 2010 Blood 115, p3346 Syntenin (IL5Ra) Geijsen N. et al., 2001 Science 293, p1136 p110 p85 IκBβ Lyn Src Syntenin ytokine a Extracellular ytoplasm ontribute to common signalling or Provide cytokine-specific signalling 5

6 The b ommon () Family of ytokines, ssembly of cytokine:receptor complexes Structural analysis of soluble cytokine:receptor complexes reveals Specific and promiscuous mechanisms of cytokine recognition Mechanisms of receptor activation a a 16 From Skiniotis G. et al., 2005 Nat. Struct. Mol. Biol. 12 p545 bsorbance 17 Recombinant soluble forms of and form a ternary complex with Recapitulates key properties of the cell surface receptor andidate for structural studies ssembly of a receptor complex T s B s Elution time s s kda - 45 kda Formation of soluble human : receptor complex, Mclure B.J. et al., 2003 Blood 101, p1308 T B = Binary omplex T = Ternary omplex B - 30 kda kda kda The :receptor complex hexamer complex with s and s ssembles via three sites of interaction Site I Extracellular Site II Site III Site III Site II Site I 18 ytoplasm Human :receptor complex, 3.3Å crystal structure, 6

7 The b ommon () Family of ytokines, The :receptor complex (2) The hexamer complex is analogous to other heteromeric cytokine receptor complexes IL-6 gp130 IL2Ra IL-2 gp130 IL6Rα gc IL2Rb receptor IL-6 receptor IL-2 receptor Human : receptor complex 3.3Å crystal structure, Hansen G. et al., 2008 ell 134, p Human IL-6: receptor complex 3.65Å crystal structure, Boulanger M.J. et al., 2003 Science300, p2101 Human IL-2: receptor complex 2.3Å crystal structure, Wang X. et al., 2005 Science310, p1159 The interaction with Site 2, critical for cytokine interaction with 20 Glu 21 of helix Hydrogen bond interaction with Tyr 421 Domain 1/4, peptide mosaic interface of (Hansen G. et al., 2008 ell 134, p496) onserved glutamate in helix onserved amongst all cytokines onserved across species Mutation disrupts interaction for all cytokines (, Lopez,.F. et al., 1992 EMBO J. 11, p909; Hercus T.R. et al., 1994 PNS 91, p5838) (IL-3, Barry, S.. et al., 1994 J. Biol. hem. 269,p8488) (IL-5, Tavernier, J. et al., 1995 PNS 92, p5194) Tyr 421 of Domain 4 FG loop Mutation abolishes interaction for all cytokines (Woodcock, J.M. et al., 1996 J. Biol. hem. 271, p25999; Hansen G. et al., 2008 ell 134, p496) Domain 1B Tyr 365 Tyr 39 Glu 21 Tyr 421 Helix His 367 Domain 4 Helix Receptor activation and the hexamer complex The structure of the hexamer complex does not explain receptor activation Extracellular 21 ytoplasm Human :receptor complex, 3.3Å crystal structure, 7

8 The b ommon () Family of ytokines, receptor activation Important event in receptor activation is transphosphorylation is specifically associated with (Quelle F.W. et al., 1994 Mol. ell Biol.14, p4335) (Watanabe S. et al., 1996 J. Biol. hem. 271, p12681) Total and phosphorylated is associated only with IP: : WB: WB: WB:p p Hansen G. et al., 2008 ell 134, p Implies functional dimerization required for receptor activation receptor activation (2) Membrane proximal domains of are widely spaced, too far for transphosphorylation? 120 Å 23 receptor activation (3) No gross changes in the structure are observed in the hexamer complex Small rotation of domain 4 upon complex formation : receptor hexamer - Monomer - Monomer B Free dimer 3 rotation 3 rotation Human :receptor complex, 3.3Å crystal structure 3XE, Hansen G. et al., 2008 ell 134, p496, Supp. 24 Human 2.7Å crystal structure 2GS, arr P.D. et al., 2006 cta rystallogr Sect F Struct. Biol. ryst. ommun. 62, p509 8

9 The b ommon () Family of ytokines, higher-order, :receptor complex The hexamer assembles into a dodecamer complex Head-to-head association of two hexamers Two novel sites of interaction D Hexamer 2 D D 25 B B Hexamer 1 Human :receptor complex, 3.3Å crystal structure, B Site 5 Site 4 The dodecamer complex The dodecamer complex provides a mechanism for functional dimerization of Ligand-dependent receptor assembly lose positioning of allows transphosphorylation, signalling a d,b c D B B D 120Å 120Å Extracellular ytoplasmic ~10Å 26 The dodecamer complex (2) The dodecamer complex assembles through novel sites of interaction between hexamers Site 4, large interaction surface, primarily :, also : Site 5, a small interaction surface between molecules (Hercus T.R. et al., 2009 Blood 114, p1289) a d,b c D Rα B B D 27 Hexamer 2 d d D D B Site 4, 5 B Hexamer 1 b b 9

10 The b ommon () Family of ytokines, Disruption of Site 4 function Disrupt dodecamer formation by mutation of residues that form Site 4 Site 4 mutant retains high affinity cytokine binding activity Hexamer 2 d B Domain 1 B Domain 1 D Site 4, 5 B Hexamer 1 b B Domain 2 Site 3 residues Site 4 residues Domain 4 28 Strand Strand G SLNVTKDGDSSLRWETMKMR EWSERSWDTES WT M M M Intact Site 4 required for function ssay Site 4 function in cell proliferation Mutation of both b strands disrupts mediated cell proliferation and IL-3 mediated cell proliferation require intact Site SLNVTKDGDSSLRWETMKMR EWSERSWDTES WT M M M % max mil2 proliferation M2 M3 WT M (ng/ml) % max mil2 proliferation M2 WT M3 M IL-3 (ng/ml) Proliferation of murine T-cell lines (TLL-2) expressing human IL3Ra, and wild type or Site 4 mutant in response to cytokine stimulation; Data normalized to IL-2 proliferation 29 ssay Site 4 function in signal transduction 30 Intact Site 4 required for function (2) Mutation of Site 4 abolishes mediated tyrosine phosphorylation of Mutation of Site 4 abolishes and 5 phosphorylation and IL-3 mediated cell signalling require intact Site nm phos-tyr IP: Total-b c IP: phos- phos-5 wild type M SLNVTKDGDSSLRWETMKMR EWSERSWDTES WT M Lysate Lysate phos- phos-5 wild type M4 Lysate Lysate Signal transduction in murine T-cell lines (TLL-2) expressing human IL3Ra, and wild type or Site 4 mutant in response to cytokine stimulation phos-tyr Total-b c IL nm IP: IP: 10

11 The b ommon () Family of ytokines, Model of receptor activation Receptor activation through the sequential assembly of a higher-order receptor complex Free Binary omplex Free Hexamer omplex Dodecamer omplex Bound Bound Signalling? Bound ctivation Signalling 31 Mechanism of receptor activation is likely to be conserved in IL-3 and IL-5 receptors Model of receptor activation (2) Receptor activation through the sequential assembly of a higher-order receptor complex Binary omplex Free β c Hexamer omplex Dodecamer omplex 32 Bound Bound ctivation Signalling 1) Does the hexamer receptor complex provide any signal transduction or biological function? 2) Mechanisms of cytokine-specific signal transduction through alpha chains? 3) How is signalling through serine phosphorylation of initiated? Blockade of receptor function Understanding receptor activation supports the development of new therapies that block cytokine function. ytokine specific agents, target Site 1 or 2 lpha subunit neutralizing antibodies; IL3Ra 7G3 Mb (Sun Q. et al., 1996 Blood 87, p83, Jin L. et al., 2009 ell Stem ell 5, p31) ytokine neutralizing antibodies or cytokine antagonists; E21R, IL-5 E13Q (Hercus T.R. et al., 1994 PNS 91, p5838, Tavernier J. et al., 1995 PNS 92, p5194) B. Pan-cytokine specific agents, target Site 2, 3 & 4 Neutralizing antibodies or small molecules directed to ; BION-1 Mb (Sun Q. et al., 1999 Blood 94, p1943, Rossjohn J. et al., 2000 Blood 95, p2491) 33 11

12 The b ommon () Family of ytokines, cknowledgements Barb Mclure, Mara Dottore, Frank Stomski, Jo Woodcock, hris Bagley entre for ancer Biology, S.. ustralia Michael Parker, Guido Hansen St. Vincent s Institute of Medical Research, Vic. ustralia Thank you!