REGULATION OF GROWTH FACTOR/RECEPTOR INTERACTIONS 1. Determined by growth factor availability and receptor expression levels 2.
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REGULATION OF GROWTH FACTOR/RECEPTOR INTERACTIONS 1. Determined by growth factor availability and receptor expression levels 2. Different modes of growth factor action - autocrine, paracrine, other 3. Secretory properties - secretory signal; proteoglycan or serum protein binding 4. More than one member of same growth factor gene family may act on the same receptor 5. Same growth factor may cluster more than one receptor member of the same receptor family; homo- vs hetero-dimers 6. Interactions regulated by alternative growth factor/receptor products Intracrine Juxtacrine Autocrine Paracrine Endocrine BLOOD VESSEL Different modes of action for growth factors (Bafico and Aaronson, Cancer Medicine, 2002) GROWTH FACTOR FUNCTIONS IN VIVO 1. Early development 2. Tissue differentiation 3. Wound healing and tissue repair 4. Immune responses 5. Stromal mediators of sex and other hormones GROWTH FACTOR FUNCTIONS IN VITRO 1. Proliferation 2. Differentiation 3. Chemo-attraction 4. Chemo-kinesis 5. Trophic action RTK Coupling to Intracellular Signaling Components GROWTH FACTOR PIP3 PIP3 RTK RAS SOS Grb2 P RAS P P PDK1 Akt p85 p110 PI3K Raf P MEK P ERK PROLIFERATION P BAD P P NF-ĸB FKHR CELL SURVIVAL P P P MDM2 GSK3 p70S6K PROLIFERATION PROTEIN SYNTHESIS Intracellular effectors of receptor tyrosine kinases Bafico and Aaronson, Cancer Medicine, 2002 MATCHING NOVEL LIGANDS AND RECEPTORS 1. Purification of cross-linked receptor 2. Expression cloning a. Transient vs stable cloning strategies to identify novel ligands or receptors 3. Educated guesses 4. Genome database analysis Stable Expression cDNA Cloning of the KGFR KGFR is an Alternative Product of FGFR-2 A MET RON SEA TRKA TRKB TRKC AXL/UFO MER TYRO3 Bafico and Aaronson, Cancer Medicine, 2002 IR IGF-1R IRR FGFR1 FGFR2 FGFR3 FGFR4 VEGFR1/FLT1 EGFR VEGFR2/KDR/FLK1 ERBB2/HER2/neu VEGFR3/FLT4 ERBB3 ERBB4 PDGF- PDGF- CSF-1R KIT/SCFR FLK2/FLT3 EPHA1 EPHA2 EPHA3 EPHA4 EPHA5 EPHA6 EPHA7 EPHA8 EPHB1 EPHB2 EPHB3 EPHB4 EPHB5 EPHB6 EPHB7 RECEPTOR ACTIVATION BY GROWTH FACTOR 1. Growth factors induce receptor clustering a. High affinity binding b. Ligand mediated receptor cross-linking c. Ligand/receptor crystal structures 2. Receptor tyrosine kinase-activation by dimer or heterodimer formation a. Activation by mabs requires the ability to cross-link b. Dominant negative receptors Tyrosine Kinase Receptor Activation by Dimerization Figure 1. Ligand Binding Stabilizes the Formation of Activated Dimers(A) Inactive receptor monomers (green) are in equilibrium with inactive (green) or active (blue) receptor dimers. The active receptor dimers exist in a conformation compatible with transautophosphorylation and stimulation of PTK activity (blue). Ligand binding stabilizes active dimer formation and hence PTK activation.(B) Inactive disulfide bridged insulin-receptor (IR) dimers (green) are in equilibrium with active dimers (blue). Insulin binding stabilizes the active dimeric state leading to PTK activation. Schlessinger J., Cell. 2000 Oct 13;103(2):211-25. MECHANISMS OF RTK REGULATION Schlessinger J., Cell. 2000 Oct 13;103(2):211-25. Cloning of an Alternative HGF Transcript Chan et al, Science, 1991 HGF NK2 is an HGF Antagonist Chan et al, Science, 1991 MECHANISMS OF RTK REGULATION Receptor RTK Signaling in Cancer • • • • Autocrine Transforming Loops Receptor Gene Amplification Receptor Gene Mutation Paracrine Acting Growth Factors in Tumor Progression GROWTH FACTOR PIP3 PIP3 RTK RAS SOS Grb2 P RAS P P PDK1 Akt p85 p110 PI3K Raf P MEK P ERK PROLIFERATION P BAD P P NFK-B FKHR CELL SURVIVAL CELL SURVIVAL Tumor-specific activating mutations P P P MDM2 GSK3 p70S6K PROLIFERATION PROTEIN SYNTHESIS WNT SIGNALING • Highly conserved multimember family of ligands. • Role in a variety of developmental processes in vertebrates and invertebrates. • Wnt receptors identified as Frizzled and LRP5/6. • Canonical and non-canonical signaling pathways. • Aberrations in canonical signaling are implicated in human cancer. WNT CANONICAL SIGNALING WNT Fz LRP5/6 LRP5/6 Fz Dsh Dsh APC Axin GSK3 ß-cat APC Axin GSK3 ß-cat ß-cat ß-cat ß-cat TCF ß-cat ß-cat TCF FRIZZLED RECEPTOR • Family of seven-membrane spanning proteins. • Couples Wnts to canonical pathway. • Genetic studies have also identified Frizzled as a receptor in planar cell polarity and PKC pathways. • Seven Frizzled homologues exist in mammals. GST-E-CADHERIN BINDING ASSAY GST E-cad GST beads GST E-cad Uncomplexed -catenin pool GST E-cad Immunoblot --catenin FRIZZLED SIGNALING FUNCTIONS pcDNA3 Wnt3a: - + HFz1 - + uncomplexed -catenin LRP RECEPTOR • • • • Structurally related to LDL receptors. Two family members: LRP5 and LRP6. LRP intracellular domain binds Axin. Wnt signaling via LRP5 is important for bone mass. LRP6 SIGNALING FUNCTIONS Liu et al, MCB, 2003 WNT INDUCES CONFORMATIONAL SWITCH IN LRP6 RECEPTOR OLIGOMERS A B Liu et al, MCB, 2003 LRP5/6 ACTIVATION MECHANISM A B Weiss and Schlessinger, Cell,1998; Liu et al. MCB, 2003 MODULATION OF WNT SIGNALING BY WNT ANTAGONISTS Cerberus WIF DKK WNT LRP5/6 Fz FRP FRP (Frizzled Related Protein) • Family of secreted heparin binding Wnt antagonists. • Conserved in vertebrate evolution. • Structurally related to the frizzled cysteine rich domain (CRD). • Temporally and spatially regulated in development. • Pro- or anti-apoptotic depending on context. TM domains Frizzled CRD Netrin-like domain FRP/Frzb CRD Carboxypeptidase Z CRD Type XVIII collagen CRD Ror Musk TK CRD Cytoplasmic domain Dickkopf (Dkk) Family • • • • Secreted molecules with novel structure. In Xenopus, Dkk-1 is a potent Wnt inhibitor. Dkk-1 is induced by genotoxic stress. Dkk-1modulates apoptosis in limb development in concert with BMP. • Some Dkks have Wnt agonist activity depending on cellular context. THE HUMAN DKK FAMILY hDkk-1 hDkk-2 Cys-1 Cys-1 hDkk-3 hDkk-4 Cys-2 Cys-2 Cys-1 Cys-1 Cys-2 Cys-2 FRP but not hDKK-1 Physically Interacts with Wnt Bafico et al, Nature Cell Biology, 2001 hDKK-1 Receptor is Detectable in a 240kD Complex Dkk-1 125I- BS3 - - + + - - + + cold Dkk-1 - + - + - + - + 220 46 30 Lysate IP:-Flag Bafico et al, Nature Cell Biol., 2001 hDKK-1 Interacts with LRP6 in a Bimolecular Complex 125I-Dkk-1 LRP6 BS3 - - + + - + - + - - + + - + - + 125I-Dkk-1 + + + + 220 46 30 Bafico et al, Nature Cell Biol., 2001 MODULATION OF WNT SIGNALING BY WNT ANTAGONISTS WNT WNT FRP FRP FRP FRP Non canonical Fz -catenin DKK LRP5/6 Fz FRP kremen FRP LRP5/6 WNT Fz -catenin ABERRATIONS OF WNT SIGNALING PATHWAY IN CANCER LRP5/6 WNT Fz Dsh Axin APC GSK3 ß-cat ß-cat ß-cat ß-cat TCF HUMAN TUMORS WITH ONCOGENIC MUTATIONS IN THE WNT CANONICAL SIGNALING • • • • • • • • • Familial adenomatosis polyposis (100%) Sporadic Colorectal >90% Hepatocellular (20-40%) Hepatoblastoma (50-90%) Uterine endometrial (>30-50%) Ovarian, endometroid (20-50%) Thyroid, anaplastic (60%) Kidney, Wilms’ tumor (15%) Melanoma; prostate; medulloblastoma (<10%) Wnt Signaling Up-regulation in Human Breast and Ovarian Cancer Cells Bafico et al, Cancer Cell, 2004 FRP1 AND DKK1 INHIBITION OF AUTOCRINE WNT SIGNALING IN HUMAN TUMOR CELL LINES Bafico et al, Cancer Cell, 2004 FRP1 AND DKK1 INHIBITION OF AUTOCRINE WNT SIGNALING IN HUMAN TUMOR CELL LINES Bafico et al, Cancer Cell, 2004 Connection Maps relevant to this lecture: Receptor tyrosine kinases Joseph Schlessinger, Epidermal Growth Factor Receptor Pathway. Sci. STKE (Connections Map)http://stke.sciencemag.org/cgi/cm/stkecm;CMP_14987. Gary L Johnson, ERK1/ERK2 MAPK Pathway. Sci. STKE (Connections Map), http://stke.sciencemag.org/cgi/cm/stkecm;CMP_10705. Joseph Schlessinger, Fibroblast Growth Factor Receptor Pathway. Sci. STKE (Connections Map), http://stke.sciencemag.org/cgi/cm/stkecm;CMP_15049. Morris F. White, Insulin Signaling Pathway. Sci. STKE (Connections Map), http://stke.sciencemag.org/cgi/cm/stkecm;CMP_12069. Gary L Johnson, JNK MAPK Pathway. Sci. STKE (Connections Map), http://stke.sciencemag.org/cgi/cm/stkecm;CMP_10827. Gary L Johnson, p38 MAPK Pathway. Sci. STKE (Connections Map), http://stke.sciencemag.org/cgi/cm/stkecm;CMP_10958 Wnt Bruce Bowerman, C. elegans Endoderm Induction Wnt Pathway. Sci. STKE (Connections Map), http://stke.sciencemag.org/cgi/cm/stkecm;CMP_6104. Norbert Perrimon and Michael Boutros, Drosophila Wnt/Fz Pathways. Sci. STKE (Connections Map),http://stke.sciencemag.org/cgi/cm/stkecm;CMP_6459. Bruce Bowerman, C. elegans T Cell Polarity Wnt Pathway. Sci. STKE (Connections Map), http://stke.sciencemag.org/cgi/cm/stkecm;CMP_10440. Randall T. Moon, Wnt/beta-catenin Pathway. Sci. STKE (Connections Map), http://stke.sciencemag.org/cgi/cm/stkecm;CMP_5533. Randall T. Moon, Xenopus Egg Wnt/beta-catenin Pathway. Sci. STKE (Connections Map), http://stke.sciencemag.org/cgi/cm/stkecm;CMP_6031.