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1. Determined by growth factor availability and receptor expression levels

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

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1. Determined by growth factor availability and receptor expression levels

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  1. 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

  2. Intracrine Juxtacrine Paracrine Autocrine Endocrine BLOOD VESSEL Different modes of action for growth factors (Bafico and Aaronson, Cancer Medicine, 2002)

  3. 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

  4. GROWTH FACTOR FUNCTIONS IN VITRO 1. Proliferation 2. Differentiation 3. Chemo-attraction 4. Chemo-kinesis 5. Trophic action

  5. RTK Coupling to Intracellular Signaling Components

  6. RAS RAS p85 PI3K p110 Grb2 SOS p70S6K GSK3 MDM2 NF-ĸB P P P P P P P P P P P GROWTH FACTOR PIP3 PIP3 RTK PDK1 Akt Raf MEK BAD FKHR ERK PROTEIN SYNTHESIS PROLIFERATION PROLIFERATION CELL SURVIVAL Intracellular effectors of receptor tyrosine kinases Bafico and Aaronson, Cancer Medicine, 2002

  7. MATCHING NOVEL LIGANDS AND RECEPTORS 1. Purification of cross-linked receptor 2. Expression cloninga. Transient vs stable cloning strategies to identify novel ligands or receptors 3. Educated guesses 4. Genome database analysis

  8. Stable Expression cDNA Cloning of the KGFR

  9. KGFR is an Alternative Product of FGFR-2

  10.   A                  PDGF- PDGF- CSF-1R KIT/SCFR FLK2/FLT3 VEGFR1/FLT1 VEGFR2/KDR/FLK1 VEGFR3/FLT4 EGFR ERBB2/HER2/neu ERBB3 ERBB4 FGFR1 FGFR2 FGFR3 FGFR4 IR IGF-1R IRR MET RON SEA TRKA TRKB TRKC AXL/UFO MER TYRO3 EPHA1 EPHA2 EPHA3 EPHA4 EPHA5 EPHA6 EPHA7 EPHA8 EPHB1 EPHB2 EPHB3 EPHB4 EPHB5 EPHB6 EPHB7 Bafico and Aaronson, Cancer Medicine, 2002

  11. RECEPTOR ACTIVATION BY GROWTH FACTOR 1. Growth factors induce receptor clusteringa. High affinity bindingb. Ligand mediated receptor cross-linkingc. Ligand/receptor crystal structures 2. Receptor tyrosine kinase-activation by dimer or heterodimer formationa. Activation by mabs requires the ability to cross-linkb. Dominant negative receptors

  12. Schlessinger J., Cell. 2000 Oct 13;103(2):211-25. 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 trans-autophosphorylation 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.

  13. Schlessinger J., Cell. 2000 Oct 13;103(2):211-25. MECHANISMS OF RTK REGULATION

  14. Cloning of an Alternative HGF Transcript Chan et al, Science, 1991

  15. HGF NK2 is an HGF Antagonist Chan et al, Science, 1991

  16. MECHANISMS OF RTK REGULATION

  17. Receptor RTK Signaling in Cancer • Autocrine Transforming Loops • Receptor Gene Amplification • Receptor Gene Mutation • Paracrine Acting Growth Factors in Tumor Progression

  18. RAS RAS p85 PI3K p110 Grb2 SOS p70S6K GSK3 MDM2 NFK-B P P P P P P P P P P P GROWTH FACTOR PIP3 PIP3 RTK PDK1 Akt Raf MEK BAD FKHR ERK PROTEIN SYNTHESIS PROLIFERATION PROLIFERATION CELL SURVIVAL CELL SURVIVAL Tumor-specific activating mutations

  19. 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.

  20. Fz ß-cat ß-cat ß-cat WNT CANONICAL SIGNALING WNT Fz LRP5/6 LRP5/6 Dsh Dsh Axin Axin APC APC GSK3 GSK3 ß-cat ß-cat ß-cat ß-cat TCF TCF

  21. 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.

  22. E-cad GST E-cad E-cad GST GST GST-E-CADHERIN BINDING ASSAY GST beads Uncomplexed -catenin pool    Immunoblot --catenin          

  23. FRIZZLED SIGNALING FUNCTIONS pcDNA3 HFz1 - + - + Wnt3a: uncomplexed b-catenin

  24. 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.

  25. LRP6 SIGNALING FUNCTIONS Liu et al, MCB, 2003

  26. WNT INDUCES CONFORMATIONAL SWITCH IN LRP6 RECEPTOR OLIGOMERS A B Liu et al, MCB, 2003

  27. LRP5/6 ACTIVATION MECHANISM A B Weiss and Schlessinger, Cell,1998; Liu et al. MCB, 2003

  28. MODULATION OF WNT SIGNALING BY WNT ANTAGONISTS Cerberus WIF DKK WNT FRP Fz LRP5/6

  29. 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.

  30. Cytoplasmic domain TM domains CRD Frizzled Netrin-like domain CRD FRP/Frzb CRD Carboxypeptidase Z CRD Type XVIII collagen CRD Ror Musk TK

  31. 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.

  32. THE HUMAN DKK FAMILY hDkk-1 Cys-1 Cys-2 hDkk-2 Cys-1 Cys-2 Cys-1 Cys-2 hDkk-3 Cys-1 Cys-2 hDkk-4

  33. FRP but not hDKK-1 Physically Interacts with Wnt Bafico et al, Nature Cell Biology, 2001

  34. 220 125I-Dkk-1 - - + + + + - - BS3 - - - - + + + + cold Dkk-1 46 30 Lysate IP:-Flag hDKK-1 Receptor is Detectable in a 240kD Complex Bafico et al, Nature Cell Biol., 2001

  35. 220 125I-Dkk-1 125I-Dkk-1 LRP6 - + - + - + - + BS3 46 30 - - - - + + + + + + + + IP:-myc IP:-HA Lysate IP:-Flag hDKK-1 Interacts with LRP6 in a Bimolecular Complex Bafico et al, Nature Cell Biol., 2001

  36. kremen WNT FRP FRP WNT DKK FRP Fz Fz LRP5/6 -catenin MODULATION OF WNT SIGNALING BY WNT ANTAGONISTS WNT FRP FRP FRP Fz LRP5/6 Non canonical -catenin

  37. ABERRATIONS OF WNT SIGNALING PATHWAY IN CANCER WNT Fz LRP5/6 Dsh Axin APC GSK3 ß-cat ß-cat ß-cat ß-cat TCF

  38. 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%)

  39. Wnt Signaling Up-regulation in Human Breast and Ovarian Cancer Cells Bafico et al, Cancer Cell, 2004

  40. FRP1 AND DKK1 INHIBITION OF AUTOCRINE WNT SIGNALING IN HUMAN TUMOR CELL LINES Bafico et al, Cancer Cell, 2004

  41. FRP1 AND DKK1 INHIBITION OF AUTOCRINE WNT SIGNALING IN HUMAN TUMOR CELL LINES Bafico et al, Cancer Cell, 2004

  42. 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.

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