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 REGULATION OF GROWTH FACTOR/RECEPTOR INTERACTIONS

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

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 VIVO

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

RTK Coupling to Intracellular Signaling Components

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

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 MATCHING NOVEL LIGANDS AND RECEPTORS

Stable Expression cDNA Cloning of the KGFR

KGFR is an Alternative Product of FGFR-2

     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 A Bafico and Aaronson, Cancer Medicine, 2002

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 RECEPTOR ACTIVATION BY GROWTH FACTOR

Tyrosine Kinase Receptor Activation by Dimerization Schlessinger J., Cell Oct 13;103(2): 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.

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

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

Autocrine Transforming LoopsAutocrine Transforming Loops Receptor Gene AmplificationReceptor Gene Amplification Receptor Gene MutationReceptor Gene Mutation Paracrine Acting Growth Factors in Tumor ProgressionParacrine Acting Growth Factors in Tumor Progression Receptor RTK Signaling in Cancer

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

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.Canonical and non-canonical signaling pathways. Aberrations in canonical signaling are implicated in human cancer.

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

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 GST E-cad Immunoblot  -  -catenin Uncomplexed  -catenin pool             

FRIZZLED SIGNALING FUNCTIONS Wnt3a: - + pcDNA3HFz1 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 AB Weiss and Schlessinger, Cell,1998; Liu et al. MCB, 2003

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

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

Dickkopf (Dkk) Family Secreted molecules with novel structure.Secreted molecules with novel structure. In Xenopus, Dkk-1 is a potent Wnt inhibitor.In Xenopus, Dkk-1 is a potent Wnt inhibitor. Dkk-1 is induced by genotoxic stress.Dkk-1 is induced by genotoxic stress. Dkk-1modulates apoptosis in limb development in concert with BMP.Dkk-1modulates apoptosis in limb development in concert with BMP. Some Dkks have Wnt agonist activity depending on cellular context.Some Dkks have Wnt agonist activity depending on cellular context.

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

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 125 I- Dkk cold Dkk BS Lysate IP:  -Flag Bafico et al, Nature Cell Biol., 2001

hDKK-1 Interacts with LRP6 in a Bimolecular Complex BS LRP I-Dkk-1 Lysate IP:  -Flag 125 I-Dkk-1 IP:  -myc IP:  -HA Bafico et al, Nature Cell Biol., 2001

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

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

HUMAN TUMORS WITH ONCOGENIC MUTATIONS IN THE WNT CANONICAL SIGNALING Familial adenomatosis polyposis (100%)Familial adenomatosis polyposis (100%) Sporadic Colorectal >90%Sporadic Colorectal >90% Hepatocellular (20-40%)Hepatocellular (20-40%) Hepatoblastoma (50-90%)Hepatoblastoma (50-90%) Uterine endometrial (>30-50%)Uterine endometrial (>30-50%) Ovarian, endometroid (20-50%)Ovarian, endometroid (20-50%) Thyroid, anaplastic (60%)Thyroid, anaplastic (60%) Kidney, Wilms’ tumor (15%)Kidney, Wilms’ tumor (15%) Melanoma; prostate; medulloblastoma (<10%)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) Gary L Johnson, ERK1/ERK2 MAPK Pathway. Sci. STKE (Connections Map), Joseph Schlessinger, Fibroblast Growth Factor Receptor Pathway. Sci. STKE (Connections Map), Morris F. White, Insulin Signaling Pathway. Sci. STKE (Connections Map), Gary L Johnson, JNK MAPK Pathway. Sci. STKE (Connections Map), Gary L Johnson, p38 MAPK Pathway. Sci. STKE (Connections Map), Wnt Bruce Bowerman, C. elegans Endoderm Induction Wnt Pathway. Sci. STKE (Connections Map), Norbert Perrimon and Michael Boutros, Drosophila Wnt/Fz Pathways. Sci. STKE (Connections Map), Bruce Bowerman, C. elegans T Cell Polarity Wnt Pathway. Sci. STKE (Connections Map), Randall T. Moon, Wnt/beta-catenin Pathway. Sci. STKE (Connections Map), Randall T. Moon, Xenopus Egg Wnt/beta-catenin Pathway. Sci. STKE (Connections Map),