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Example RTK: EGF receptor

Example RTK: EGF receptor. Epidermal Growth Factor Ubiquitous Development Determination of muscle, brain, kidney (differentiation) Patterning of ectoderm, gastric mucosa, airway (proliferation) Formation of segments, stomatogastric neural connections (migration)

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Example RTK: EGF receptor

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  1. Example RTK: EGF receptor • Epidermal Growth Factor • Ubiquitous • Development • Determination of muscle, brain, kidney (differentiation) • Patterning of ectoderm, gastric mucosa, airway (proliferation) • Formation of segments, stomatogastric neural connections (migration) • Greatest postnatal production by kidney, salivary gland & mammary gland • Tubule integrity and regeneration in kidney • Reduces acid production in stomach • Commercial use in wound healing and cosmetics • Anti-EGF cancer therapy

  2. In vitro effects • Sequential growth processes • Glycolysis & nutrient transport • Protein synthesis • RNA synthesis • DNA synthesis • Suppresses contact inhibition Somewhat cell type dependent Somewhat dependent on cofactors Serum, insulin, ascorbate 1 hr 3-10 hr 8-12 hr Transport Gylcolysis Protein RNA DNA Cell division

  3. Classical model • Ligand activates receptor • Receptor activates signaling cascade • Signaling cascade mediates biological outcome Cell Division EGF EGFR Grb2/Sos Ras MEK MAPK Inhibitors & knockout models block function Overexpression or constitutively active intermediates induce function But: identified signaling cascades get tied to multiple, even antagonistic functions

  4. EGF Receptor composition • ErbB1 • EGF ~3nM • TGF-a ~0.5 nM • 104-105 receptors/cell • KO embryonic lethal with defective neural structuring • ErbB3 • Lacks kinase domain • Slow internalization • ErbB4 • EGF >1 uM • Neuregulin ~5 nM • Activates PI3-K • ErbB2 • No known ligands • High kinase activity • Slow internalization • Functional receptors may be homo- or hetero-dimers, and isoform composition varies by cell type and developmental stage • Cell regulates its own response to EGF/TGF/NRG

  5. Receptor internalization Bound receptor is internalized over 0.5-2 hr Spends 20-45 minutes in processing Bound Receptor Internal Receptor EGF/TGF in compartment EGF TGFa Lysosomal Degradation Most of the ligand is released as fragments Probably the receptor is degraded, too But some TGFa is released intact TGFa-bound ErbB1 gets recycled Ebner & Derynck, 1991

  6. Internalized receptor processing • TGF dissociates at higher pH • Dissociated receptors recycle to surface • Associated receptors continue to signal • EGFR respond differently to EGF/TGFa • Affinity • Ligand/receptor stability • Time course EGF TGF pH dependent ligand dissociation Ebner & Derynck, 1991

  7. Unique tyrosine coding • ErbB isoforms share some adapter modes • Grb2/Shc • STAT • Each is unique • PI3-K • Src • Cbl Schulze, Deng & Mann, 2005

  8. Multiplex evaluation of pY affinity • Jones, et al. 2006. Nature 439:168 • Microarray containing 160 proteins with known SH2 and PTB domains • 66 Peptide fragments ~13 AA surrounding pY • Probe microarrays with 500-fold range of peptide concentration • Calculate 10,000 kD’s

  9. Multiplex evaluation of pY affinity Jones, et al., 2006

  10. Affinity-based interaction maps • SH2/PTB around box edge • Lines indicate binding of specific pY to SH2/PTB at concentration threshold • Venn indicate overlap among isoforms

  11. Downstream specificity • Receptor affinity 3nM  [EGF]~1e-13 M (15 ng/L) • Observed [EGF] ~ 1-100,000 ng/L • ie: [EGF] ranges from less than minimal to 1000x max • Cell volume ~ 10-11-10-12 L • EGF receptor content ~500 nM, 2e5-6 molecules • Effector (Grb2) concentrations ~200 nM, 1e5-6 molecules • Most sensitive pY interactions ~500 nM • Lower abundance species saturated • Higher abundance effectors limited by receptor • Competition for binding. Kinetics. • Individual residues interact with 1-20 targets • ie: effectors compete for access to favored pY’s Solve Kd=(R-b)(E-b)/b R+b=Rtot; E+b=Etot Max Grb2 bound: 90 nM

  12. Combinatorial mechanisms • Effectors may have multiple activation mechanisms • Multiple interactions stabilize effector • Competition among effectors • Competitionacross receptors • eg: PLC • SH2 domainsphos-tyrosine • PH domainsPIP3 Lemmon & Schlessinger, 2010

  13. Network model • “Bow tie” structure • Actual outcome depends on multiple states • No single master control: redundancy

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