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

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

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

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

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

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 pH dependent ligand dissociation EGF TGF Ebner & Derynck, 1991

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

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

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

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

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

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

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