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
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 hr
Spends 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
pH dependent ligand dissociation
EGF
TGF
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 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 ~ 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
Competition across 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