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

1 hr 3-10 hr 8-12 hrTransportGylcolysis

ProteinRNA

DNACell division

Somewhat cell type dependentSomewhat dependent on cofactors

Serum, insulin, ascorbate

Classical model

• Ligand activates receptor• Receptor activates signaling cascade• Signaling cascade mediates biological outcome

EGF EGFR Grb2/Sos Ras MEK MAPKCell

Division

Inhibitors & knockout models block functionOverexpression 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

•ErbB2•No known ligands•High kinase activity•Slow internalization

•ErbB3•Lacks kinase domain•Slow internalization

•ErbB4•EGF >1 uM•Neuregulin ~5 nM•Activates PI3-K

•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 internalizationBound receptor is internalized over 0.5-2 hr

Spends 20-45 minutes in processing

Most of the ligand is released as fragmentsProbably the receptor is degraded, too

But some TGFa is released intact TGFa-bound ErbB1 gets recycled

EG

F/T

GF

in c

ompa

rtm

ent

EGFTGF

Bound Receptor

Internal Receptor

Lysosomal Degradation

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 EGF

TGF

Ebner & Derynck, 1991

pH dependent ligand dissociation

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)/bR+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