Receptor Tyrosine Kinases

• RTKs are critical for mediating signalling from ligands (growth factors, mitogenic factors, and factors important in survival like insulin) • extracellular/cytoplasmic ligand-binding domain with intrinsic (built-in) tyrosine kinase binding activity which is stimulated by ligand binding due to Receptor Dimerization (two receptors binding together). Now adaptor proteins are recruited. • exterior ligand-binding region + single transmembrane alpha-helix + cytoplasmic protein tyrosine kinase with activation loop • Ras (small monomeric G protein) acts as a GTPase switch protein to signal further downstream kinases • aberrant signalling in any part of this pathway is the root for cancers • these receptors actually phosphorylate themselves (their cytoplasmic domain has the ability to phosphorylate itself and other proteins directly through transcription factors, and can also activate G proteins which activate kinases in a series of kinases.)

• Dimerization: Ligand binding to ligand-binding region of RTK changes shape of receptor, which causes the two receptors to come together and dimerize (so you have two receptors and 2 ligands) • Phosphorylation: dimerization allows for trans-autophosphorylation of BOTH cytoplasmic domains (=the tyrosine kinases– specifically their Activation Loop. aka, ATP–>ADP) • Phosphorylation of additional tyrosine residues(AAs)==> Phosphotyrosines (has lots of Ps on domain). Serve as docking sites for downstream signal-transduction proteins containing SH2 (src homology 2) domains (adaptor domain) - you now have activated RTK - the recruited adapter proteins contain unique domains that recognize specific sequences

• Noticed that if we were to inject a tumour cell extract from unhealthy chicken sarcoma tissues into healthy chicken, this chicken would develop tumours and get cancer. Therefore transmissible agent was determined to be a retrovirus (Rous Sarcoma Virus) • They were able to sequence the genome of that virus, and find a gene that encodes a tyrosine kinase (src) (so there was a kinase domain plus more domains labelled SH3 and SH2 in the genome– which is highly conserved and found in humans) -Normal version of virus = c-Src = cellular src which is inhibited by phosphorylation • Viral version (v-Src) which is turned on all the time, which leads to it promoting tumours

= monomeric G protein, GTPase superfamily, lipid-anchored protein = downstream effector of RTK signalling - similar to Galpha but smaller - its activity is not directly linked to cell-surface receptors. Doesn’t have intrinsic GTPase activity (aka, can’t activate itself, needs middle men to regulate its activity) - so uses GAP to help speed up GTP hydrolysis (INACTIVATES) - and GEF to turn GDP–> GTP (ACTIVATES)

Ras binds GTP but no hydrolysis (so it’s constitutively on) because there’s been a mutation of glycine-12 which prevents binding of GAP

• Ras is inactive and anchored to membrane by prenylation • FGF (hormone) binds FGF receptor monomers (FGFR) • binding of FGF causes receptor dimerization, kinase activation, and phosphorylation of cytosolic receptor tyrosine residues —> active FGF receptor dimer • this sets up a docking scaffold for adapter proteins containing SH2 and SH3 domains • SH2 is critical for recognizing and binding to the tyrosines on the activated receptor tyrosine kinase • example of adaptor protein recruited: GRB = Growth factor Receptor Bound protein = adapter protein with no enzymatic activity

• assembly of a multiprotein complex at the cytosolic face of the plasma membrane == GRB2 has SH2 domain that binds Phosphotyrosine (on receptor), and two SH3 domains bind Proline in Sos • Sos connects to Ras • the binding of GRB2 acts as a middle man btwn kinase (receptor) and Sos • Ras is still inactive, but is now primed

• Sos is a GEF— it promotes dissociation of GDP from Ras; GTP binds and active Ras dissociates from Sos • Ras-GTP is now active and triggers the downstream kinase cascade (Ras-GTP –> Raf –> MEK–>MAPK= mitogen-activated protein kinase

• Ras (lipid-anchored to membrane on cytosolic face) activated by exchange of GDP to GTP • Active Ras recruits, binds at Raf’s N-terminal regulatory domain, and activates Raf (inactive Raf is in cytosol bound to inhibitory protein 14-3-3) • GTP hydrolysis leads to dissociation of Ras from Raf (now Ras is inactive again) • Raf activates MEK by phosphorylation • MEK activates MAPK by dephosphorylating itself and phosphorylating MAPK (2Ps) • Active MAP kinase translocates to nucleus; activates many transcription factors, which go and alter genes controlling cell-cycle, cell growth, cell differentiation - MAPK aka, ERK= extracellular signal-regulated kinase

• everything in the pathway is a proto-oncogene (=disregulation is biased to being turned on, which makes cell likely to grow in uncontrollable manner) except for NF1 which is a tumour-suppressor gene • Usually NF1 binds to activated RAS and helps hydrolyze GTP to turn RAS off. But recessive loss-of-function mutation in GTPase activating protein (NF1) leads to constitutive (permanent) activation of RAS. • loss of GAP (because of glycine-12 mutation) leads to sustained RAS activation of down stream signal transducing proteins

EGF receptor is a type of RTK. - an oncogenic mutation (Val->Gln) in Her2 receptor (a type of EGF receptor) causes dimerization and activation of receptor in absence of ligand (==>Neu oncoprotein -neuroblastoma) –> uncontrolled cell growth in brain • deletion (oncogenic mutation) that causes loss of extracellular ligand binding domain in EGF receptor leads to spontaneous dimerization and thus constitutive activation of cytoplasmic kinase domain (==>ErbB oncoprotein)

• part of epidermal growth factor (EGF) family • 4 RTKs: HER1 to HER4 • HER2 doesn’t bind a ligand directly, but can form heterodimers (aka, HER1 and HER2, etc.) • this dimerization is essential for transmitting the signal • all 4 can bind EGF

• 25% of breast cancer patients have increased HER2 expression (>100 fold) due to HER2 gene amplification • the cells are stimulated by very low concentration of EGF • HER2 is targeted by a monoclonal antibody called Herceptin • a cancer cell will also have many more receptors for Herceptin to bind onto because the gene has been amplified.

• HER2 therapy designed to bind to HER2 and tumour cells to flag them for destruction by immune system (using antibody-dependent cytotoxicity and increased endocytosis of HER2) • also blocks downstream HER2 signalling to inhibit proliferation of cells by physically blocking either homodimerization of heterodimerization

a deletion that causes the loss of the extra-cellular binding domain in the EGF receptor