Bioc 325 Lecture 10: Receptor Tyrosine Kinases (RTKs)

Receptor Tyrosine Kinases (RTKs)  Introduce the family of tyrosine kinase-linked receptors (RTK): Ligand binding-Receptor activation Signal initiation and transmission (players) Diverse functions of RTK Implication in biological processes Overview of RTKs ...

Receptor Tyrosine Kinases (RTKs)  Introduce the family of tyrosine kinase-linked receptors (RTK): Ligand binding-Receptor activation Signal initiation and transmission (players) Diverse functions of RTK Implication in biological processes Overview of RTKs  Cell surface receptors that are directly linked to intracellular enzymes (kinases)  Majority of growth factor receptors (PDGF, NGF, EGF, FGF,VEGF), insulin, and IGFI are part of this family  Receptor can either be a single polypeptide or dimer or can be 2 single peptides that dimerize upon ligand activation Insulin and IGF1 can cross bind the tyrosine kinase is cytoplasmic —> leads to the propagation of the signal   Common structure ◦ Ligand binding domain, N-terminal extracellular ONE transmembrane α helix ◦ Cytosolic C-terminal domain with intrinsic tyrosine kinase activity. ligand binds —> autophosphorylation of the R (this is intrinsic) Receptor Tyrosine Kinases (RTKs) Mechanism of Activation Ligand Receptor-Ligand complex A dimeric ligand protein is formed by di-sulfide bonds between two identical protein monomers The ligand has two identical receptor binding sites and can cross link two adjacent receptors upon their binding This initiates the intracellular signaling process ligands are usually part of the mb, have proteases that cleave it and can go bind to the R —> heterodimerization —> activation of the intrisinc kinase —> many signaling processes and becomes a docking site Receptor Tyrosine Kinases (RTKs) Mechanism of Activation Receptor-Ligand Binding lots of phosphorylation sites cleave and relieve the ligand which can bind to the R (different than others but can all result in the same thing) but both require the shedding of the ligand (gets chopped by shedder proteases) Receptor Tyrosine Kinases (RTKs) Mechanism of Activation Receptor Dimerization and Kinase Activation heterodimerization is a type of phorphorylation conformation change which can cross sites become ~p to make it active docking sites Dimerization leads to autophosphorylation of the receptor (cross-phosphorylation). Each receptor cross phosphorylates a specific tyrosine residue on its counterpart, which fully activates its kinase activity Docking of signaling molecules at RTK Phosphorylated-tyrosine residues become docking sites for adapter proteins ^ this is where the signal transduction initiaites this can be Grb2 for example (binding thru SH2 domain) Receptor Tyrosine Kinases (RTKs) Mechanism of Activation The binding of SH2-containing signaling proteins to an activated PDGF receptor  The phosphorylated tyrosines can be specifically identified by SH2 domains on other proteins, including adapter proteins  The activated receptor can then phosphorylate these bound proteins leading to intracellular signal transmission PI3 kinase binds to the R and becomes activated cross talking here with GPCR can have abs directed important bz they can be sites for specifically to these tyr ~p regulation of these Rs sites —> so can distinguish bw GPCRs or RTKs or non-RTK kinases (Src) 2 insulin molecules bind and induce conformational change in receptor Autophosphorylates, catalyzes phosphorylation of target proteins, IRS1 Multiple cellular responses initiated insulin is GF and main function is to activate glucose transport (so dual IRS-1 activates PI3k —> binds to the action). phosphonisotides (substrate) leading to activation of Akt Akt recycles Glut4  Receptor Tyrosine Kinases (RTKs) Transduction of response to signal through the cell Protein kinase cascades are valuable signal transducers: Signal amplified at every step Information received at plasma membrane is communicated to the nucleus Multiple steps provide specificity Various target proteins provide diversity in response  Receptor Tyrosine Kinases (RTKs) RTK can activate different downstream pathways Grb2 —> Ras-GEF —> Ras becomes activated (oncogenic gene) and activates the MAP IRS1 kinase pathway. these are communicators /mediators bw the cytoplasm and nuclear SOS PI3 kinase —> AKT —> PDK1 Raf MEK AKT  growth factor and cancer inducer (oncogenic)  Monomeric (small) GTPase switch protein ◦ unlike hetrotrimeric G proteins, Ras does not directly bind to receptors.  Its activation is enhanced by GEF (guanine nucleotide exchange factor) ◦ GDP-GTP exchange  Deactivation of Ras-GTP complex requires GAP (GTPase-activating proteins), which increases intrinsic GTPase activity 100 fold  GAP can regulate RAS activity  Once activated, Ras propagates signaling further inside the cell via a kinase cascade that culminates in the activation of members of the MAP kinase family.  MAP kinases phosphorylate Transcription Factors (TFs) that regulate genes involved in the cell cycle and in differentiation. leading to an increase in number of cells and differentiation (turn them into cancerous)  Mutant RTKs or Ras/MAP kinase signaling proteins are associated with nearly all cancers. Dominant Ras mutations that block GAP binding and lock Ras in the "on" state promote cancer. so it is an imp pathway that shouldnt be active all the time. we have 2 checkpints (we dont want it active all the time and inactive all the time)  Ras is tethered to the mb exanged GDP to GTP and so this becomes active binds to R via its SH2 domain and to SOS by its SH3 domain SOS RAS can be considered a signal amplifier any problems in the mb can lead to problems in many pathways  In Step 1, EGF binding causes receptor dimerization and autophosphorylation on cytosolic tyrosines.  In Step 2, the adaptor protein GRB2 binds receptor phosphotyrosine residues via its SH2 domain. GRB2 contains SH3 domains that allow the GEF protein known as Sos to bind to the membrane complex. son of sevenless , a form of GEF  Sos then recruits Ras to the complex. In the last step of Ras activation (Step 3), Sos promotes GTP exchange for GDP on Ras. SOS recruits Ras and exchanges GDP to GTP  The activated Ras-GTP complex then dissociates from Sos, but remains tethered to the inner leaflet of the cytoplasmic membrane via a lipid anchor sequence. The active form of Ras then activates the MAP kinase portion of the signaling pathway (discussed in later lecture). the complex dissociates and we now have an active Ras —> activates MAP kinase pathway When hormone binds to the extracellular domain the receptors dimerize/aggregate http://faculty.plattsburgh.edu/donald.slish/animations.html When the receptors dimerize, the tyrosine kinase domains phosphorylate the C terminal tyrosine residues http://faculty.plattsburgh.edu/donald.slish/animations.html Mechanism of Tyrosine Kinase Receptors This phosphorylation produces binding sites for proteins with SH2 domains. GRB2 is one of these proteins. GRB2, with SOS bound to it, then binds to the receptor complex. This causes the activation of SOS. http://faculty.plattsburgh.edu/donald.slish/animations.html Mechanism of Tyrosine Kinase Receptors SOS is a GEF. When this is activated, it causes certain G proteins to release GDP and exchange it for GTP. Ras is one of these proteins. When Ras has GTP bound to it, it becomes active. http://faculty.plattsburgh.edu/donald.slish/animations.html Mechanism of Tyrosine Kinase Receptors Activated Ras then causes the activation of a cellular kinase called raf-1 MAP kinase kinase kinase http://faculty.plattsburgh.edu/donald.slish/animations.html Mechanism of Tyrosine Kinase Receptors Raf-1 kinase then phosphorylates another cellular kinase called MEK. This cause the activation of MEK MAP kinase kianse http://faculty.plattsburgh.edu/donald.slish/animations.html  Mechanism of Tyrosine Kinase Receptors MAPK: MAP kinase Activated MEK then phosphorylates another protein kinase called MAPK causing its activation. This series of phosphorylating activations is called a kinase cascade. It results in amplification of the signal http://faculty.plattsburgh.edu/donald.slish/animations.html Mechanism of Tyrosine Kinase Receptors Among the final targets of the kinase cascade are transcriptions factors (fos and jun showed here). Phosphorylation of these proteins causes them to become active and bind to the DNA, causing changes in gene transcription http://faculty.plattsburgh.edu/donald.slish/animations.html ^ Raf ^ Mek ^ MAPK