Enzyme-Coupled Receptors PDF

Session Learning Outcomes (SLOs) SLO# 1 : Define kinases and phosphatases and their role in signal transduction. SLO# 2 : Explain the different mechanisms by which cells regulate and/or terminate responses to chemical signals Enzyme-coupled receptors Enzyme-coupled recepto...

Session Learning Outcomes (SLOs) SLO# 1 : Define kinases and phosphatases and their role in signal transduction. SLO# 2 : Explain the different mechanisms by which cells regulate and/or terminate responses to chemical signals Enzyme-coupled receptors Enzyme-coupled receptors are transmembrane proteins that display their ligand-binding domain on the outer surface of the plasma membrane The cytoplasmic domain of the receptor either acts as an enzyme itself or forms a complex with another protein that acts as an enzyme. Receptor tyrosine kinases (RTKs) are the largest class of enzyme-coupled receptors made up of those with a cytoplasmic domain that functions as a tyrosine protein kinase, phosphorylating specific tyrosine on selected intracellular proteins aminoacid Receptor Tyrosine Kinases Receptor tyrosine kinases (RTKs) regulate cell differentiation and proliferation. The ligands for RTKs are soluble or membrane-bound peptide/protein hormones including nerve growth factor (NGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and insulin. IT ligand binding causes receptor dimerization. In other cases (e.g., the insulin RTK), binding occurs to pre-existing dimers. important  ENZYME-COUPLED RECEPTORS Activated RTKs Recruit a Complex of Intracellular protein Signaling Proteins small 6 10 s Most RTKs Activate the Monomeric GTPase Ras RTKs Activate PI 3-Kinase to Produce Lipid Docking Sites in the Plasma Membrane Some Receptors Activate a Fast Track to the Nucleus Protein Kinase Networks Integrate Information to Control Complex Cell Behaviors Three ways in which signaling proteins can cross-link receptor chains A. Dimer After thebinding ofligand B. Monomer but brought together by proteoglycan. C. Cluster on membrane  RTKs exhibit intrinsic tyrosine kinase activity located within their cytosolic domains. The binding of ligand activates the kinase domains which cross-phosphorylate the two monomers of the dimeric receptor. Phosphorylation first occurs at a regulatory site known as the activation lip (autophosphorylation). Phosphorylation of the lip causes conformational changes that allow the kinase domain to phosphorylate other tyrosine residues in the receptor. The phosphotyrosine residues in activated RTKs interact with selected intracellular proteins in signal transduction proteins. Activated receptor tyrosine kinase assemble a complex of intracellular signaling proteins. Autophosphorylation activate the kinase domain phosphorylate thetwo cross receptor monomersofthe dimeric Phosphorylated tyrosine serves as docking sites for proteins with SH2 domains The binding of SH2-containing intracellular signaling proteins to an activated PDGF receptor I iii in  ENZYME-COUPLED RECEPTORS Activated RTKs Recruit a Complex of Intracellular Signaling Proteins Most RTKs Activate the Monomeric GTPase Ras RTKs Activate PI 3-Kinase to Produce Lipid Docking Sites in the Plasma Membrane Some Receptors Activate a Fast Track to the Nucleus Protein Kinase Networks Integrate Information to Control Complex Cell Behaviors  in theci before nucleus Nearly all RTKs signal via Ras/MAP kinase pathways. They also may signal via other pathways. For example, the insulin receptor uses the Ras/MAP kinase pathway to regulate gene expression and the PI-3 kinase pathway to regulate enzyme activity (e.g., glycogen synthase).  The activation of Ras by an activated receptor tyrosine kinase 1.Ras: a small protein that is bound by a lipid tail to the cytoplasmic face of the plasma membrane. The binding of Ras-activating protein makes Ras exchange its bound GDP to GTP. The activated Ras then stimulates the next steps in the signaling pathway. EdingGDP 19 of binding and GTP 2. Adaptor: protein for helping to build a large signaling aggregate by coupling the receptor to other proteins. Here the adaptor recruits and stimulates Ras-activating protein. 5142 SH3  The MAP-kinase serine/threonine phosphorylation pathway activated by Ras 1.MAP kinase: protein kinase that performs a crucial step in relaying signals from cell- e surface receptors to the nucleus. It is the final kinase in a 3-kinase sequence (mitogen- activated protein kinase). Final kinase in 3 kinasesequence 2. MAP kinase phosphorylates various downstream target proteins. These target can include other protein kinase, and most important, gene regulatory proteins that control gene expression. Changes in gene expression and protein activity result in complex changes in cell behaviors such as proliferation and differentiation. activate many protein effector factors release growth wound forhealing  RTK Activation of Ras The mechanism of EGF activates Ras: 1- EGF binding causes receptor dimerization and Hormone autophosphorylation on cytosolic tyrosines. 11 w̅ 2ws Iii c 2- The adaptor protein GRB2 binds receptor phosphotyrosine residues via its SH2 domain.  adapp ein 3- GRB2 contains SH3 domains that allow the GEF protein known as Sos to bind to the membrane complex. Sos then recruits Ras to the complex. SH2 receptorphosphotyrosine residues SH3 SOS GEFprotein  4- Sos promotes GTP exchange for GDP on Ras. 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. lipid anchor sequence The active form of Ras then activates the MAP kinase portion of the signaling pathway. ▪ Activation of both Ras and G a is triggered by hormone binding to an appropriate cell- surface receptor. ▪Ras and Ga Subunits Belong to the GTPase Superfamily of Intracellular Switch Proteins ▪ Ras is a monomeric (small) GTPase switch protein that unlike trimeric G proteins does not directly bind to receptors. areadaptor There andRas activating protein  ▪ Ras typically relies on guanine nucleotide-exchange factors (GEFs) for binding GTP, and on GTPase-activating proteins (GAPs) for stimulation of GTP hydrolysis. inpresent of gos Becoming into an activeform cdc25 RasGRF GEF Sos present of GAP intoinactive form in Becoming NFD GTPase Activating protein P120RasGAP Such as SOS, Cdc25, Ras GRF Such as, p120 Ras GAP, Neurofibromin (NF1)  ii ▪ 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. win ▪ MAP kinases phosphorylate TFs that regulate genes involved in the cell cycle and in differentiation. ▪ RTK-Ras/MAP kinase signaling controls cell division, differentiation, and metabolism. win ▪At high ligand concentration, some cell-surface receptors are internalized by endocytosis, reducing the number of receptors on the surface and making cells less minatio sensitive to ligand. op iwis ▪ Many internalized RTKs are degraded in lysosomes. In this case, resensitization It depends on synthesis of additional receptor molecules.  Thispathwayaim celldevisionproliferati anddifferentiationso anymutationwillcan ▪ Mutant RTKs or Ras/MAP kinase signaling proteins are cancer associated with nearly all cancers. active pathwayisalways mutation the Im ▪ Dominant Ras mutations that block GAP binding and lock Ras in the "on" state promote cancer. DYF.pro Ysis  Ras Activation of MAP Kinase 0ᵗʰ kinase Raf Mapkinasekinase MAPkinase Ras activates MAP kinase via a phosphorylation cascade that athina b beforenucleus proceeds from: Ras Raf kinase MEK kinase MAP kinase  The final steps of RTK-Ras/MAP kinase signaling: MAP kinase phosphorylates and activates the p90RSK kinase in the cytoplasm. Both kinases enter the nucleus where they phosphorylate some transcription factors and then activate the expression of genes that propel cells through the cell cycle. activate transcription I factors expression activate gene  SUMMARY ▪ Receptor tyrosine kinases (RTKs), bind to peptide hormones, may exist as dimers or dimerize during binding to ligands. ▪ Ligand binding leads to activation of the kinase activity of the receptor and autophosphorylation of tyrosine residues ▪ Ras is an intracellular GTPase switch protein that acts downstream from most RTKs. Like Gsα , Ras cycles between an inactive GDP- bound form and active GTP-bound form. ▪ Unlike GPCRs, which interact directly with an associated G protein, RTKs are linked indirectly to Ras via two proteins, GRB2 and Sos. adaptor off ▪ The SH2 domain in GRB2, an adapter protein, binds to specific phosphotyrosines in activated RTKs. ▪ Activated Ras promotes formation of signaling complexes at the membrane containing three sequentially acting protein kinases. ▪ Raf is recruited to the membrane by binding to Ras -GTP and then activated. It then phosphorylates MEK, a dual specificity kinase that phosphorylates MAP kinase. I ▪ Phosphorylated MAP kinase dimerizes and translocates to the nucleus where it regulates gene expression. ▪ RTKs, GPCRs, and other receptor classes can activate MAP kinase pathways. XV  ENZYME-COUPLED RECEPTORS Activated RTKs Recruit a Complex of Intracellular Signaling Proteins Most RTKs Activate the Monomeric GTPase Ras RTKs Activate PI 3-Kinase to Produce Lipid Docking Sites in the Plasma Membrane Some Receptors Activate a Fast Track to the Nucleus Protein Kinase Networks Integrate Information to Control Complex Cell Behaviors RTKs can activate the PI-3-Akt signaling pathway An extracellular survival signal, such as IGF (insulin-like growth factor) family, activate an RTK, which recruits and activates PI- 3-kinase pathway to promote cell growth and survival Ghibate apoptosis  Signaling via Phosphatidyl-inositol 3-phosphates 1. PI3-kinase (phosphoinositide 3-kinase) is an enzyme which can phoshphorylate inositol phospholipid in the plasma membrane. 2 activation ofinositol 3 phospholipid SH2 kin Activation protein by domain Activationbyprotein kinase2 RTKs also can signal via formation of phosphoinositide compounds. Like GPCRs, they signal via the IP3/DAG pathway. However, RTKs activate the PLCγ isoform of phospholipase C, not PLCß as occurs with GPCRs. PLCγ binds to activated RTKs via SH2 domains.  Signaling via Phosphatidyl-inositol 3-phosphates 2. These phosphorylated lipids become docking sites for specific intracellular signaling proteins, which relocate from cytosol to membrane, where they can activate one another. O SH2 domain from dissociate membra plasma the Activated Akt promotes cell survival: 1. One of the relocated signaling proteins in the PI-3-kinase pathway is the serine/threonine protein kinase Akt (also called protein kinase B (PKB)). This pathway called PI-3-kinase-Akt singnaling pathway 2. Akt promotes the growth and survival of many cell types often by inactivating the signaling proteins it phosphorylates. It also stimulates cells to grow in size phosphorylation but cause inactivation ifsiitifEs ftp.osisippiplois iq survive Figure 16-34 Essential Cell Biology (© Garland Science 2010) 3. Mechanism of PI-3-kinase-Akt singnaling to promote cell survival: Akt phosphorylates and inactivates a cytosolic protein, Bad, to inhibits apoptosis in cells The PI 3-kinase/protein kinase B signaling pathway can stimulate cells to survive and grow phosphorylated phospholipid inositol a protein 2 pinase  Activation of Protein Kinase B Signaling downstream of PI 3-phosphates is conducted by PKB. PKB is recruited to the membrane via binding to PI 3-phosphates. There it is phosphorylated and activated by the PDK1 & PDK2 kinases. PDK1 also is recruited to the membrane via binding to PI 3-phosphates. Activated PKB then enters the cytosol, where it phosphorylates target proteins. In insulin receptor signaling, PKB phosphorylates and inactivates glycogen synthase kinase, stimulating glycogen synthesis. PKB also is a potent inhibitor of apoptosis. PI 3-phosphate signaling ultimately is terminated by cleavage of 3-phosphates from phosphoinositides by the PTEN phosphatase. PTEN is inactive in many advanced cancers.  in sin Insulin stimulation of muscle cells and adipocytes leads to activation of protein kinase B, which promotes glucose uptake and glycogen synthesis, resulting in a decrease in blood glucose. Akt also stimulates cells to grow in size by activating Tor: 1.The binding of growth factor to and RTK activates the PI-3-kinase-Akt signaling pathway 2.Akt then indirectly activate Tor (by phosphorylating and inhibiting a protein that helps to keep Tor shut down) 3. Tor, a serine/threonine kinase, stimulates protein synthesis and inhibits protein degradation Figure 16-35 Essential Cell Biology (© Garland Science 2010) Inhibition of signaling through normal receptor tyrosine kinases by an excess of mutant receptors As a tool for determining normal function of receptor Mutant proteins can help to determine exactly where an intracellular signaling molecule binds researchersusereceptorprotein withspecificaminoacidsmutated change toalanine to identify molecu whereanintracellularsignaling binds signalingmolecule cannolongerbindto ▪ ñI the pathway’: ‘Jamming To test the function: use the recombinant DNA technology to insert the gene in the form of constantly active form, to see if this mimics the effect of the extracellular signal e.g. A constitutively active form of Ras transmits a signal even in the absence of an extracellular signal molecule  ENZYME-COUPLED RECEPTORS Activated RTKs Recruit a Complex of Intracellular Signaling Proteins Most RTKs Activate the Monomeric GTPase Ras RTKs Activate PI 3-Kinase to Produce Lipid Docking Sites in the Plasma Membrane Some Receptors Activate a Fast Track to the Nucleus Protein Kinase Networks Integrate Information to Control Complex Cell Behaviors Some Enzyme-linked receptors activate a fast track to the nucleus 1. Some receptors use a more direct route to control gene expression 2. Cytokines binds to receptor, which activates a type of regulatory proteins called STATS (signal transducers and activators of transcription) Janus kinase (JAK) of formation dimer ayyaP jack Tieus 3. The signaling pathway of the cytokines: Binding of a cytokine to its receptor causes associated tyrosine kinase (called janus kinases, or JAKs) to cross-phosphorylate and activate one another and then they phosphorylate the receptor protein on tyrosine Tyrosine kinase 4. Gene regulatory proteins, called STATs, attach to the phosphotyrosine site of the receptor to be activated STATs dissociate from the receptor, dimerize, migrate to the nucleus activate the transcription of specific target genes 5. example: hormone prolactin which stimulates breast cell to make milk Cytokines can activate the JAK-STAT (Janus kinase - Signal Transducer and Activator of Transcription) pathway through a series of molecular events: 1. Cytokine Binding: Cytokines, which are signaling proteins, bind to their specific cell surface receptors. 2. Receptor Dimerization: The binding of cytokines induces a conformational change in the receptor, leading to the dimerization (pairing) of receptor subunits. 3. Activation of JAKs: The receptor dimerization brings Janus kinases (JAKs), associated with the cytoplasmic domain of the receptors, into close proximity. The JAKs then transphosphorylate each other, becoming activated. 4. STAT Binding and Phosphorylation: Activated JAKs phosphorylate specific tyrosine residues on the cytoplasmic tail of the receptor. This phosphorylation creates docking sites for Signal Transducer and Activator of Transcription (STAT) proteins. 5. STAT Activation: STAT proteins, present in the cytoplasm, bind to the phosphorylated docking sites on the receptor. 6. STAT Translocation: The STAT proteins are then phosphorylated by JAKs, leading to their activation. Activated STATs form dimers and translocate into the nucleus. 7. Gene Transcription: Once in the nucleus, STAT dimers bind to specific DNA sequences, regulating the transcription of target genes. This process leads to changes in gene expression, ultimately influencing various cellular responses. In one cell surface → nucleus signaling pathway, binding of interferon  to its cell-surface receptor induces membrane recruitment and activation of a cytosolic protein-tyrosine kinase called JAK. The activated JAK then phosphorylates Stat1, a member of transcription factors. The signal transduction pathways used by RTKs are summarized AKT  ENZYME-COUPLED RECEPTORS Activated RTKs Recruit a Complex of Intracellular Signaling Proteins Most RTKs Activate the Monomeric GTPase Ras RTKs Activate PI 3-Kinase to Produce Lipid Docking Sites in the Plasma Membrane Some Receptors Activate a Fast Track to the Nucleus Protein Kinase Networks Integrate Information to Control Complex Cell Behaviors Signaling pathways can be highly interconnected (cross-talk) Protein kinases in both pathways phosphorylate many proteins, including proteins belonging to the other pathway Some intracellular signaling proteins serve to integrate incoming signals activatethesame moleculeimegrate integrate Figure 16-43 Essential Cell Biology (© Garland Science 2010)  SUMMARY insulin ▪ Receptor tyrosine kinases (RTKs), bind to peptide hormones and may exist as dimers or dimerize during binding to ligands. ▪ Ligand binding leads to activation of the kinase activity of the receptor and autophosphorylation of tyrosine residues. ▪ Ras is an intracellular GTPase switch protein that acts downstream from most RTKs. Like Gsα , Ras cycles between an inactive GDP-bound form and active GTP-bound form. ▪ Unlike GPCRs, which interact directly with an associated G protein, RTKs are linked indirectly to Ras via two proteins, GRB2 and Sos. ▪ The SH2 domain in GRB2, an adapter protein, binds to specific phosphotyrosines in activated RTKs. ▪ Normally, Ras activation and the subsequent cellular response are induced by ligand binding to an RTK. However, in cells that contain a constitutively active Ras, the cellular response occurs in the absence of ligand binding. ▪ MAP kinase, activated via the RTK-Ras pathway, translocates to the nucleus, where it phosphorylates various transcriptional activators and repressors. MAP kinase phosphorylation promotes the activity of some transcription factors and inhibits the activity of others.