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Lecture 6

Lecture 6 - Feb 25 - BIO 2B03.docx

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Gautam Ullal

BIO 2B03 2013 Lecture 6 – Feb 25 Signal Transduction  Cell-Cell Signaling – transmitting information from one cell to another and inducing a change in behaviour (response) o Signal may be received by different cells and elicit different responses  Eg/ Class Example – Dictyostellium cells – loose aggregate forms; can grow, fall over into “slug”; mature cell forms stalk and body (spore cells located); cAMP signal, G-protein receptor; response to signaling – chemotaxis (neutrophil attracted to bacterial cells, find bacteria and eliminate them from the body)  Cells communication by: o Direct cell-to-cell contact, junctions o Cell-to-cell via plasmodesmata in plants o Extracellular signaling molecules  Signaling molecules: o Produced by signaling cells o Induce a specific response in target cells with receptors for ligand  Responses include: o Changes in gene transcription, cell division, growth, differentiation, changes in shape, movement, changes in metabolism  Signal Transduction – process of converting extracellular signals into a cellular response; interpreting the signal o Fast response – changes in enzyme activation  Activated enzyme can change cell movement, shape, part of metabolic pathway etc  Fast – just need to modify protein (already in the cell); just needs to be switched on o Slow response – changes in gene transcription  Slow – many processes before cellular change can occur  Receptor must be activated; gene expression activation (mRNA made and translated); fold and modify protein  Signaling molecules and cell-surface receptors o Signaling cells – source of signal or ligand o Target cells – receptors that receive signal o Ligand + receptor – specificity and affinity are determined by molecular complementarity o Signal transduction pathway (STP) = signal  cellular response  Types of Intercellular Signaling a) Endocrine Signaling  Signaling molecules = hormone (soluble extracellular molecules)  Target = distant  Soluble signal released by signaling cell into circulatory system; signal can go all around the body; only cells with appropriate receptor can respond  distant cells can communicate  Inefficient – make a lot of signal but only a fraction of cells can respond b) Paracrine Signaling  Signaling molecules = eg/ growth factors, neurotransmitters (soluble extracellular molecules)  Target = proximate (two adjacent cells) 1 BIO 2B03 2013 a. Other Proximal Signaling  Integral membrane proteins o Signaling molecule = membrane bound (not soluble signal) o Target = neighbor; two cells are physically associated with one another through signal-receptor interaction (adhesion)  Cell-to-cell signaling via plasmodesmata in plants o Target = neighbor o Establishment of vascular system  Gap junctions direct cell-cell contact in animal cells o Target = neighbor c) Autocrine Signaling  Signaling molecule = growth factors (disruption may lead to tumorigenesis) (soluble extracellular molecules)  Target = self  Cells respond to signals they produce themselves  Common to induce cell growth and cell division (eg/ cell grows larger, reaches threshold size, signals to self to undergo mitosis)  Neighboring cells will not be signaled because they may not have receptors or molecular complementarity  Mechanisms of signal transduction are highly conserved 1. Signal synthesis 2. Release 3. Transport 4. Receptor binding – activation (receptor will respond; changes shape, localization on cell etc; change leads to STP) 5. Initiation of a signal transduction pathway (STP) 6. Changes in cellular function:  Two major types of responses: (there may be more than one response)  Changes in protein activity  Change in protein levels (via transcription) 7. Removal of signal – if signal isn’t removed, have uncontrolled communication system  Specificity of a signal response achieved by: o Ligand binding specificity – binding to receptor  A receptor generally binds only one signaling molecule or a small family of close related molecules  Related cells may have similar receptors – may respond to one signal but with different response (eg/ different changes in behaviour) o Effector specificity – intracellular response  There is a unique cellular response to ligand-receptor association on a specific cell 2 BIO 2B03 2013  Some signaling molecules bind to receptors on different cells inducing alternate responses via alternate STPs  Ligand Binding and Effector Specificity o Signaling molecule = ligand or 1 messenger o Ligand binding to report – molecular complementarity o Induces conformational change in receptor o Initiates a cascade of reactions o Produces cellular response o Molecules associated with one another – molecular complementarity; shapes fit, non-covalent bonds o Figure – yellow space filling = residues important for interaction with hormone; change one of them, can no longer bind to signal, eliminate signal transduction pathway; pink = emphasis on AA residues, important for signal-receptor binding  Cellular response may not require binding to all receptors o Typical cell has 10 to 10 receptors o KD = dissociation constant of Receptor-Ligand complex  Also [L] where half receptors occupied  Lower K D higher affinity o Ligand [L] needed to induce maximal cell response is lower than amount needed to saturate receptors o Response Level of Cell – out of all the ligand, what fraction are bound to a signal  Large physiological response, even when not all receptors occupied – eg/ 100 cells, 50% receptors filled, 80% are responding to signal  Cell-surface receptors; seven different types (different in morphology and location) o G-Protein Coupled Receptors (GPCR)  Ligand binding activates G protein  Causes activation of 2 enzyme (effector)  Second messengers generated  Largest class o Receptor Tyrosine Kinases  Ligand causes receptor dimerization  Phosphorylates self and other substrates and changes their activity o Tyrosine Kinase-Linked Receptors = Cytokine Receptor  Ligand causes receptor dimerization  Dimer now interacts and activates cytosolic kinas  Phosphorylation can change activity of a cellular protein  Types of enzymes in signaling pathways – protein kinases and GTPase Switch Proteins Signaling pathways  Objectives o Describe key steps I signal transduction pathways  Cytokine receptor and JAK-STAT  Receptor tyrosine kinase (RTK) and Ras 3 BIO 2B03 2013  G-protein coupled receptor (GPCR) o Identify similarities and differences between these pathways o Predict and interpret the effects of mutations in signal transduction pathways (STP) 1. Signaling growth and differentiation: Formation of red blood cells  Eg/ Cytokine receptors and JAK-STAT pathway  Cytokine – (small secreted signal proteins)  Erythropoietin (EPO) signals erythroid progenitor cells in the blood marrow to reproduce; without Epo, erythroid progenitors undergo apoptosis (cell death)  Leads to an increase in RBCs  Low O 2evels cause synthesis and release of Epo  Oxygen sensitive transcription factor in kidneys  Ligand = cytokine, Epo  Receptor = Epo receptor (EpoR)  Intracellular Transduction – JAK kinases and STAT transcription factors  Cellular Response – transcription of STAT target genes  Dimerization and phosphorylation are key steps in activation of the cytokine pathway o Phosphorylation leads to conformational change in activation lip of JAK kinase that increases enzyme activity o Phosphorylation of tyrosine’s in EpoR occurs o Phosphorylation of tyrosine’s on EpoR at docking site allows binding of the STAT transcription factor to Phos-EpoR SH2 domain – recognizes phosphorylated tyrosine  P-Try containing peptide – Pro-Asn-pTyr-Glu- Glu-Ile-Pro  Yellow = backbone; green = R-groups; blue = phosphate  Tight fit (molecular complementarity) is only achieved when tyrosine is phosphorylated: P- Tyr and Ile fit into binding pockets  Protein-Protein Interactions – based on molecularlar complementarity o Domains identified that recognize specific sequenes on their target proteins o Protein interactions are requred for the assembly of signallign complexes  Eg/  A) SH2 domains recognize phospho-tyrosine  C) PDC domains recognizing hydrophobic C-Terminus  Target genes of STAT transcrption fators o In erytrhoid progenitors, STAT5 increases transcription of Lcl-x which prevents death of these cells; instead they become mature erythroid cells (production of erythrocytes) 4 BIO 2B03 2013 o Mutations in pathway:  Turning off the signal – eg/ from EpoR pathway  A) SHP1 Phosphatase; short term regulation  B) Protein degradation by SOCS; long term regulation 2. Signaling cell proliferation, cell differentiation, cell survival/apoptosis, cell metabolism – eg/ receptor tyrosine kinase (RTKs) pathway and activation of Ras G-protein  Ligands/Signals include hormones: o Nerve growth factor (NGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF) insulin  Activate intrinsic kinase activity of transmembrane cell surface receptors  From signal perception to cellular response  Ligand – growth hormone  Receptor – RTK  Intracellular transduction o Adaptors = GRB2 o Ras proteins (GTPase) o Ras effectors = GEF and GAP o Cascade of kinases o NAP Kinas o Transcription Factors – modified by phosphorylation  Cellular Response – Transcription 5 BIO 2B03 2013  RTK activation o RTK – monomeric, single pass, transmembrane receptor o Ligand binding  dimerization of receptor o EGF = hormone; interacts with 2 receptors and causes them to dimerize  1 molecular of EGF binds to receptors – changes conformation to form dimerization loop  Two hydrophobic helices interact with each other o Receptor autophosphorylation of tyrosine residues o Phosphotyrosines serve as docking sites for adapter proteins o Adaptor plus Ras protein couples RTKS to other components of signaling pathway o Lateral diffusion of receptors – receptors brought close; kinases can phosphorylate each other o Inactive docking site (masked)  active docking site (visible)  Adaptor Proteins – contain protein-protein interaction domains (eg/ SH2, PTB) but do not have enzymatic or signaling activity o Couple RTKs to other proteins in pathway o Scaffold proteins – many protein-protein interaction domains to assemble many proteins o Signal from membrane into nucleus to change level of transcription o Proteins bind to docking site  Do not bind in absence of phosphates  would remain in cytosol and not accumulate on underside of membrane o Adaptor Protein: GRB2  One SH2 domain (Src Homology)  Recognizes phosphorylated tyrosine  Binds residues C-terminal to P-Tyr on RTK  Two SH3 domains  Recognizes Pro-rich sequences  Binds SOS via SH3 domains  Causes relocation of SOS from cytosol to membrane  SH3 domain – binds proline-rich peptides 6 BIO 2B03 2013 Red = SH3 domain Yellow = backbone Green = proline side chains  GTPase Switch Proteins or G-Proteins o GTP-binding proteins  Active in GTP-bound state – arms interacting by non-covalent bonds; terminal phosphate when GTP bound to binding site  Inactive in GDP-bound state – terminal phosphate missing when GDP in binding site o Signals stimulate  GDP release  Leading to binding of GTP (GTP concentration high in cell) o Cycle GTP and GDP – when no nucleotide in binding pocket – GTP in high abundance (and higher affinity than GDP) will enter nucleotide binding site  GTP hydrolyzed to GDP  GDP low affinity for binding site leaves  Ras GTPase - member of large superfamily of GTP-binding switch proteins with intrinsic GTPase activity – monomeric GTPase
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