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Cell Signalling.doc

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Western University
Biology 2382B
Sashko Damjanovski

Cell Signalling Signal Transduction: conversion of 1 signal into another - Involves growth factors, cytokines, hormones, ECM, neurotransmitters, etc. - Complex mechanism that can lead to many diseases (cancer) For cell signalling to occur you need: 1. Signal/signalling molecule: Ligand, primary messenger 2. epinephrine, acetylcholine, steroids, peptides, hormones 3. Receptors: cell surface receptors, intracellular receptors 4. Intracellular signaling and effector proteins : G proteins, protein kinase and phosphatases 5. Second messengers: calcium, cAMP, cGMP, IP3, DAG, NO - cell surface receptors are proteins, transmembrane proteins that bind hydrophilic signal molecules (hydrophilic molecules cannot cross membrane) - intracellular receptors (hydrophobic) require a carrier protein which comes in close proximity of cell, it then releases molecule and crosses plasma membrane Nuclear receptor superfamily - lipid soluble hormones bind to intracellular receptors which constitute the nuclear receptor superfamily of transcription factors - steroid hormones are synthesized by adrenal glands - all have Ligand binding domain and DNA binding domain - some are found in cytoplasm (estrogen receptor, progesterone, glucocorticoid) and some in nucleus (thyroxine receptor and retinoic acid receptor) Example: GR= glucocorticoid receptor - in the absence of hormone, the receptor is trapped in the cytoplasm by inhibitor protein (I.e. HSP90) - hormone binding to nuclear receptor releases the inhibitor protein, allowing the receptor to enter the nucleus - receptor binds to a response element of the target gene and stimulates preinitiation complex assembly required for transcription (mRNA synthesis) - bind ligands in cytosol and act as transcription factors Intercellular signaling 1. Endocrine: signaling to distant cells (Signaling molecules are synthesized and secreted by endocrine cells and transported through the circulatory system to target cells) 2. Paracrine: signaling to nearby cells (signaling molecules released by a cell affect only those target cells in close proximity ex. Neurotransmitters) 3. Autocrine: signaling to cell itself (cells respond to substances that they themselves release) Signaling by cell-surface receptors (Signal Transduction) - synthesis and release the signaling molecule by the signaling cell - transport and binding the signal to a specific receptor to the target cell (CHANGE IN CONFORMATION) - initiation one or more intracellular signal transduction pathway - Short term cellular response: changes in the activity/function of specific enzymes and other proteins that pre-exist in the cell - Long term cellular response: changes in the amounts of specific proteins produced by the cell, most commonly by modification of transcription factors that stimulate/repress gene expression. Cells are dependant on multiple extracellular signal molecules - survival - growth and division - differentiate - death (apoptosis) Ligand receptor interactions - binding specificity is based on the molecular complementarity between the interacting surfaces of a receptor and Ligand - triggers conformational change in receptor which activates it - often signaling molecules (ligands) induce receptor dimerization - Kd: measure of affinity of a receptor to its ligand (ligand concentration required to bind 50% of the cell surface receptors) - lower Kd= lower ligand concentration required to occupy 50% of receptors - if receptor and ligand bind easily then you will need little ligand present in ECM for receptor to bind  low Kd because better binding - if receptor and ligand don’t bind easily then you need to saturate ECM with ligand in order to initiate better binding  high Kd Functional expression assay to identify a cDNA encoding a cell receptor - cultured cells do not express receptor for ligand - transfect cells with cDNA library and screen for cellular phenotype associated with ligand - identify incorporated cDNA by PCR followed by sequencing - the receptor protein can be deduced from the cDNA sequence - can perform further studies by mutating specific AA to determine essential ligand binding domain G Protein Coupled receptor system - receptor with 7 membrane domains - trimeric G protein - cytosolic domains interact with G proteins - 3 subunits: Galpha, Gbeta and Ggamma - Gα and Gy are lipid anchored proteins at the cytoplasmic face of plasma membrane - Gα-GDP is inactive, Gα-GTP is active FRET experiment with G proteins - cells are transfected with genes encoding 2 fusion proteins: - Ga is fused to CFP (cyan fluorescent) - Gb is fused to YFP (yellow fluorescent) - GPCR that regulate adenylyl cyclase: cAMP, epithelial cell chloride channels, PKA, CREB - GPCR that activate phospholipase C : Calcium, IP3, DAG, PKC, NO, cGMP, PKG Regulation of Adenylyl Cyclase Example 1: Cholera Toxin - toxin enters small intestine using a GM1 receptor - toxin maintains G stimulatory alpha in active state (GTP)  mass activation of adenylyl cyclase  increase in cAMP levels - increase of cAMP levels leads to hyperactivity of CTFR ion channel - Chlorine pumped out  sodium follows and water loss to intestine  diarrhea and dehydration Example 2: Whooping cough (Pertussis Toxin) - toxin enters ciliated epithelial cells in LUNGS - toxin maintains G alpha inhibitory in inactive state (GDP) - activation of Adenylyl Cyclase - increase in cAMP levels  increase activity in ion pumps  mucous secretion and electrolyte/H20 accumulation in lungs Fight or Flight Response - cAMP activates protein Kinase A (PKA) catalytic subunits - 1 molecule of epinephrine can generate a lot of molecules of cAMP (amplification of signal Adrenaline rush  glucose liberation  heart muscle contraction) Regulation of glycogen metabolism by cAMP Activation of gene transcription by GPCR - cAMP and PKA can induce not only short term effects but also long term effects - gene regulated by PKA contains a specific nucleotide sequence known as cAMP response element (CRE) - Activation of CREB transcription factor (CRE-binding protein) by phosphorylation in nucleus CREB signaling pathway - involves G-protein activation - increase in cAMP levels - activation of PKA - PKA translocation to nucleus - Phosphorylation of CREB - P-CREB binds as a dimer to cAMP response element (CRE) - P-CREB also binds CBP/P300 co-activator - CBP/P300 recruits transcriptional machinery Phospholipase C and second messengers - Phospholipase C (PLC) cleaves PIP2 into DAG and IP3 - PLC is an effector which is activated by G proteins - DAG is cytosolic leaflet (stays there) IP3/DAG signaling pathway and Calcium - signal molecule binds its GPCR - activates G alpha proteins - stimulation of PLC occurs - PLC cleaves PIP2 into DAG and IP3 - IP3 shuttles to ER membrane and opens Calcium channels - Levels of calcium ions increase  bind protein kinase C - PKC translocates to PM where it interacts with DAG - Active PKC phosphorylates numerous substrates including players that activate MAPK NO, cGMP and PKG (smooth muscle pathway) - stimulation of acetylcholine G protein coupled receptors on endothelial cells induce an increase in cytosolic calcium and subsequent synthesis of NO - after diffusing into surrounding smooth muscle cells, NO activates an intracellular guanylate cyclase (NO receptor) to synthesize cGMP - the resul
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