Cell Structure and Function Test 3 extra notes from textbook Lecture 21 Cell Signaling Many extracellular signaling molecules are synthesized and released by signaling cells within an organism In all cases signal molecules produce a response ONLY in target cells that have receptors for signaling molecules Most receptors bind a single molecule or a group of related molecules The signaling molecule acts as a ligand binds to a structurally complementary site on extracellular or membrane spanning domains of receptor o Binding conformational change in receptor that is transmitted through membrane spanning domain to cytosolic domain subsequent activation inhibition of other proteins in cytosol or attached to plasma membrane o Whole process Signal Transduction Activation of receptors on Target cell o When a signaling molecule arrives at a target cell it binds to a receptor o Activated by binding of secreted or membrane bound molecules o Some activated by changes in concentration of a metabolite O2 or nutrients or by physical stimuli light touch heat o Ligand Gprotein coupled receptor triggers intracellular protein G protein to exchange one bound GDP nucleotide for GTP o GTP binding conformational change Affects interaction of G protein with downstream signal transduction proteins Cell surface receptors generation of second messengers o The response of a cell or tissue to specific external signal dictated by a cell s complement of receptors that can recognize signals signal transduction pathways and intracellular processes affected by those pathways o Receptor binds to ligand with specificity G protein Coupled receptors GPCRs Modular system o Binding of ligands to many cell surface receptors leads to short lived increase decrease in concentration of certain low molecular weight intracellular signaling second messengers bind to other proteins modifying their activity o Cyclic AMP cAMP rise activation of protein kinase various changes in cellular metabolism in different types of cells o camp can also regulate activity of ion channels o second messengers diffuse through cytosol way faster than proteins employment in pathways where the downstream target is located in intracellular particle or organelle distant from plasma membrane o advantage of second messengers they can facilitate amplification of receptor an extracellular signal o activation of single cell surface receptor molecule increase in thousands of camp molecules Ca2 ions in cytosol activation of downstream proteins Seven TM domain receptors o ALL GPCR signaling pathways share the following common elements Receptor that contains 7 membrane spanning domains A couple trimeric G protein functions as a switch by cycling between active and inactive forms A membrane bound effector protein Feedback regulation and desensitization of signaling pathway Structure model of the epinephrine and B2 adrenergic receptor complex o Epinephrine binds in the middle of the plane of the membrane interacting with amino acids in the interior facing side of several of the membrane spanning alpha helices G protein On Off switch o GPCRs activate exchange of GTP for GDP on the alpha subunit of a o Intracellular signaling Beta and gamma subunits remain bound trimeric Gprotein together o Resting state when no ligand is bound to receptor G alpha subunit has a bound GDP and is complexed with Gbeta and Ggamma o Binding of ligand changes conformation of its cytosol facing loops and enables receptor to bind to Galpha subunit GTP rapidly binds to empty guanine nucleotide site in Galpha subunit conformational change of its switch segments weaken Galpha binding with both receptor and Gbetagamma subunit G protein activation o in most cases Galpha GTP remains anchored in membrane interacts with and activates and associated effector protein active state GTP hydrolyzed to GDP in minutes catalyzed by intrinsic GTPase activity of Galpha conformation of Galpha switched back to GDP Galpha inactive blocking of further activation of effector proteins reduces duration of effector activation and avoids cellular o in some cases it can inhibit an associated effector protein Experimental Approach FRET o Activation of G proteins occurs within seconds of ligand binding in overreaction amoeba cells o cAMP acts as an extracellular signaling molecule and binds to a G protein coupled receptor it isn t a second messenger o Amoeba cells were transfected with genes encoding 2 fusion proteins Galpha fused to cyan fluorescent protein and a Gbeta fused with another GFP variant yellow fluorescent protein YFP o Irradiation of resting cells with light causes emission of yellow light YFP o If ligand binding leads to dissociation of Galpha and Gbetagamma subunits fluorescent energy can t occur irradiation of cells o Drop in yellow fluorescence dissociation of Galpha CFP fusion protein from Gbetagamma YFP fusion protein within seconds of cAMP emission of CFP addition Toxins target cell signal systems cholera toxin o Some bacterial toxins contain a subunit that penetrates plasma membrane of target mammalian cells and in cytosol catalyzes a chemical modification of Galpha proteins that prevents hydrolysis of bound GTP to GDP o Galpa remains active continuous activation of adenylyl cyclase in absence of hormone stimulation excessive rise in intracellular cAMP loss of electrolytes and h2o in intestinal lumen watery diarrhea Mechanism of cAMP second messenger production o Galpha and Gbetagamma of trimeric G proteins tethered to membraned by covalently attached lipid molecules o Following ligand binding exchange of GDP with GTP dissociation o Free Galpha GTP binds to and activates an effector protein o Hydrolysis of GTP terminates signaling reassembly of trimeric G of G protein subunits protein resting state o Binding of another ligand molecule repetition of cycle Gsalpha GTP activation of AC o Gsalpa GTP interact with adenylyl cyclase o Multipass transmembrane protein o 2 regions of Gsalpha switch II helix and alpha3 beta5 loop contact adenylyl cyclase fragments responsible for activation of enzyme Gsalpha GTP o one of the segments of Galpha protein conformation different in GTP bound and GDP bound states o GTP induced conformation of Gsalpha dissociation from Gbetagamma conformation essential for binding of Gsalpha to adenylyl cyclase Mechanism of cAMP second messenger production o Ligand binding to Gsalpha coupled receptors activation of adenylyl cyclase o Ligand binding to Galphai inhibition of enzyme