Cell Structure and Function: Test 3 extra notes from textbookLecture 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 receptoro 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 membraneo Whole process= Signal Transduction• Activation of receptors on Target cello When a signaling molecule arrives at a target cell, it binds to a receptor o Activated by binding of secreted or membrane bound moleculeso 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 GTPo GTP binding = conformational change Affects interaction of G protein with downstream signal transduction proteins• Cell surface receptors- generation of second messengerso 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 pathwayso Receptor binds to ligand with specificity• G- protein Coupled receptors (GPCRs)- Modular systemo 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 activityo Cyclic AMP ( cAMP)  rise= activation of protein kinase= various changes in cellular metabolism in different types of cellso 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 receptoro advantage of second messengers they can facilitate amplification of an extracellular signalo activation of single cell surface receptor molecule= increase in thousands of camp molecules/ Ca2+ ions in cytosol= activation of downstream proteins• Seven TM domain receptorso 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 complexo 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 switcho GPCRs activate exchange of GTP for GDP on the alpha subunit of a trimeric Gproteino Intracellular signaling  Beta and gamma subunits remain bound togethero Resting state  when no ligand is bound to receptor G alpha subunit has a bound GDP and is complexed with Gbeta and Ggammao 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 activationo 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 overreactiono in some cases it can inhibit an associated effector protein• Experimental Approach: FRETo Activation of G proteins occurs within seconds of ligand binding in amoeba cellso 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 YFPo If ligand binding leads to dissociation of Galpha and Gbetagamma subunits= fluorescent energy can’t occur = irradiation of cells= emission of CFPo Drop in yellow fluorescence= dissociation of Galpha- CFP fusion protein from Gbetagamma-YFP fusion protein within seconds of cAMP addition• Toxins target cell- signal systems- cholera toxino 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 GDPo 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 productiono Galpha and Gbetagamma of trimeric G proteins tethered to membraned by covalently attached lipid moleculeso Following ligand binding exchange of GDP with GTP dissociation of G protein subunitso Free Galpha-GTP binds to and activates an effector protein o Hydrolysis of GTP= terminates signaling= reassembly of trimeric G protein= resting stateo Binding of another ligand molecule= repetition of cycle• Gsalpha- GTP activation of ACo Gsalpa-GTP interact with adenylyl cyclaseo Multipass transmembrane proteino 2 regions of Gsalpha, switch II helix and alpha3-beta5 loop contact adenylyl cyclase fragments responsible for activation of enzyme Gsalpha-GTPo 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