Class Note for BIOC 460 at UA 4
Class Note for BIOC 460 at UA 4
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Date Created: 02/06/15
Bioc 460 Dr Miesfeld Spring 2008 Signal Transduction 1 Supplmental Reading Key Concepts Components of signaling pathways Small molecules function as diffusible signals First messengers Second messengers Receptor proteins are the gatekeepers of the cell Gprotein coupled receptors Heterotrimeric G proteins Gag activation of adenylate cyclase Gaq activation of phospholipase C KEY CONCEPT QUESTIONS IN SIGNAL TRANSDUCTION What is the role of receptor proteins in signal transduction What is the role of second messenger molecules in cell signaling pathways Biochemical Applications of Signal Transduction Botulinum Botulism Toxin A blocks neuromuscular signaling through TOX39nTypeA acetylcholine receptors and is lethal if ingested in contaminated food The toxin is a protease that prevents acetylcholine release from nerve cells Botox is a recombinant form of Botulism Toxin A used to treat migraine headaches and to temporarily smooth out facial wrinkles 7amp9 Components of Signaling Pathways Biological diversity is based on complexity and with this complexity comes the need for high level organization With the evolution of multicellular organisms cell signaling processes became much more complex to allow cells in different tissues to communicate physiological changes that affect the entire organism The molecular basis ofthis communication process is a biochemical circuit or relay consisting of protein structural changes that are initiated by the binding of small biomolecules to large receptor proteins which trigger a cellular response In many cases the primary cell signaling event is the specific binding ofa hydrophilic biomolecule to a receptor protein imbedded in the plasma membrane However some signaling events involve activation of soluble proteins such as the intracellular receptors which are activated by steroid hormones or direct activation of a signaling protein such as guanylyl cyclase which is one of the targets of nitric oxideBiomolecules with this property are called receptor ligands and are represented by hormones such as insulin and neurotransmitters like acetylcholine The name given to the receptor protein reflects the ligand that activates it For example insulin binds the insulin receptor and acetylcholine binds to the acetylcholine receptor Another major form of cellcell communication is mediated by lipophilic hydrophobic receptor ligands derived from cholesterol which pass through the plasma membrane and bind to intracellular receptor proteins Examples of intracellular receptors are the estrogen receptor the glucocorticoid receptor and the vitamin D receptor 1 of13 pages As illustrated in figure 1 in addition to receptors target proteins and ligands which function in signaling pathways as first messengers signal transduction also involves intracellular biomolecules called second messengers and a variety of intermediary signaling proteins that function to transmit amplify and terminate the signal Small biomolecules function as diffusible siqnals lnsulin is an example of a first messenger because it binds directly to the insulin receptor Second messengers such as calcium cyclic AMP cAMP and cyclic GMP cGMP diacylglycerol DAG and inositol 145trisphosphate IP3 function to amplify the biochemical signal First Messengers The simplest types of first messengers are small diffusible biomolecules that act at a distance Bioc 460 Dr Miesfeld Spring 2008 Figure 1 O Firstmessenger l Upstream 39 Receptor protein ll magma a Upstream membrane Signa ng rr proteins Signaling Pathway Second V E E S messengers B E A Downstream signaling p L proteins 39 v 0 r g 2quot vs f G Downstream 0 Enzyme ym kglem Protein protein Target Proteins through endocrine mechanisms or that function W locally as paracrine or autocrine signals figure 2 Cells aleacrrejtory Molecular structures of several first messengers important in 39 Hmone biochemistry are shown in figure 3 Human growth hormone 39 4 and insulin are peptide hormones cortisol is a steroid that is Circulale derived from cholesterol epinephrine also known as l39i 51 ENDOCRINE adrenaline is a metabolite ofthe amino acid tyrosine acetylcholine is a neurotransmitter synthesized from choline and acetyl CoA in neurons and nitric oxide is a soluble gas 39 N that activates signaling pathways by diffusing across cell membranes and directly activating signaling proteins Figure 3 First Messengers insulin Human Growth Hormone Cortisol Nitric Oxide 1 l 39 IV Rewrite n I i d Targ t UTOC tissues A EllLquot Wells Growth Kj i factor PliRAczriE C39H t T l Adrenaline iHa H3C CH3 N Atotylcholinc 0 CH1 0 2 of 13 pages Bioc 460 Dr Miesfeld Spring 2008 Nitric oxide NO which is made in g nerve cells by the enzyme NO fa o 39f gug synthase through the deamination has NO bound of the amino acid Larginine The kissing bug Rhodnius proixus 7 quot delivers NO to their victims by 39 injecting hemecontaining proteins Low pHinthe saliva 539 Higher pH in the host blood gt NO Histaminex Q called nitrophorins that carry NO 7 ml ll into the wound along with their 4 A The hemegroup quot AC saliva see figure 4 NO binds to Plndshistamine guanylyl and activates guanylyl cyclase Kissing Bug m place OfNO L Cyc39ase which generates cyclic GMP that functions as a second messenger in the vasodilation response In J CGMP GTP this context the uan l lc clase WNW 39 u g y l y 2 739 r 739 V 4 enzyme IS the receptor for the AntiInflammation I ampVaOdIatlonJ NO signal quot r i I J quotMVquot Second messengers The primary role of second messengers is to amplify the receptorgenerated signal to more quickly obtain a maximal response One of the best characterized second messengers is cyclic AMP cAMP which is produced by the enzyme adenylate cyclase from ATP Figure 5 shows structures of some second messengers As shown in figure 6 receptor activation of adenylate cyclase generates large amounts of cAMP which in turn binds to and activates downstream signaling proteins such as cAMP dependent protein kinase A PKA lmportantly the intracellular concentration of cAMP is carefully controlled by the relative levels of receptoractivated adenylate cyclase and soluble forms of cAMP phosphodiesterase PDE which converts cAMP to AMP figure 7 Fidure 5 Second Messengers Figure 6 Receptor activation of adenvlate CVClase o t 0 PH P R2 Receptor Activation Ho 7 3 030 L 0H0 0 XR1 0 4 H 0 olt HZC O AMP O 1 Adenylate cyclase i 0 0 Activation 0 0 Hzc OH 2 CAMP Diacy glyceml 39 phosphodiesterasePDE lnositol 145triphosphate lnsP ATP pp m N JR Enzymel K1 j N PKA 39 2 22 o N N39 Enzyme2 39 H es P 7 H Enzyme 3 oH OH Ph Cyclic adenosine monophosphate CAMP 1 o if 3 of 13 pages Figure 7 Interconversion of ATP CVClic AMP CAMP and AMP NHZ Idenylnte cyclase 0 if 9 OETO TCquotO CH2 0 K V ATP OH O H Bioc 460 Dr Miesfeld Spring 2008 NHv cAMP phosphodiesterase 39 quot2 cI i c C e I 339539cyclchMP P 5 AMP 0Po o 0n Another second messenger important in signal transduction is cyclic GMP which is produced from GTP by the enzyme guanylyl cyclase figure 8 The cGMP analog Sildenafil also know as Viagra is used to treat sexual dysfunction by inhibiting the activity of cGMP phosphodiesterase PDE The molecular structure of sildenafil is similar to cGMP and binds tightly to cGMP PDE Notice that the NO signal neuronal input must be intact to initiate the vasodilation process Fidure 8 Viadra brolonds vasodilation bv preventing the breakdown of CVCIic GMP followind nitric oxide stimulation Neuornal Activated GTP input by nitric oxide l Activated PPi NO guanyy ACTIVGTEd Nitric Cydase oxide N0 cGMP gt cGMP PTOte39 v Vasodilation kmase G Increased blood flow cGMP gt Viagra Phospho V39agm quot diesteraseE 39 I Inactivated Drug by Viagra GMP treatment Figure 9 Three other second messengers important to Wl metabolic control are diacylglycerol DAG Q inositol 145trisphosphate IP3 and calcium ion Ca2 As illustrated in figure 9 the intracellular levels of DAG P3 and Ca2 are controlled by the activity of a membrane associated enzyme called phospholipase C PLC Receptormediated activation of phospholipase C leads to cleavage of the membrane phospholipid phosphatidylinositol 45bisphosphate PIP2 to form DAG and IP3 We will come back to this signaling pathway at the end of the lecture when we examine an signaling through the x1 adrenergic receptor see figure 23 PIPz V Activated l protein kinaseC 3 1 I L gquot Activation of CaZ channels in the endoplasmic reticulum j Enzyme dam 3L e ran x Calmodulin 4 of 13 pages Bioc 460 Dr Miesfeld Spring 2008 Receptor proteins are the Gatekeepers of the cell Using bioinformatic approaches it has been estimated that 510 of the estimated 32000 genes in the human genome encode proteins involved in signal transduction The majority ofthese genes encode receptor proteins ion gated channels peptide hormones and protein kinases Another abundant class of signaling proteins encoded in the human genome are G proteins which are small GTPases involved in transmitting intracellular signals from G protein coupled receptors to signaling enzymes such as adenylate cyclase and phospholipase C Figure 10 illustrates how these five classes of receptors function in signal transduction Five maior classes of receptor proteins have been characterized 1 G protein coupled receptors 2 Receptor tyrosine kinases 3 Tumor Necrosis Factor TNF family receptors 4 Ion channel receptors 5 Intracellular receptors nuclear receptors Figure 10 Receptor TNF family Ion channel tyrosine kinases receptors receptors 6 protein 39 coupled receptors 39 u 1H1 s E ng Ada tor Adaptor Heterotrimeric p proteins lon flow proteins G protein Intracellular receptors Gene regulation G protein coupled receptors G protein coupled receptors consist of a central core that encodes seven consecutive transmembrane D helices The bundled arrangement ofthe D helices orients the amino terminus of the receptor protein towards the extracellular space with the carboxy terminus exposed to the cytosol Figure 11 shows a diagram of the seven transmembrane 0t helical regions of G protein coupled receptors 5 of 13 pages Bioc 460 Dr Miesfeld Spring 2008 Figure 11 Membrane organization of G protein coupled receptors iiimrtii As shown in figure 12 ligand activation of a Gprotein coupled receptor causes a conformational change in the associated heterotrimeric G protein complex which consists of three subunits called the Ga GI3 and Gv subunits Receptor activation by hormone ligand binding stimulates the release of GDP and subsequent binding of GTP to the Ga subunit The activated GaGTP subunit dissociates from the GM complex and both the GQGTP and GIiv components regulate downstream signaling proteins Fi ure12Activation ofG rotein cou led rece tor si nalin b li and bindin D Receptor Activation G protein coupled receptor GTEGDP Exchangequot M Subunit dissociation Gum heterotrimeric complex Gm GOL GTP Regulation of effector proteins Heterotrimeric G proteins Signaling through G protein coupled receptors requires a physical interaction between the 43 kDa G a 37 kDa GIS and a 8 kDa Gv subunits Figure 13 illustrates how the subunit composition of the heterotrimeric G protein determines what downstream signaling pathways will be activated by GTP binding and subunit dissociation 6 of 13 pages Bioc 460 Dr Miesfeld Spring 2008 Figure 14 shows the molecular structure of a typical heterotrimeric complex in the inactive conformation GDP bound to the Ga subunit Note that the Ga and GY subunits are tightly associated with each other through alpha helices creating a GM interface that makes additional high affinity contacts with the Ga subunit Figure 13 Figure 14 plasma membrane G proteins are inactive with GDP bound to the oc subunit 0c subunit k I Activate 39 Jtd Regulate ion channels adenylate St39mmate CGMP Stimulate Phospholipase A cyclase phospho este39ase Regulation receptor kinases H Regulate Q 4 Rhosi nalin Inhibit g g Stimulate YSUbumt adenylate phospholipase C cyclase Figure 15 H 2 Active 7 Active Gq GTP quot effeCtF r r roteln How does the G protein signal get turned off H O a y i I 2 v 4 The GTPase aclt39v39tyln Ga ISUbunltS IS IEffectoror RGS protein stimulation stimulated by interactions With the effector Pi of intrinsic Gd GTPase activity protein for example phospholipase C or quot adenylate cyclase which therefore limits the amount of time the second messenger signal is nactive transduced figure 15 Once the GTP is G WOW I hydrolyzed the inactive GelGDP complex reassociates with GM and the receptor It has been found that the GTPase activity of Ga can Reassociation emu also be stimulated by interactions with a class of 5 with Gmand GPCR regulatory proteins called RGS proteins regulator of G protein signaling RGS proteins are members of a family of signaling proteins called GAPs GTPase activating proteins 7 of 13 pages Bloc 460 7 Dr Mlesfeld Spring 2008 Gs activation of adenylate cyclase Now that we have seen how ligand activation ofthe receptor stimulates signal transduction through the heterotrimeric G protein we need to look at an entire signaling pathway to see how it all ts together We will do this by rst examining Gsa signaling through the 2 adrenergic receptor and glucagon receptor and then compare this to an signaling through the 11 adrenergic receptor Interestingly in human liver cells all three G protein coupled receptors stimulate glucose release into the blood even though different hormones epinephrine or glucagon and downstream effector proteins adenylate cyclase or phospholipase C are utilized As illustrated in gure 16 glucagon and epinephrine activate a cAMPmediated signaling pathway by stimulating adenylate cyclase AC activity utilizing Gsa In contrast activation of the X1 adrenergic receptor by epinephrine is dependent on an which stimulates phospholipase C PLC activity leading to the production ofthe second messengers DAG and IP3 Signal transduction through the 32 adrenergic and glucagon receptors represents activation ofa shared pathway by different hormones In contrast epinephrine signaling through the x1 and p2 adrenergic receptors using an and Gsa respectively is an example ofsignal transduction through parallel pathways Figure 16 77 Emilj am ay 739 Epinephrine s gp ephrine Elucagon gig z 39 39 quot39 r gt r 39 5552 A i l in Glnmgon 6quot quot3 39quotquot receptor A j GT G rezeplor 5a W Gsu ATP Gm CM 2 klfs PIPZ r quot Wu Glycogen e n Degradation Glucose Export Epinephrine also called adrenalin belongs to a class of rst messengers called catecholamr39nes It is considered the ght or ightquot hormone because it is released from the adrenal medulla under times of acute stress Epinephrine signaling stimulates glycogen breakdovm in liver cells glycogen is a storage form of glucose resulting in glucose export to muscle cells that use the glucose for energy conversion to activate muscle contraction ght or ight energy Glucagon is a peptide hormone that signals low glucose levels in the blood and also activates glucose export 8 am pages Bioc 460 Dr Miesfeld Spring 2008 Activation of adenylate cyclase leads to EQM increased levels of CAMR Figure 17 Complex between Gm and Adenylate Cyclase shows the molecular structure of Gsa Aden I I GTP bound to a portion of adenylate I cyclasyey cyclase containing the two cytoplasmic r subunits domains of the protein In order to trap the proteins in the active conformation for xray crystallographic analysis the Gsa subunit has a GTP analogue bound to the active site and an adenylate cyclase activator called forskolin is bound to the enzyme A short helical region in the ng subunit called switch ll undergoes a conformational change in the presence of GTP and is critical for stimulation of adenylate cyclase activity As we will discuss in some detail in lecture 34 glycogen is a storage form of glucose Glycogen synthesis anabolism and degradation catabolism are highly regulated by hormone signaling Regulation of glycogen breakdown in liver cells by epinephrine and glucagon is dependent on the presence of 32 adrenergic and glucagon receptors respectively As shown earlier in figure 6 activation of adenylate cyclase activity by hormone signaling leads to production of the second messenger cyclic AMP cAMP which then binds to and activates a downstream signaling protein called protein kinase A PKA How does cAMP binding activate the phosphorylating function of protein kinase A As illustrated in figure 18 enzymatically inactive PKA exists as an R202 tetramer in cells consisting of two regulatory subunits R and two catalytic subunits C In the absence of cAMP the enzyme active sites in the C subunits are buried by the protein interface between R and C subunits When two cAMP molecules bind to each R subunit they induce a conformational change in the R subunits that cause them to dissociate from the tetrameric complex as an R2cAMP4 dimer This general mechanism of kinase activation by subunit dissociation or by a ligandinduced protein conformational change that removes a linked autoinhibitory domain is a common theme in regulatory enzymes Figure 19 shows the molecular structure of cAMP and one R subunit of PKA with two cAMP bound Figure 18 Figure 19 c O gt d 0 lt2 Q Inactive holoenzyme R dimer Active Spacefill of CAMP C monomers PKA regulatory subunit with two CAMP bound 9 of 13 pages Bioc 460 Dr Miesfeld Spring 2008 Figure 20 illustrates the primary PKA signaling events in liver cells that are stimulated by epinephrine through the 52 adrenergic receptor Two PKAtarget proteins are phosphorylase kinase and glycogen synthase which regulate glycogen metabolism in opposing ways lecture 34 PKA phosphorylation of phosphorylase kinase is an activating signal that stimulates the phosphorylation of glycogen phosphorylase and subsequent removal of glucose units from glycogen Another important target of PKA is the transcription factor CREB which binds to DNA and regulates the transcription of specific genes in this case the PEPCK gene which is responsible for initiating glucose synthesis via the gluconeogenic pathway Don39t worry about the details at this point instead focus on the phosphorylation cascade that links upstream receptor activation to downstream effects We will be referring back to this figure when we get to lecture 35 Figure 20 B2 adrenergic Q receptor W K gt Via ET GsCL ctivation of 3313ch 39 I adenylate cyclase Gsa 39 ATPT PP39 39 CAMP PDE Activation of PKA CAMP 7 AMP activity by CAMP KN H20 f quot A Lquot Hquot 739 Activation of Inhibition of i t Activation of CREB regulatory J glycogen phosphorylase activity synthase 39139 I kinase Em quot v a F 2 es 7 w m A e 3 ATP ADPglquot K ATP ADP at PEPCK ene x i 39 lt TP ADP F H39 h t A 94730006 lg ra es r If F ofglycogen Low rates q V x rm synthesis ofglycogen P r l quotI K 7 synthesis E x Glycogen ZATP ZADP phosphorylase increased expression Glucose i P Glycogen 0f PEPCK enzyme Release of stored glucose 39 Protein KEY AC adenylate cyclase PDE phosphodiesterase PKA A protein kinase A GS glycogen synthase PhK phosphorylase kinase Ph 7 glycogen phosphorylase CREB CAMP response element binding protein PEPCK phosphoenolpyruvate carboxykinase Pi Glucose synthesis Glucose export to tissues 10of13 pages Btu 46D 7 Dr Mtesretd Sprtng 2mm Gg acttyatton or phosphottpase c tn addttton to 2 adrenergtc receptors human ttyercetts atso contatn at adrenergtc receptors whtch bmd eptnephrtne atoett at a reduced amntty and stgnat gtycogen degradatton through a second messenger pathway mked to a phosphorytatton cascade tn thts stgnattng pathway at adrenergtc receptors are coupted to a heterotnmertc e protetn contatntng quwmch acttyates the enzyme phospholipase 5 through a mechantsm yery stmttarto 35 sttmutatton oradenytate cyctase acttytty Phosphottpase c ts a membrane assoctated protetn that catatyzesthe hydrotysts orphosphatidylinositol 45Joisphosphate PR to form the second messengers DAG and P3 Three tsoronns orphosphottpase c haye oeen tdenttrted and tt has been round that qu acttyates the p tsororm caHed Ptop The cteayage reactton or F th oy PLOp ts shown tn rtgure 21 Note that DAG rematns assoctated wtth the ptasma membrane because of tts hydrophobtc tatL Whereast H33 t5 Waterso ub e and dtffuses away from the membrane Fto re 21 The Svmhests of P3 amp DAG CeH Membvane FOHV Odd chums w T ucE c t t H7 777H Diucvlglvcelot t tt Umwho tpcsc tt tt ctt DAG Phosphottdyltnosttot Ch 0 a o 45 bisphosphoie mpg 0quot l 0quot tnosttct 145 OH tttsphosphate ups The qu stgnattng pathway tn ttyercetts ts charactertzed oy downstream stgnattng eyents that resutt from the acttyatton ofPLOp and generatton orDAe and Pa As shown tn gure 22 Pa otndtng to catctum channets tocated on the endoptasmtc rettcutum causes a raptd tncrease tn tntracettutar Caz teyets see atso gure 9 The newty reteased Caz then otnds to an enzyme caHed protein kinase c PKC and sttmutates tts assoctatton wtth DAG at the ptasma memorane resutttng tn acttyatton orthe PKC kmase tunctton tn addmon CaZ39 otnds to calmodulin whtch acttyates phosphoryase knase and another enzyme caHed calmodulin dependent kinase Ftnatty ooth PKC and catmoduttn dependent kmase phosphorytate and tnacttyate gycogen synthase whereas catmoduhnractwated phosphorytase kmase sttmuates gycogen degradatton oy acttyattng gtycogen phosphorytase tt ortapages Bioc 460 Dr Miesfeld Spring 2008 Figure 23 xi adrenergic receptor GTP GDP Glycogen synthase enzyme is inactivated by phosphorylation at mulitple sites lP3mediated calcium a release from the ER 39 Phosphorylase kinase activates glycogen phosphorylase dimer mm7sim i lm i flip V39Cl Ji ZADP ZATP Glycogen 7 GlucoselP Pi Release of Protein KEY stored glucose PLC phospholipase C GS glycogen synthase CAM calmodulin CAMK CAMdependent kinase PhK phosphorylase kinase Ph glycogen phosphorylase Stimulation of the intrinsic GTPase activity in un by PLC5 throws the G protein signaling switch to the off position leading to the dissociation of un and PLC5 The duration ofthe epinephrine signal is controlled by receptor desensitization PKC phosphorylates 01 adrenergic receptors and by the activity of an P3 phosphatase which dephosphorylates and inactivates IP3 The P3 phosphatase therefore serves the same functional role in controlling second messenger signaling as does cAMP phosphodiesterase In summary G protein coupled receptor signaling involves five basic mechanistic steps Receptormediated activation of GDPGTP exchange in Ga subunits Gastimulation of an effector enzyme that generates 2nd messengers Activation of a phosphorylation cascade by 2nd messenger signaling Inactivation of G by effector stimulation of the intrinsic GTPase activity Signal duration is controlled by loss of 2nd messengers and receptor desensitization UlPSDNT 12 of13pages Bioc 460 Dr Miesfeld Spring 2008 ANSWERS TO KEY CONCEPT QUESTIONS Receptor proteins function as aatekeepers to intracellular sionalino pathways Receptor proteins bind hormones and ligands that function as rst messengers in signaling pathways Ligand binding results in protein conformational changes in the receptor that transmit the signal to other signaling proteins through a variety mechanisms There are ve major classes of receptor proteins G protein coupled receptors receptor tyrosine kinases Tumor Necrosis Factor TNF family receptors ion channel receptors and intracellular receptors Most receptors function at the plasma membrane and bind to extracellular ligands intracellular receptors are ligandregulated transcription factors that bind ligands after they cross the membrane Second messenqer molecules such as cAMP cGMP DAG IP3 and Ca2 function in cell signaling pathways by activating enzymes and ion channels that amplify the initiating siqnal Second messengers are most often generated by enzymatic reactions representing upstream events in signaling pathways Cyclic AMP and GMP are the products of adenylate cyclase and guanylyl cyclase reactions respectively whereas DAG and P3 are generated from Ple by the enzyme phospholipase C Calcium ions are released from the endoplasmic reticulum through ion gated channels The levels of second messengers in the cell are controlled by enzymes such as phosphodiesterases and phosphatases that rapidly remove them from the signaling pool 13 of 13 pages
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