Comparative Animal Physiology (BIOL 3060) Lecture Notes Fall 2024 PDF

Comparative Animal Physiology (Biol 3060) Professor: Dr. Michael Cardinal-Aucoin Fall 2024  Signal Transduction and GPCRs GPCR signaling cAMP PKA Biol 3060 - Dr. M. Cardinal-Aucoin 2  Extracellular signal molecules fall into 2 broad classes, corr...

Comparative Animal Physiology (Biol 3060) Professor: Dr. Michael Cardinal-Aucoin Fall 2024  Signal Transduction and GPCRs GPCR signaling cAMP PKA Biol 3060 - Dr. M. Cardinal-Aucoin 2  Extracellular signal molecules fall into 2 broad classes, corresponding to 2 fundamentally different classes of receptors. 6 major classes of chemical messengers: 1. Peptides (e.g. oxytocin) 2. Purines (e.g. adenosine) Large and Cannot Bind to cross receptor on 3. Amines (e.g. hydrophilic. membrane. cell surface. norepinephrine) 4. Eicosanoids (e.g. prostaglandins) 5. Gases (e.g. NO) Bind to Small and Can cross intracellular 6. Steroids (e.g. estrogen) hydrophobic. membrane. receptor. Biol 3060 - Dr. M. Cardinal-Aucoin 3 4 main types of ligand-triggered signal receptor responses: Transmembrane cell Cytosolic/nuclear surface receptor receptor (Class 1) (Class 2) Enzyme-linked receptor Biol 3060 - Dr. M. Cardinal-Aucoin 4 1st class: large hydrophilic signaling molecule – cell-surface receptor large hydrophilic signaling molecule cell-surface receptor nucleus cytosol signaling cascade Most extracellular signaling molecules are hydrophilic and are therefore unable to cross the plasma membrane directly. These extracellular signaling molecules bind to cell-surface receptors, which in turn transduce (convert) extracellular signals into intracellular signals. These intracellular signals initiate signaling cascades that lead to a cellular response. Biol 3060 - Dr. M. Cardinal-Aucoin 5  Intracellular signaling cascades include multiple signal transduction steps extracellular signaling molecule extracellular medium cell-surface receptor cytosol Primary signal transduction step small molecule (2nd messenger) protein The primary signal transduction step: a receptor protein located on the cell surface receives the extracellular signaling molecule and generates a new intracellular signal molecule in response. There are 2 types of these intracellular signal molecules: (1) low-molecular-weight molecules (2nd messengers) – cyclic AMP, Ca2+ (2) proteins Biol 3060 - Dr. M. Cardinal-Aucoin 6  Intracellular signaling cascades include multiple signal transduction steps Intracellular signaling cascade Primary signal transduction step Secondary (downstream) signal transduction steps: Modulation The intracellular signaling Secondary molecules (2nd messengers or by other signal factors proteins) initiate signaling transduction steps cascades. Amplification, distribution, and divergence of signal. Each step in these signaling cascades can be influenced Metabolism Cytoskeleton (modulated) by other events inside or outside the cell. Transcription Biol 3060 - Dr. M. Cardinal-Aucoin 7  extracellular signaling extracellular molecule (ligand) medium cell-surface receptor cytosol Primary signal transduction step small molecule (2nd messenger) protein The primary signal transduction step: a receptor protein located on the cell surface receives the extracellular signaling molecule (ligand) and generates a new intracellular signal molecule in response. There are 3 classes of ligand-triggered cell-surface receptors: (1) G-protein-linked receptors Most cell surface receptors in animals. (2) ion-channel-linked receptors (3) enzyme-linked receptors Most cell surface receptors in plants. Biol 3060 - Dr. M. Cardinal-Aucoin 8 Types of Receptors 1. Intracellular – Binds to hydrophobic ligands and alters transcription 2. Ligand-gated ion channels – Lead to changes in membrane potential (instantaneous) 3. Receptor-enzymes – Lead to changes in intracellular enzyme activity 4. G-protein-coupled – Activation of membrane-bound G-proteins – Lead to changes in cell activities Biol 3060 - Dr. M. Cardinal-Aucoin 9 G-Protein-Coupled Receptors Ligand binds to transmembrane receptor. Receptor interacts with intracellular GTP binding- proteins. Subunits of G-protein dissociate and activate other membrane- associated proteins and produce second messengers. Biol 3060 - Dr. M. Cardinal-Aucoin 10 There Are Three Major Classes of Cell-Surface Receptor Proteins Ligand-gated ion channels (neurotransmitters) Receptor activates trimeric G protein Alter enzymes, ion channels, genes Receptor is an enzyme or activates an enzyme Involve phosphorylation/ kinase activity Biol 3060 - Dr. M. Cardinal-Aucoin 11  (1) G-protein-linked receptors Signalling Receptor G protein Effector Pathway Response (2ndmessenger) AC cAMP Genes GPCR G-protein PLC-β IP3/DG Enzymes Ion Channels Other Shutting Off Response Biol 3060 - Dr. M. Cardinal-Aucoin 12  (1) G-protein-linked receptors There are two major types of GTP-binding proteins: 1. Trimeric GTP-binding proteins (G proteins) 2. Monomeric GTPases (Regulated by GAPs and GEFs) Trimeric Monomeric Biol 3060 - Dr. M. Cardinal-Aucoin 13  (1) G-protein-linked receptors Phosphorylation is the largest class of molecular switches “On” switch: protein kinases ~520 human protein kinases “Off” switch: phosphatases ~150 human phosphatases Attach phosphate to hydroxyl group (-OH) of specific amino acids - serine/threonine kinases - tyrosine kinases Biol 3060 - Dr. M. Cardinal-Aucoin 14 (1) G-protein-linked receptors Largest family of cell-surface receptors Single polypeptide with 7 transmembrane domains, Gprotein binding domain (cytoplasmic) and for many a ligand binding domain (extracellular) ~4% of the human genome is comprised of genes for these receptors 7800 GPCRs in humans Almost half of all known drugs work through GPCRs or their pathways Biol 3060 - Dr. M. Cardinal-Aucoin 15 (1) G-protein-linked receptors Mediate most responses to signals from external world (senses) and many other cells (hormones, neurotransmitters, local mediators) Activated by proteins, small peptides, small organic molecules, amino acids, fatty acids, light, molecules that we taste and smell Activation by ligand or light triggers conformational change and activation of G protein Biol 3060 - Dr. M. Cardinal-Aucoin 16  (1) G-protein-linked receptors GPCRs are either associated with a heterotrimeric G protein, or become associated upon activation. G proteins have 3 subunits ,  and   and  are covalently linked to lipid in membrane  is a GTPase - molecular switch Biol 3060 - Dr. M. Cardinal-Aucoin 17  (1) G-protein-linked receptors extracellular signaling molecule (ligand) extracellular medium (1) G-protein- (2) (3) linked receptor inactive G protein activated G inactive protein effector enzyme (1) G-protein-linked receptor binds its ligand (an extracellular signaling molecule). (2) Ligand binding induces an interaction of the receptor with the inactive G (GTP- binding) protein. (3) Binding to the receptor activates G protein. Biol 3060 - Dr. M. Cardinal-Aucoin 18  (1) G-protein-linked receptors extracellular signaling molecule (ligand) extracellular medium (1) G-protein- (2) (3) (4) linked receptor inactive G protein activated G activated effector (5) protein enzyme inactive effector 2nd messenger enzyme (4) The activated G protein leaves the receptor and binds the inactive effector enzyme. (5) Binding to the activated G protein activates the effector enzyme that generates a specific 2nd messenger. Biol 3060 - Dr. M. Cardinal-Aucoin 19 (1) G-protein-linked receptors The basic mechanism of G- protein activation is shared by all GPCRs. Effectors differ depending on the specific receptor: Adenalyl cyclase (make cAMP) Phospholipase C-β (makes IP3) Others Biol 3060 - Dr. M. Cardinal-Aucoin 20  Secondary (downstream) signal transduction steps: The intracellular signaling molecules (2nd messengers or proteins) initiate signaling cascades. Intracellular signaling cascade Primary signal transduction step These intracellular signaling cascades are formed by intracellular signaling Modulation Secondary proteins that act as a series of by other signal molecular switches. factors transduction steps 3 classes of molecular switches: (1) Protein phosphorylation – dephosphorylation switches. (2) GTP binding – hydrolysis switches. Metabolism Cytoskeleton (3) Assembly – disassembly of Transcription multiprotein complexes. Biol 3060 - Dr. M. Cardinal-Aucoin 21  (1) G-protein-linked receptors Signalling Receptor G protein Effector Pathway Response (2ndmessenger) AC cAMP Genes GPCR G-protein PLC-β IP3/DG Enzymes Ion Channels Other Shutting Off Response Biol 3060 - Dr. M. Cardinal-Aucoin 22  G-protein-coupled receptors extracellular signaling molecule (ligand) extracellular medium (1) G-protein- (2) (3) linked receptor inactive G protein activated G inactive protein effector enzyme (1) G-protein-coupled receptor binds its ligand (an extracellular signaling molecule). (2) Ligand binding induces an interaction of the receptor with the inactive G (GTP- binding) protein. (3) Binding to the receptor activates G protein. Biol 3060 - Dr. M. Cardinal-Aucoin 23  G-protein-coupled receptors extracellular signaling molecule (ligand) extracellular medium (1) G-protein- (2) (3) (4) linked receptor inactive G protein activated G activated effector (5) protein enzyme inactive effector 2nd messenger enzyme (4) The activated G protein leaves the receptor and binds the inactive effector enzyme. (5) Binding to the activated G protein activates the effector enzyme that generates a specific 2nd messenger. Biol 3060 - Dr. M. Cardinal-Aucoin 24 Biol 3060 - Dr. M. Cardinal-Aucoin 25  The result/function depends on the effector. Biol 3060 - Dr. M. Cardinal-Aucoin 26  (1) G-protein-linked receptors Signalling Receptor G protein Effector Pathway Response (2ndmessenger) AC cAMP Genes GPCR G-protein PLC-β IP3/DG Enzymes Ion Channels Other Shutting Off Response Biol 3060 - Dr. M. Cardinal-Aucoin 27  Many G proteins act on the enzyme Adenylyl Cyclase (effector) Adenylyl cyclase makes cAMP. cAMP is synthesized from ATP by enzyme adenylyl cyclase (AC) (second messenger) What does cAMP do? Bind and open Ca2+ channels Bind and activate other proteins cAMP is degraded by phosphdiesterases into 5’-adenosine monophosphate (AMP) Biol 3060 - Dr. M. Cardinal-Aucoin 28  2nd messenger: cyclic AMP (cAMP) NH2 O O O N N cAMP is formed from ATP by -O – P – O - P - O – P - O – CH a cyclization reaction that 2 N N removes two phosphate - O - O - O O groups from ATP. ATP OH OH NH2 P P O N N P - O – CH2 Adenylate cyclase N N O - O cAMP O OH NH2 O N N -O – P - O – CH cAMP phosphodiesterase 2 N N O - O The degradation reaction AMP breaks cAMP down to AMP. OH OH Biol 3060 - Dr. M. Cardinal-Aucoin 29  AC is the effector of many G proteins Activated  subunits bind AC and can either activate or inhibit its activity Activate: stimulatory G protein  subunit (Gs or Gs) Inhibit: inhibitory G protein  subunit (Gi or Gi) Biol 3060 - Dr. M. Cardinal-Aucoin 30  Metabolic responses to hormone-induced rise in cAMP in various tissues e.g. stress response Hormone inducing Tissue Metabolic response rise in cAMP Adrenaline Muscle, Breakdown of glycogen (the polymerized storage Liver form of glucose) to make more glucose, an immediately usable form of metabolic fuel. Adipose Breakdown of triacylglycerols (the storage form of (fat storage fatty acids) to make more fatty acids, an immediately cells) usable form of metabolic fuel. Heart Increase in contraction rate, which increases the blood supply to the tissues. NH2 Adrenaline-mediated rise in cAMP is O N N important in mediating the body’s response to P - O – CH2 stress, such as fright or heavy exercise, when N N O - O all tissues have an increased need for glucose and fatty acids. cAMP O OH Biol 3060 - Dr. M. Cardinal-Aucoin 31 Adrenaline-induced activation of adenylate cyclase is mediated by β-adrenergic Gs- protein-linked receptors and heterotrimeric Gs proteins. ligand-binding domain extracellular transmembrane domain space Plasma membrane cytosol β γ Gs-protein - binding domain sα GDP The Gs-protein-linked receptors contain: - the extracellular ligand-binding domain. - seven transmembrane α helices. - the cytosolic Gs-protein-binding domain. Biol 3060 - Dr. M. Cardinal-Aucoin 32 Adrenaline-induced activation of adenylate cyclase is mediated by β-adrenergic Gs- protein-linked receptors and heterotrimeric Gs proteins. extracellular space Plasma membrane cytosol β γ receptor-binding domain sα guanine nucleotide-binding domain GDP The heterotrimeric Gs (s = stimulatory) proteins: - consist of 3 subunits, designated sα, β, and γ. - are called heterotrimeric G proteins to distinguish them from other guanine nucleotide-binding proteins, such as the Ras proteins. - the sα subunit binds guanine nucleotides (GDP or GTP), which regulate G protein activity. Biol 3060 - Dr. M. Cardinal-Aucoin 33  Adrenaline-induced activation of adenylate cyclase inactive β-adrenergic Gs- protein-linked receptor β γ inactive heterotrimeric sα Gs protein GDP inactive adenylate cyclase When no adrenaline is bound to a β-adrenergic Gs- protein-linked receptor: - the receptor, heterotrimeric Gs protein and adenylate cyclase are all inactive and are not in contact with each other. - the sα subunit is bound to GDP in a complex with β and γ. Biol 3060 - Dr. M. Cardinal-Aucoin 34  Adrenaline-induced activation of adenylate cyclase adrenaline active β-adrenergic Gs- protein-linked receptor β γ sα inactive heterotrimeric inactive adenylate cyclase Gs protein GDP Step 1 : Binding of adrenaline to the β-adrenergic Gs- protein-linked receptor activates the receptor by inducing a conformational change ( ) in the receptor. Biol 3060 - Dr. M. Cardinal-Aucoin 35  Adrenaline-induced activation of adenylate cyclase active β-adrenergic Gs- protein-linked receptor β γ sα GDP inactive adenylate cyclase inactive heterotrimeric Gs protein Step 2 : The activated receptor binds to the sα subunit of the inactive heterotrimeric Gs protein. Biol 3060 - Dr. M. Cardinal-Aucoin 36  Adrenaline-induced activation of adenylate cyclase β γ sα GDP GTP inactive adenylate cyclase active heterotrimeric Gs protein Step 3 : Binding to the receptor induces a conformational change in the sα subunit of the heterotrimeric Gs protein, such that the sα subunit of the Gs protein exchanges its GDP for GTP. Biol 3060 - Dr. M. Cardinal-Aucoin 37  Adrenaline-induced activation of adenylate cyclase β γ sα sα dissociates GTP inactive adenylate cyclase from β and γ Step 4 : The activated GTP-bound sα subunit dissociates from β and γ, which remain together. Biol 3060 - Dr. M. Cardinal-Aucoin 38  Adrenaline-induced activation of adenylate cyclase β γ sα GTP inactive adenylate cyclase Step 5 : The dissociation of the GTP-bound sα subunit from β and γ induces a conformational change in the sα subunit, which then diffuses along the cytosolic surface of the plasma membrane until it binds to the adenylate cyclase. Biol 3060 - Dr. M. Cardinal-Aucoin 39  Adrenaline-induced activation of adenylate cyclase active adenylate cyclase β γ sα GTP ATP cAMP Step 6 : Binding to the GTP-bound sα subunit stimulates adenylate cyclase, which catalyses the synthesis of cAMP from ATP. Biol 3060 - Dr. M. Cardinal-Aucoin 40  Adrenaline-induced activation of adenylate cyclase inactive β-adrenergic Gs- protein-linked receptor β γ inactive heterotrimeric sα Gs protein GDP inactive adenylate cyclase GTP hydrolysis / reassociation with βγ Step 7 : The activity of the GTP-bound sα subunit is terminated by hydrolysis of bound GTP, and the inactive sα subunit (now with GDP bound) then reassociates with the βγ complex, ready for the cycle to start anew. Biol 3060 - Dr. M. Cardinal-Aucoin 41  In adipose tissues (fat storage cells), the cAMP level can be both up-regulated and down-regulated by the action of different hormones. Adrenaline Prostaglandin cAMP level Adenylate cyclase activity Receptor β-adrenergic α-adrenergic active adenylate inactive adenylate cyclase cyclase sα (s = stimulatory) iα (i = inhibitory) α –subunit of the G protein sα iα GTP GTP ATP cAMP ATP cAMP β- and γ- subunits identical Biol 3060 - Dr. M. Cardinal-Aucoin 42  A major target of the cAMP second messenger is PKA cAMP activates PKA PKA = Protein Kinase A (PKA) What does PKA do? Biol 3060 - Dr. M. Cardinal-Aucoin 43 Cyclic-AMP Signaling Once adenylate cyclase is activated it produces cAMP. cAMP binds to an enzyme, protein kinase A (PKA), activating it. PKA then activates or inhibits other enzymes by adding a phosphate group, a process called phosphorylation. Biol 3060 - Dr. M. Cardinal-Aucoin 44  What happens once cAMP is produced? 2 regulatory subunits and 2 catalytic subunits 2 cAMP bind to each of the two regulatory subunits of PKA regulatory subunits dissociate releasing active catalytic subunits Biol 3060 - Dr. M. Cardinal-Aucoin 45  What happens once cAMP is produced? The diverse effects of cAMP in animal cells are mediated by the action of cAMP-dependent protein kinase (aka protein kinase A). Regulatory subunit Site B for binding cAMP Catalytic subunit R C PKA R C Catalytic subunit Site B for binding cAMP Regulatory subunit The inactive form of protein kinase A is a tetramer, consisting of two regulatory ( R ) and two catalytic ( C ) subunits. Each R subunit has site B for binding cAMP. Biol 3060 - Dr. M. Cardinal-Aucoin 46  Regulation of protein kinase A Site B for binding cAMP Site A for binding cAMP R C 1 R C R C R C cAMP Site A for binding cAMP Site B for binding cAMP Step 1 : Binding of cAMP to site B induces a conformational change that unmasks the second site for binding cAMP, site A. Biol 3060 - Dr. M. Cardinal-Aucoin 47  Regulation of protein kinase A C R C 2 R Enzymatically R active C subunits R C cAMP C Phosphorylates various protein Step 2 : targets. Binding of cAMP to site A, in turn, leads to release of the catalytic subunits C, which are then enzymatically active. Biol 3060 - Dr. M. Cardinal-Aucoin 48  Fast and slow responses to adrenaline-induced, cAMP- and protein kinase A-mediated signaling AC 1. Fast response (< sec to minutes): ATP cAMP Protein kinase A phosphorylates many enzymes, increasing or decreasing their enzymatic activities within seconds. C R 2. Slow response (minutes to hours): R Protein kinase A phosphorylates some C transcriptional activators that stimulate the transcription of cAMP-inducible genes within Enzymatically active minutes or hours. C subunits of protein kinase A Biol 3060 - Dr. M. Cardinal-Aucoin 49  1. Fast response Hormone inducing Tissue Metabolic response rise in cAMP Adrenaline Muscle, Breakdown of glycogen (the polymerized storage Liver form of glucose) to make more glucose, an immediately usable form of metabolic fuel. cAMP-activated cAMP-activated protein kinase A protein kinase A Inhibition Activation glucose Synthesis Glycogen Degradation (glucose glucose monomers Enzyme: polymer) Enzyme: monomers Glycogen synthase Glycogen phosphorylase Biol 3060 - Dr. M. Cardinal-Aucoin 50  1. Fast response: activation of glycogen degradation Protein kinase A (catalytic subunit C) Activation cAMP Activation (1) Inactive Active Glycogen phosphorylase P KINASE ATP ADP (1) Catalytic subunit of cAMP- activated protein kinase A phosphorylates and activates glycogen phosphorylase kinase. Biol 3060 - Dr. M. Cardinal-Aucoin 51  1. Fast response: activation of glycogen degradation Protein kinase A (catalytic subunit C) Activation cAMP Activation (1) Inactive Active Glycogen phosphorylase P KINASE ATP ADP Activation (2) Inactive Active Glycogen phosphorylase P ATP ADP (2) Phosphorylated and activated Activation glycogen phosphorylase kinase then phosphorylates and activates glycogen phosphorylase, which catalyzes the breakdown of glycogen to glucose. Glycogen Degradation (glucose polymer) glucose monomers Biol 3060 - Dr. M. Cardinal-Aucoin 52  1. Fast response: inhibition of glycogen synthesis Protein kinase A (catalytic subunit C) Activation cAMP Inactivation Active Inactive Glycogen P synthase ATP ADP Inhibition Synthesis Glycogen glucose (glucose monomers polymer) Catalytic subunit of cAMP-activated protein kinase A phosphorylates glycogen synthase. RESULT: Elevation of cAMP and activation of the catalytic subunit of protein kinase A blocks glycogen synthesis at the same time as it stimulates glycogen breakdown. Biol 3060 - Dr. M. Cardinal-Aucoin 53  Signal Amplification Activated PKA in turn will phosphorylate target proteins leading to a particular response. Where do you see opportunities for amplification in this pathway? Biol 3060 - Dr. M. Cardinal-Aucoin 54  - [Adrenaline] = 10 10 M Amplification Signal transduction cascades amplify, Adenylate cyclase distribute, and Amplification diverge the signals - received, making [cAMP] = 10 6 M them stronger and distributing them Protein kinase A to various Amplification intracellular Glycogen targets. phosphorylase kinase Amplification Activated Glycogen Signal phosphorylase amplification: Amplification x 100,000,000 - [Glucose] = 10 2 M Protein kinase A : Glycogen phosphorylase kinase : Glycogen phosphorylase = 1 : 10 : 240 Biol 3060 - Dr. M. Cardinal-Aucoin 55  PKA and the action of Glucagon in the Liver Glucagon > GPCR > G protein > AC > cAMP > PKA > 3 actions PKA phosphorylates phosphorylase- Glycogen kinase which phosphorylates GP Phosphorylase activating it. Other steps Glycogen Glucose Other steps Glycogen PKA phosphorylates GS, Synthase inhibiting it. PKA enters nucleus, phosphorylates proteins including CREB > transcription of genes involved in gluconeogenesis Biol 3060 - Dr. M. Cardinal-Aucoin 56  Cholera Toxin Stimulates a cAMP pathway Cholera toxin (Vibrio cholerae) - alters Gs which prevents GTP hydrolysis - cAMP levels remain elevated in intestinal cells - opens Cl- channels > massive efflux of Cl- and water into the gut Author: Mclaneb1 https://commons.wikimedia.org/wiki/File:CholeraToxin.png Biol 3060 - Dr. M. Cardinal-Aucoin 57 2. Slow response: regulation of transcription Most PKA-mediated cell responses are rapid (seconds to minutes) – cytoplasmic (eg enzymes). PKA can also modulate gene transcription (hours). In nucleus, PKA phosphorylates CREB (cAMP Response Element Binding protein) which binds CBP > CRE > transcription of genes with CRE. Brief signal >>>> Long term change Biol 3060 - Dr. M. Cardinal-Aucoin 58  2. Slow response: regulation of transcription cAMP C Enzymatically R active C subunits of protein kinase A R C Step 1 : cytosol The free catalytic subunit of PKA 1 translocates into the nucleus nucleus via NPC. C NPC Biol 3060 - Dr. M. Cardinal-Aucoin 59  2. Slow response: regulation of transcription All genes regulated by cAMP contain a cis-acting DNA sequence, called cAMP-response element (CRE) in their promoter or enhancer regions. ‒ Conserved sequence 5'-TGACGTCA-3’ The phosphorylated form of a transcription factor called CRE-binding (CREB) protein recognizes and binds to the CRE. CRE cis-acting cAMP-inducible regulatory region target gene Cis-acting: non-coding DNA acts to regulate transcription of neighbouring genes. Biol 3060 - Dr. M. Cardinal-Aucoin 60  2. Slow response: regulation of transcription cytosol nucleus Step 2 : C Within the nucleus, the catalytic subunit of Active 2 Inactive PKA phosphorylates P and activates CREB ADP ATP protein. CREB CRE cis-acting cAMP-inducible regulatory region target gene Biol 3060 - Dr. M. Cardinal-Aucoin 61  2. Slow response: regulation of transcription cytosol nucleus C Step 3 : Active Inactive Phosphorylated CREB P proteins form a dimer ADP ATP that binds to CRE. CREB 3 P P CRE cis-acting cAMP-inducible regulatory region target gene Biol 3060 - Dr. M. Cardinal-Aucoin 62  2. Slow response: regulation of transcription cytosol Step 4 : A transcriptional co- nucleus C activator called CBP/300 binds to phosphorylated serine in the CRE-bound Active Inactive phosphorylated CREB P protein. ADP ATP CREB CBP/300 4 P P CRE cis-acting cAMP-inducible regulatory region target gene Biol 3060 - Dr. M. Cardinal-Aucoin 63  2. Slow response: regulation of transcription cytosol Step 5 : CBP/300 links CREB nucleus protein to the basal C transcriptional machinery, thereby permitting CREB protein to stimulate Active Inactive transcription of cAMP- P inducible genes. ADP ATP CREB CBP/300 P P CRE 5 mRNA Basal transcriptional machinery Biol 3060 - Dr. M. Cardinal-Aucoin 64  Stopping the Response Activated G proteins are inactivated by GTP hydrolysis  Subunit GTP hydrolysis slow rate of GTP hydrolysis increased by interaction with effector or regulator of G protein signaling (RGS) proteins, which act as GAPs. cAMP is broken down by phosphodiesterase Biol 3060 - Dr. M. Cardinal-Aucoin 65 Termination of Signaling Serine/threonine phosphatases rapidly dephosphorylate (remove a phosphate) the phosphorylated proteins, terminating the response. Biol 3060 - Dr. M. Cardinal-Aucoin 66