Chapter 15 Signal Transduction and G Protein Coupled Receptors

Signal transduction? Signal transduction (also known as cell signaling) is the transmission of molecular signals from a cell's exterior to its interior. Signals received by cells must be transmitted effectively into the cell to ensure an appropriate response. This step is initiated by cell-surface receptors.

Chapter 15 Signal Transduction and G Protein Coupled Receptors 15.1 Signal Transduction: From Extracellular Signal to Cellular Response 15.2 Studying Cell-Surface Receptors and Signal Transduction Proteins 15.3 G Protein Coupled Receptors: Structure and Mechanism 15.4 G Protein Coupled Receptors That Regulate Ion Channels 15.5 G Protein Coupled Receptors That Activate or Inhibit Adenylyl Cyclase 15.6 G Protein Coupled Receptors That Trigger Elevations in Cytosolic and Mitochondrial Calcium

Signal Transduction and G Protein Coupled Receptors 15.1 Signal Transduction: From Extracellular Signal to Cellular Response All cells respond to extracellular signals/stimuli that activate plasma membrane or cytosolic receptors. Activated receptors function as transcription factors or activate G protein switches that regulate a variety of downstream pathways or induce generation of intracellular second messengers that do so. How????

Overview of cell signaling Extracellular signaling molecules synthesized, packaged into secretory vesicles, and secreted by specialized signaling cells within multicellular organisms Signal produces a specific response only in target cells expressing receptor proteins that bind the signal Hydrophobic signals vs. Hydrophilic signals How different?

Types of extracellular signaling. Extracellular molecule signaling three classifications; based on distance over which the signal acts: (a) Endocrine: (epinephrine, insulin) Signaling molecules synthesized and secreted by signaling cells (e.g., cells in endocrine glands) Transported through the circulatory system Affect distant target cells expressing the receptor (b) Paracrine: (neurotransmitters, growth factors) Signaling molecules secreted by a cell affect only nearby target cells expressing the receptor Some may bind to ECM released only when ECM is degraded (c) Autocrine: (growth factors) Cells respond to signals they secrete. (Tumor cells may overproduce and respond to growth factors.) (d) Membrane protein signals: signal neighboring cells by direct contact with surface receptors.

How to regulate the function of protein? Kinase/Phosphatase switch GTPase switch

Regulation of protein activity by a kinase/phosphatase switch. Cell-surface receptor signaling involves kinase phosphorylation and phosphatase dephosphorylation to regulate target protein activity. Protein kinase transfers terminal phosphate from ATP to specific Ser/Thr or Tyr OH (phosphorylated residue is part of a specific kinase target motif) Protein phosphatase hydrolyzes P off protein restoring Ser/Thr or Tyr OH Protein kinases and phosphatases Regulated by signaling processes Modify specific protein targets containing target motifs Effect phosphorylation (and reversal by dephosphorylation) on protein activation or deactivation protein-specific. Example target protein (reversed in other proteins): unphosphorylated inactive phosphorylated active

GTPase switch GTPases play an important role in: Signal transduction at the intracellular domain of transmembrane receptors, including recognition of taste, smell and light. Protein biosynthesis (a.k.a. translation) at the ribosome. Control and differentiation during cell division. Translocation of proteins through membranes. Transport of vesicles within the cell. (GTPases control assembly of vesicle coats.)

GTPase switch proteins cycle between active and inactive forms. GTP-binding proteins signal transduction pathway on-off switches GTPase protein superfamily (GTP-binding, G protein): ON/active GTP bound OFF to ON promoted by GEFs (guanine nucleotide exchange factors) GEFs catalyze dissociation of bound GDP and replacement by GTP (not phosphorylation of GDP) OFF/inactive bound GDP ON to OFF GTPase activity GTP GDP + Pi (ON to OFF) Accelerated by GAPs (GTPase-activating proteins) and RGSs (regulators of G protein signaling) GTPase switch proteins two large signaling classes: Heterotrimeric activated by direct interaction with surface receptors (GEFs) Monomeric activated by GEFs that are activated by surface receptors or other proteins

What s the switch? How does it have a funciton? Force Change? What?

Switching mechanism of monomeric G proteins. G protein ON-OFF transition conformational changes: Involve switch I and switch II (e.g., Ras monomeric G protein) Promotes binding to downstream signaling proteins (a) Active/ON state bound GTP (b) Inactive/OFF state bound GDP Intrinsic GTPase activity hydrolyzes GTP to GDP (removes GTP γ phosphate)

Ras signaling A) Endogenous Ras activity B) Loss of Ras function Ras Ras Raf Raf rl/mapk rl/mapk Cell growth Postmitotic cell survival Photoreceptor differentiation Reduced growth apoptosis

Ras-Raf-MAPK pathway! RAS: Rat Sarcoma Ras is a family of related proteins which is expressed in all animal cell lineages and organs. All Ras protein family members belong to a class of protein called small GTPase, and are involved in transmitting signals within cells (cellular signal transduction).

Ras-Raf-MAPK pathway! RAF is an acronym for Rapidly Accelerated Fibrosarcoma RAF kinases are a family of three serine/threoninespecific protein kinases that are related to retroviral oncogenes. The mouse sarcoma virus 3611 contains a RAF kinase-related oncogene that enhances fibrosarcoma induction. Activation of RAF kinases requires interaction with RAS-GTPases. * A serine/threonine protein kinase (EC2.7.11.1) is a kinase enzyme that phosphorylates the OH group of serine or threonine

Ras-Raf-MAPK pathway! Mitogen-activated protein kinases (MAPKs) are a highly conserved family of serine/threonine protein kinases involved in a variety of fundamental cellular processes such as proliferation, differentiation, motility, stress response, apoptosis, and survival.

Ras-Raf-MAPK pathway https://www.youtube.com/watch?v=r7goz9vfcy8

Gain of function in Ras

Think why this signaling is necessary

Second messenger? Wide spread the signal!

Second messenger Primary messengers? Second messengers are molecules that relay signals received at receptors on the cell surface such as the arrival of protein hormones, growth factors, etc. to target molecules in the cytosol and/or nucleus.

Four common intracellular second messengers. Intracellular second messengers transmit signals through the cytosol. camp: Generated from ATP by adenylyl cyclase Activates PKA cgmp: Generated by guanylyl cyclase Activates PKG and specific cation channels IP3 and DAG: Both made from PIP2 by phospholipase C IP3 opens channels to release Ca 2+ from the ER DAG with Ca 2+ activates PKC Calcium ions (Ca 2+ ) (not shown): Released from intracellular stores or transported into the cell Activates calmodulin, specific kinases (PKC), and other regulatory proteins

A signal transduction pathway involving a G protein, a second messenger, a protein kinase, and several target proteins Generalized signal transduction pathway: Step 1: Hormone binding to its cell-surface receptor Step 2: Activated receptor (GEF) activates trimeric G protein Step 3: G protein alpha subunit binds to and activates second messengergenerating enzyme. Step 4: Activated enzyme generates multiple second messenger molecules. Step 5: Second messenger activates a protein kinase. Step 6: Kinase phosphorylates and changes activity of one or more target proteins. Step 6a: Cytosolic target proteins induce changes in cellular function, metabolism, or movement. Step 6b: Target transcription factors induce changes in gene expression.

Beta-adrenergic receptor The adrenergic receptors (or adrenoceptors) are a class of G protein-coupled receptors that are targets of the catecholamines, especially norepinephrine (noradrenaline) and epinephrine (adrenaline) Many cells possess these receptors, and the binding of a catecholamine to the receptor will generally stimulate the sympathetic nervous system. The sympathetic nervous system is responsible for the fight-or-flight response, which includes dilating the pupils, increasing heart rate, mobilizing energy, and diverting blood flow from non-essential organs to skeletal muscle.

G protein G proteins, also known as guanine nucleotide-binding proteins, are a family of proteins that act as molecular switches inside cells, and are involved in transmitting signals from a variety of stimuli outside a cell to its interior. Their activity is regulated by factors that control their ability to bind to and hydrolyze guanosine triphosphate (GTP) to guanosine diphosphate (GDP). When they are bound to GTP, they are 'on', and, when they are bound to GDP, they are 'off'. G proteins belong to the larger group of enzymes called GTPases. glycosylphosphatidylinositol-linked proteins (GPI)

AC Adenylyl cyclase (EC4.6.1.1, also commonly known as adenyl cyclase and adenylate cyclase, abbreviated AC) is an enzyme with key regulatory roles in essentially all cells.

G-protein coupled receptor https://www.youtube.com/watch?v=nt2r5r0zo5u

GPCR?

Signal Transduction and G Protein Coupled Receptors 15.2 Studying Cell-Surface Receptors and Signal Transduction Proteins Near-maximal response of a cell to a particular ligand generally occurs at ligand concentrations at which less than 100 percent of its receptors are bound to the ligand. Signal receptors and pathways are targeted by numerous drugs. Think this! Receptors and signaling pathway intermediates are studied with a variety of experimental approaches including affinity chromatography, Western blotting, immunoprecipitation, and pull-down assays. Think this!

Binding assay Experiment to determine the affinity of a receptor for a ligand: Labeled ligand added at various concentrations (x axis) to cells that do (experimental cells) and do not (control cells) express the receptor Incubation in ligand 1 hr (allows binding) at 4 C (Low temperature prevents endocytosis of the cell-surface receptors.) Amount of bound ligand (label) is measured (control cell label binding subtracted from binding to receptor-expressing cells). Plot bound ligand per cell as a function of the ligand concentration (red)

Binding assay Results: At relatively high ligand concentrations number of receptor-bound ligand molecules approaches number of total cell-surface receptors K d (half maximal) ligand binding = 1 nm ligand Parallel physiological response experiments results (blue): 18 percent receptors bound to ligand 50 percent of the maximal physiological response 50 percent receptors bound to ligand near maximal physiological response Conclusion: relative physiological response is greater than ligand binding.

Is the study of receptor-ligand binding important?

Structures of the natural hormone epinephrine, the synthetic agonist isoproterenol, and the synthetic antagonist alprenolol. Synthetic analogs of natural hormones are widely used both in research on cell-surface receptors and as drugs two classes: May bind much more tightly to the receptor than does the natural hormone May be more stable Agonist mimics function of a natural hormone Binds to and activates receptor Induces the normal cellular response to the hormone Antagonist inhibits function of natural hormone Binds to the receptor ligand-binding site but induces no response Blocks natural hormone binding Reduces normal physiological activity of the hormone Epinephrine receptor drugs: Structurally similar to natural hormone epinephrine Isoproterenol Agonist Binds ~10x more strongly (10x lower K d ) than epinephrine Used in treating bronchial asthma, chronic bronchitis, and emphysema Alprenolol Antagonist Antagonist of cardiac muscle cell epinephrine-responsive G protein coupled receptor (β 1 -adrenergic receptor) Receptor activation increases the heart contraction rate Antagonists (beta-blockers) slow heart contractions. Used in treatment of cardiac arrhythmias and angina

Proparanolol Agonist or antagonist? Propranolol is a medication of the beta blocker type. It is used to treat high blood pressure, a number of types of irregular heart rate, thyrotoxicosis, capillary hemangiomas, performance anxiety, and essential tremors Structure? PTSD treatment: Propranolol is being investigated as a potential treatment for PTSD. Propranolol works to inhibit the actions of norepinephrine, a neurotransmitter that enhances memory consolidation.

Now you can understand the signaling!!! Now you can think for the process!

How to study the downstream pathway? Binding or not? Phosphorylation or not?

Experiment Hematopoietic stem cell PDGF RacGTP Hematopoietic stem cells (HSCs) or hemocytoblasts are the stem cells that give rise to all the other blood cells through the process of haematopoiesis. Platelet-derived growth factor (PDGF) is one of numerous growth factors that regulate cell growth and division. In particular, PDGF plays a significant role in blood vessel formation (angiogenesis), the growth of blood vessels from already-existing blood vessel tissue. Rac is a subfamily of the Rho family of GTPases, small (~21 kda) signaling G proteins (more specifically a GTPase).

Signal transduction 1. Binding of Rac to PAK? 2. Phosphorylation? *p21-activated kinases (PAKs)

Pull down assay

Then let s study the downstream of PAK! What s the PAK? How to test the phosphorylation?

Activation by the hormone erythropoietin (Epo) of three signal transduction proteins via their phosphorylation Western blotting with antibody specific for phosphorylated site reveals signal-dependent phosphorylation of target proteins.

Then let s study the downstream of PAK! Why? Nature Reviews Cancer 14, 13 25 (2014)

Angiogenesis

How many GPCR do the cells have?

Signal Transduction and G Protein Coupled Receptors 15.3 G Protein Coupled Receptors: Structure and Mechanism The large diverse family of G protein-coupled receptors (GPCRs) respond to a variety of extracellular signals and activate trimeric G proteins. G proteins function as On-Off switches for intracellular signaling pathways by activating or inactivating ion channels or effector enzymes that generate second messenger molecules. GPCR signaling pathways regulate a wide range of cellular activities from metabolism to gene expression.

General structure of G protein coupled receptors. The same orientation in the membrane N-terminus outside, C-terminus in cytosol Contain seven transmembrane α-helical regions (H1 H7) Have four extracellular segments (E1 E4) Four cytosolic segments (C1 C4) Ligands have not been identified for many orphan receptors.

3D structure of GPCR Pocket? Conformational change? What induces?

Binding of ligands to GPCRs (a) β 1 -adrenergic receptor bound to antagonist cyanopindolol (ligand): H 1-7 transmembrane helices E2 one of 4 extracellular loops C2, C4 two of four cytosolic domains (b) Hormone-binding pocket: Side chains of 15 amino acids in four transmembrane α helices (3, 5, 6, and 7) and one extracellular loop E2 make noncovalent contacts with the ligand. Examples of specific binding interactions: N atom in both in cyanopindolol and in epinephrine forms an ionic bond with the carboxylate side chains of helix 3 aspartate 121 (D) and helix 7 asparagine 329 (N). (c) Glucagon (29-amino-acid peptide) binding to the glucagon receptor: Glucagon C-terminus (red) binds to the receptor N-terminal domain Glucagon N-terminus thought to insert into a binding pocket that is in the center of the seven transmembrane α helices

General mechanism of the activation of effector proteins associated with G protein coupled receptors Ligand-activated G protein coupled receptors (GEFs) catalyze exchange of GTP for GDP on the α subunit of a heterotrimeric G protein Box trimeric G protein G α and G βγ subunits tethered to the membrane by covalently attached lipid molecules (wiggly black lines) Step 1: Ligand binding induces receptor activation conformational change. Step 2: Activated receptor binds to trimeric G protein. Step 3: Activated receptor GEF activity stimulates G α subunit release of GDP. Step 4: GTP binding changes G α conformation Dissociates G βγ (G βγ subunit activates other effector enzymes in some pathways.) Activates G α Step 5: G α GTP activates effector enzyme. Step 6: G α intrinsic GTPase activity hydrolyzes GTP to GDP dissociates G α and turns off effector enzyme. (G protein active for minutes or less)

How to show the activation of G- protein activation in the cell?

Activation of a G protein occurs within seconds of ligand binding to its cell-surface G protein coupled receptor Forster resonance energy transfer (FRET) technique: G α -cyan fluorescent protein (CFP, excited by 440-nm light, fluoresces 490-nm light) G β -yellow fluorescent protein (YFP, excited by 490-nm light emitted from CFP, fluoresces 527-nm light) (a, left) Inactive G α G βγ complex CFP and YFP close enough for energy transfer Excitation of CFP with 440-nm light causes fluorescence energy transfer from activated CFP to YFP emission of 527-nm (yellow) light instead of 490-nm (cyan) light (a, right) activated G-protein dissociation of the G α and G βγ subunits CFP and YFP not close enough for energy transfer Excitation of CFP with 440-nm light Loss of 527-nm light emission emission of 490-nm (cyan) light instead (b) Results: plot of yellow light (527 nm) emission from a single transfected amoeba cell before and after addition of extracellular camp (arrow) Conclusion: Extracellular signal-gpcr interaction stimulates G protein activation within seconds.

Structure of the β 2 -adrenergic receptor in the inactive and active states and with its associated heterotrimeric G protein, G s Ligand binding converts β 2 -adrenergic receptor into a conformation that binds its trimeric G protein.

GPCR Robert Lefkowitz and Brian Kobilka awarded 2012 Nobel Prize in Chemistry for work on β 2 - adrenergic receptor, including structure determination

Major Classes of Mammalian Heterotrimeric G Porteins and their Effectors

GPCR https://www.youtube.com/watch?v=glu_t6dqulu

Signal Transduction and G Protein Coupled Receptors 15.4 G Protein Coupled Receptors That Regulate Ion Channels The cardiac muscarinic acetylcholine GPCR regulates a K + channel. Light stimulation of the photosensitive rhodopsin GPCR closes cgmp-gated Na + /Ca 2+ channels by regulating a cgmp pathway in retinal cells. Several mechanisms act to terminate visual signaling. Adaptation to a wide range of ambient light levels is mediated by movements of the G protein transducin and the inhibitor protein arrestin into and out of the rod-cell outer segment.

In heart muscle, the muscarinic acetylcholine receptor activates its effector K + channel via the G βγ subunit of a G i protein G βγ subunit (rather than G αi -GTP) binds to and opens a K + channel (effector protein). Increased K + exit hyperpolarizes the cardiac muscle cell membrane reduces heart muscle contraction frequency

Rod cell

Rod cell Rod cells are photoreceptor cells in the retina of the eye that can function in less intense light than the other type of visual photoreceptor, cone cells. Rods are usually found concentrated at the outer edges of the retina and are used in peripheral vision.

Cone cell Cone cells, or cones, are one of three types of photoreceptor cells in the retina of mammalian eyes (e.g. the human eye). They are responsible for color vision and function best in relatively bright light, as opposed to rod cells, which work better in dim light. Cone cells are densely packed in the fovea centralis, a 0.3 mm diameter rod-free area with very thin, densely packed cones which quickly reduce in number towards the periphery of the retina. There are about six to seven million cones in a human eye and are most concentrated towards the macula.

Human rod cell. Rhodopsin GPCR senses light in rod cells. (a) Rod cell schematic diagram: Rhodopsin located in the flattened membrane disks of the cell outer segment Synaptic body synapses with one or more interneurons (b) EM of the region of the rod cell bracketed region in (a) junction of the inner and outer segments

Vision depends on the light-triggered isomerization of the retinal moiety of rhodopsin. Rhodopsin: Activated by absorbing energy from photon of light Opsin protein lysine 296 amino group covalently attached to the light-absorbing pigment 11-cisretinal Light absorption: Causes rapid photoisomerization of the bound cis-retinal to the all-trans isomer Triggers rhodopsin conformational change to the activated unstable intermediate meta-rhodopsin I Activated rhodopsin activates G t protein (transducin)

Transducin Transducin (G t ) is a protein naturally expressed in vertebrate retina rods and cones and it is very important in vertebrate phototransduction. It is a type of heterotrimeric G-protein with different α subunits in rod and cone photoreceptors. Rhodopsin Transducin

The light-activated rhodopsin pathway and the closing of cation channels in rod cells. Dark-adapted rod cells: High level of cgmp keeps cgmp-gated nonselective cation channels open Open channels depolarize the plasma membrane to ~ 30 mv, considerably more positive (less negative) than resting potential ( 60 to 90 mv) typical of neurons and other electrically active cells. Stimulates neurotransmitter release

Membrane potential? -30 mv Light on t

Membrane potential? Light on How to go back? -30 mv PDE inactive t

Inhibition of rhodopsin signaling by rhodopsin kinase. Feedback repression of overactivated rhodopsin Process of rhodopsin phosphorylation and inactivation by arrestin completed very quickly, within 50 milliseconds

Now you can imagine your own cell signaling Rhodopsin Transducin Arrestin

Dark- and light-adapted rod cells Sensitivity and protein location Dark adaptation? Light adaptation? Transducin and Arrestin location?

Schematic illustration of transducin and arrestin distribution in dark-adapted and light-adapted rod cells. (a) Dark (vision most sensitive to very low light levels) Transducin most localized in outer segment membranes Arrestin most localized in inner segment region of the cell (b) Bright light (vision is relatively insensitive to small changes in light): Transducin moved from outer segment to inner segment Arrestin moved from inner segment to outer segment Coordinated movement of transducin and arrestin: Contributes to ability to perceive images over a 100,000-fold range of ambient light levels Protein movement mechanism may involve microtubules and motor proteins

Visual signaling https://www.youtube.com/watch?v=jipe3in2ecq

Signal Transduction and G Protein Coupled Receptors 15.5 G Protein Coupled Receptors That Activate or Inhibit Adenylyl Cyclase GPCRs activate G proteins that activate or inhibit adenylyl cyclase generation of camp from ATP and are regulated by feedback repression. camp activates protein kinase A (PKA), which phosphorylates-regulates multiple target proteins including enzymes in cells. Epinephrine activation of its GPCR in liver and muscle cells stimulates glycogen breakdown into glucose by inhibiting glycogen synthesis and stimulating glycogen breakdown via a kinase cascade. PKA activation can stimulate gene expression.

Synthesis and hydrolysis of camp by adenylyl cyclase and PDE. Adenylyl cyclase (AC) catalyzes formation of cyclic camp (second messenger) bond from ATP precursor camp phosphodiesterase (PDE) catalyzes hydrolysis of cyclic bond AMP (not second messenger)

Hormone-induced activation and inhibition of adenylyl cyclase in adipose cells.

Activation of the catalytic domain of mammalian adenylyl cyclase by binding to G αs GTP. (a) Mammalian adenylyl cyclase (AC) membrane-bound enzyme: Two similar catalytic domains each convert ATP to camp on the cytosolic face of the membrane Two integral membrane domains each contains six transmembrane α helices

Structure of PKA and its activation by camp (a) PKA: Two catalytic (C) kinase subunits transfer terminal phosphate from ATP to target protein specific Ser/Thr-OH Two regulatory (R) subunits (-) camp bind and inhibit catalytic subunit phosphorylation activity (+) camp release active catalytic subunits

GPCR & camp https://www.youtube.com/watch?v=0na2xhniaow

Amplification of an extracellular signal by a signal transduction pathway involving camp and PKA.

Activation of CREB transcription factor following ligand binding to G αs -coupled GPCRs camp-pka regulates gene expression through CREB. Step 1: Receptor stimulation leads to rise in camp. Step 2: camp activates PKA. Step 3: PKA catalytic subunits translocate into the nucleus. Step 4: PKA phosphorylates-activates the CREB transcription factor. Step 5: Activated CREB forms complex with the co-activator CBP/P300 and other proteins. CREB complex binds to CRE regulatory elements in promoters of multiple genes. CREB complex binding stimulates transcription of the various target genes controlled by a CRE.

Signal Transduction and G Protein Coupled Receptors 15.6 G Protein Coupled Receptors That Trigger Elevations in Cytosolic and Mitochondrial Calcium GPCR-G protein activation of phospholipase C generates IP 3 (soluble ) and DAG (membrane bound) second messengers from PIP 2. IP 3 triggers the opening of IP 3 -gated Ca 2+ channels in the endoplasmic reticulum and elevation of cytosolic free Ca 2+, which activates PKC and calmodulin. Neural and hormonal stimulation coordinately regulate glycogen breakdown through Ca 2+ and camp. Acetylcholine activation of its GPCR on endothelial cells induces generation of the NO gaseous signal, which stimulates smooth muscle relaxation and vasodilation.

Synthesis of second messengers DAG and IP 3 from phosphatidylinositol (PI).

The IP/DAG pathway and the elevation of cytosolic Ca2+. (a) Opening of endoplasmic reticulum Ca 2+ channels: Step 1: GPCR activation of either the G αo or G αq subunit activates phospholipase C (PLC). Step 2: PLC cleaves PI(4,5)P 2 yields IP 3 and DAG Step 3: IP 3 diffuses through the cytosol IP 3 interacts with and opens IP 3 -gated Ca 2+ channels in the ER membrane Step 4: Ca 2+ ions move down concentration gradient through the channel into the cytosol. Step 5: Ca 2+ binding activates PKC and its recruitment to the plasma membrane. Step 6: DAG activates membrane-associated PKC. Step 7: Activated PKC-Ca 2+ leaves membrane to phosphorylate various cellular enzymes and transcription factors, activating proteins involved in cell growth and metabolism. What s the advantage of DAG?

Oscillations in the cytosolic Ca 2+ concentration following treatment of human HeLa cells with histamine. What happens? High cytosolic Ca 2+ level >10-4 M Ca 2+ : Decreases Ca 2+ channel affinity for IP 3 channels close (Ca 2+ feedback inhibitor of IP 3 -gated Ca 2+ channels) Inhibits further IP 3 -induced release of Ca 2+ from ER store, even in elevated IP 3 Ca 2+ ATPase pumps Ca 2+ back into ER lowers cytosolic Ca 2+ concentration Low cytosolic Ca 2+ concentration potentiates IP 3 reopening of IP 3 -gated Ca 2+ channels if signal persists

Integrated regulation of glycogenolysis by Ca 2+ and camp/pka pathways

Glycogen breakdown

Integrated regulation of glycogenolysis by Ca 2+ and camp/pka pathways Neuronal stimulation Cytosolic Ca 2+ increase Ca 2+ binds to and activates glycogen phosphorylase kinase (GPK) increases glycogen breakdown Epinephrine binding to β- adrenergic receptors camp increase camp activates PKA PKA phosphorylatesactivates GPK increases glycogen breakdown PKA phosphorylatesinhibits glycogen synthase (GS) inhibits glycogen synthesis

Integrated regulation of glycogenolysis by Ca 2+ and camp/pka pathways Hormonal stimulation of two β-adrenergic receptor pathways Epinephrine camp increase activation of PKA PKA Phosphorylates-activates GPK increases glycogen breakdown Phosphorylates-inhibits GS inhibits glycogen synthesis Vasopressin IP 3 increase cytosolic Ca 2+ increase Ca 2+ binds to and activates glycogen phosphorylase kinase (GPK) increases glycogen breakdown enhances PKC activation by DAG DAG increase activation of PKC PKC phosphorylatesinhibits GS inhibits glycogen synthesis Different pathways => camp and Calcium regulations!

The Ca 2+ /nitric oxide (NO)/cGMP pathway and the relaxation of vascular smooth muscle Endothelial cells signaling to smooth muscle cells: Acetylcholine activation of its GPCR Step 1: G-protein activation of PLC Step 2: PLC generation of IP 3 (+ DAG) cytosolic Ca 2+ increase activates calmodulin Step 3: Ca 2+ -calmodulin activates NO synthase Step 4: NO synthase generates NO (nitric oxide, gaseous signal molecule) Step 5: NO diffuses locally into smooth muscle cells activates guanylyl cyclase (NO receptor) Step 6: Guanylyl cyclase generates cgmp (second messenger) Step 7: cgmp activates protein kinase G (PKG). PKG phosphorylates target proteins decreases cytosolic Ca 2+ smooth muscle relaxation

Discussion with friends Ras Signaling 1. Here is the Rassignaling. Please find Ras-Raf-MAPK pathway and explain the result. www.abcam.com 2. Find one example drug which regulates the GPCR signaling. Impaired Memory Consolidation in Rats Produced with -Adrenergic Blockade Larry Cahill,* Christian A. Pham,* and Barry Setlow 3. Find this paper and explain the figure 1 with the betaadrenergic pathway. 4. Find the mechanism of Viagra and its side effects in terms of the molecular signaling.

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