Signal Transduction I: Ion Channel-Linked Receptors and G-Protein Coupled Receptors

Second messenger: cyclic AMP (cAMP)

-cAMP was the first substance recognized as a second messenger and has proven to be ubiquitous in biology -Receptors which associate with Gs-type G-protein stimulate adenylyl cyclase which increases cAMP -Receptors which associate with Gi-type G-protein, inhibit adenylyl cyclase which decreases cAMP -cAMP exerts its effect in most cases by activating protein kinase A (PKA) -PKA in turn phosphorylates existing proteins or stimulates gene expression to affect cell metabolism, function and morphology -cAMP is rapidly converted to AMP by phosphodiesterase

G proteins that regulate ion channels

Gαi and βγ control an ion channel in the heart -Acetylcholine (ACh) from the vagus nerve acts on pacemaker cells, causing decreased heart rate -This is mediated by muscarinic ACh receptors, which are 7-pass receptors that work through G proteins -This GPCR activates Gαi and liberates βγ subunits, which bind to a plasma membrane K+ channel, open i, increase K+ efflux from myocytes, hyperpolarize the myocytes, and make them harder to "fire" -The K+ channel recluses when Gαi inactivates itself by hydrolyzing its bound GTP, returning to its inactive state in which the beta-gamma subunit is re-associated with the alpha subunit

Ion Channel-Linked Receptors

-Act as gates in the cell membrane -Ligand binding opens the gate, which lets in specific ions through a channel in the receptor -The receptors are protein multimers that form the membrane-spanning pore Example: Nicotinic Acetylcholine (nACh) Receptor: -The nACh receptor is a ion-channel-coupled receptor (In contrast, muscarinic ACh receptors are G protein coupled receptors, GPCRs) -Found at the neuromuscular junction and other parts of the nervous system -ACh binding increases Na+ and K+ permeability causing depolarization, the end-plate potential, that opens voltage-gated Na+ channel, which triggers action potential and muscular contraction -The arrow poison Curare and some snake toxins block binding of ACh to its nicotinic receptor. In the case of the neuromuscular junction, this induces paralysis and death. -Myasthenia graves (MG): autoantibodies to ACh receptor More Examples of Ion Channel-Linked Receptors -IP3 Receptor (ER; Ca+2 channel) -Ryanodine Receptor (ER; Ca+2 channel) -ACh Receptor superfamily: serotonin, GABA, glycine receptors

Examples of cAMP-mediated responses

-Adrenaline (epinephrine)- targets heart issue-major response is increase in heart rate and force of contraction -Adrenaline- targets skeletal muscle- major response is glycogen breakdown -Adrenaline, ACTH, glucagon- targets fat tissue- major response is fat breakdown -ACTH- targets adrenal gland tissue- major response is cortisol secretion

Generation of AA and Eicosanoids

-Agonist binds to a specific GPCR -Receptor activates a G-protein of the Go or Gi/Go family -Activated G-protein then activates membrane-bound phospholipase A2 (PLA2) -PLA2 hydrolyzes specific phospholipids to release AA -Enzymes in three different pathways catalyze the stereospecific insertion of oxygen into AA to produce several different eicosanoids -AA and the eicosanoids can either act as intracellular second messengers, or diffuse out of the cell and produce profound paracrine effects on nearby cells, as happens in the inflammatory response

Second messengers: Arachidonic Acid and Eicosanoids

-Arachidonic Acid (AA) is a polyunsaturated essential fatty acid 20:4 (ω-6) and is one of the lipid "tails" in some membrane-bound phospholipids that can be enzymatically released -Eicosanoids are metabolites of AA

Second messenger: calcium

-Calcium ion (Ca++) is an important versatile second messenger in many pathways since cells can regulate its concentration and move it around between different cellular compartments -Ca++ is present in the cytoplasm at low concentration (nM) but at much higher concentration outside the cell (mM) and in the ER and mitochondria within the ell -To raise cytoplasmic Ca++, the cell opens a channel which can be ligand-gated, or G protein-linked, or voltage-dependent -Ca++ interacts with various kinds of Ca++ responsive proteins to affect cellular functions -To remove Ca++ and terminate Ca++ signal in the cytoplasm, the cell uses one or more active transport mechanisms, including Ca-ATPases and Na/Ca exchangers

Bacterial toxins that target Gs and Gi proteins

-Cholera toxin causes "ADP ribosylation" of the Gαs, which abolishes the GTPase activity of Gαs, locking it in the active state in which it continuously stimulates adenylyl cyclase, causing excessive outflow of Cl- and water into the gut -Pertussis toxin ADP ribosylates Gαi, which inhibits the function of αi in lung and airway cells, leaving it unable to inhibit adenylyl cyclase. This stimulates coughing.

Transduction Pathways for Cell Membrane Receptors

-Ion channel-linked receptors, aka "ligand-gated ion channels" or "inotropic receptors" -G-protein-linked receptors -Enzyme-linked receptors, aka "catalytic receptors"

Enzymes which synthesize eicosanoids from AA

-Cyclooxygenase enzymes (COX1 & 2) produce prostaglandins, prostacyclins, and thromboxanes -5-lipoxygenase enzymes produce leukotrienes and some "HETE" compounds (hydroxyeicosatetraenoic acid) -Epoxygenase enzymes produce other HETE molecules and "EET" compounds (cis-epoxy-eicosatrienoic acid)

Action of DAG

-DAG is a lipid that remains membrane associated after its produced by PLC-β -DAG helps recruit and activate a particular protein kinase, protein kinase C (PKC), which translocates from the cytosol to the plasma membrane -Activated PKC phosphorylates other proteins and alters their functional state

Eicosanoids-Actions

-Eicosanoids have powerful effects on processes such as inflammatory and allergic responses, platelet aggregation, vascular smooth muscle and the vascular endothelium, and gastric acid secretion -The cyclooxygenase enzyme (COX) comes in two isoforms, COX-1 and COX-2. One of the actions of aspirin is to inhibit both isoforms

G-Proteins

-GTP/GDP- binding proteins -Activated by the cell surface receptors and carry the signals from the receptors to intracellular effector proteins -Can (1) bind GTP, (2) hydrolyze GTP, and (3) switch between an active GTP-bound state and an inactive GDP-bound state -Have an intrinsic GTPase activity that hydrolyzes GTP to GDP, inactivating the G Proteins

G proteins that regulate membrane-bound enzymes

-Gαs or simply Gs: stimulates adenylyl cyclase activity which produces cAMP -Gαi or Gi: inhibits adenylyl cyclase activity -Gαq or Gq: stimulates phospholipase C which produces the IP3 and DAG -Gαo or Go: activates phospholipase A2 to produce arachidonic acid (AA)

Action of IP3

-IP3 receptor on the endoplasmic reticulum (ER) is a calcium (Ca++)-specific ion channel -IP3 binds to IP3 receptors which trigger the opening of the Ca++ channel -Ca++ stored inside the ER rushes out into the cytosol through the open channels -The increased Ca++ signals to other proteins, participating in many cellular processes including the activation of PKC

Structure of GPCR receptors

-The receptors that couple to G proteins have a characteristic structure that spans the membrane 7 times. These are referred to variously as "seven-pass", "7TM" (seven transmembrane), "serpentine", or G-protein-coupled receptors (GPCR)

G-proteins- Subunit Isoforms

-α Isoforms: at least 16 different isoforms are known in mammalian tissues, each specific to particular receptors and activating particular downstream effectors -βγ isoforms: there are at least 5 different β and 11 different γ subunits in mammalian tissue -Implications: The 16α, 5 β, and 11 γ subunits can potentially form several hundred different combinations, making G proteins ideally suited to link a diversity of receptors to a diversity of effectors

G-Protein: Activation

1. Agonist binding 2. Receptor-G-Protein interaction 3. GDP release 4. GTP binding 5. α dissociates from βγ Note: α and γ subunits are still attached to the plasma membrane by lipid anchors, moving freely laterally to interact with other membrane proteins

G-Protein: Inactivation

6. Gα and βγ each activate target proteins 7. Hydrolysis of GTP by Gα's 8. Inactive Gα re-associates with βγ The activation cycle ends when α hydrolyzes the GTP and re-associates with βγ to restore the inactive configuration. In this way, G proteins work like a switch---turned on or off by signal-receptor interactions on the cell's surface

cAMP Mediates Glycogen Breakdown

Epinephrine promotes glycogen breakdown via cAMP-PKA in skeletal muscle and liver: 1. Activates GPCR 2. Increases cAMP 3. Activates PKA 4. Activates glycogen phosphorylase 5. Glycogen breakdown

G Protein Downstream Transduction Pathways

G proteins are activated by a receptor, and their free α-GTP and free βγ subunits carry "the message" to, and regulate, a specific set of target proteins in the plasma membrane including: -Ion Channels -Membrane-bound enzymes

Ca++ responsive protein: Calmodulin

In some cases, Ca exerts directly (as in muscle). More often, however, it works in conjunction with a ubiquitous calcium-binding protein called calmodulin -Calmodulin is the most widespread and common member of a family Ca-binding proteins that includes troponin C in the muscle -Ca-Calmodulin can bind to and activate a multitude of other proteins. Included among these are: --5 different Ca++/calmodulin-dependent protein kinases (CaM-kinases) --Myosin light chain kinase (MLCK)

Second messengers: IP3 and DAG

Inositol triphosphate (IP3) and Diacylglycerol (DAG) The pathway leading to the production of IP3 and DAG -A ligand such as angiotensin-II binds to a GCPR that is coupled to heterotrimeric Gq protein -The activated Gq then binds to and activates the membrane bound enzyme phospholipase C-β (PLC-β) -PLC-β cleaves the membrane phospholipid phosphatidyl-ionositol 4,5-bisphosphate (PIP2) -The second messengers IP3 and DAG are produced

G protein transduction pathways

Second messengers produced downstream of G proteins -Cyclic nucleotides (cAMP, cGMP) -IP3 (inositol triphosphate) and DAG (diacylglycerol) -Intracellular Ca2+ -Arachidonic acid and metabolites (eicosanoids)

G Protein-Linked or Coupled Receptors (GPCR)

Why do you care about G Protein-Coupled receptors (GPCR) as a physician? -The largest family of cell membrane receptors: >1000 GPCRs in humans -Respond to numerous signals: hormones, neurotransmitters and light (photons) -Mediate virtually every important physiological process, from immune function to taste and smell to the fight-or-flight response in humans - > One-third of all drugs used today work through GPCRs -Naturally occurring small molecules which bind to GCPRs include adrenaline (epinephrine), dopamine, prostaglandins, and adenosine -Drug-like small molecules which bind to GPCRs include caffeine, morphine, heroine, and histamine -Mutations in the GPCR genes are being increasingly recognized as important in the pathogenesis of various disease

Cyclic-AMP-Synthesis and breakdown

cAMP cycle: GPCR->Gs->adenylyl cyclase->cAMP cAMP phosphodiesterase breaks down cAMP to 5'-AMP

Go activates

membrane-bound phospholipase A2

Gq activates

phospholipase C