Signal Transduction I: Ion channel-linked receptors & GPCRs

1. bind GTP 2. hydrolyze GTP 3. switch between active GTP-bound state and inactive GDP-bound state

what can GTP/GDP-binding proteins do?

the binding of ACh to its nicotinic receptor --> in the case of the neuromuscular junction, can induce paralysis & death

what do Curare (arrow poison) and some snake toxins block? what can this cause?

membrane-bound phospholipase A2 to produce second messengers arachidonic acid and eicosanoids

what does G(o) activate?

phospholipase 3 to produce IP3 and DAG [see slide picture]

what does G(q) activate?

ACh binding increases Na+ & K+ permeability causing depolarization that opens voltage-gated Na+ channels --> triggers action potential & muscular contraction

what does nACh do?

1. cyclooxygenase enzymes 2. 5-lipoxygenase 3. epoxygenase

what enzymes synthesize eicosanoids from AA?

corticosteroids

what inhibits arachidonic acid in cell membrane?

aspirin and NSAIDs

what inhibits cyclooxygenase pathway?

intrinsic GTPase activity that hydrolyzes GTP --> GDP which inactivates G proteins

what intrinsic activity do GTP/GDP-binding proteins have? what does this result in?

polyunsaturated essential fatty acid --> one of the lipid tails in membrane-bound phospholipids that can be enzymatically released

what is arachidonic acid (AA)?

can form many different combos --> G-proteins are ideally suited to link a diversity of receptors to a diversity of effectors

what is the implication of the numerous different subunit isoforms of the G-protein?

- GPCRs - respond to many signals: hormones, NTs, and light - mediate nearly every important physiologic process

what is the largest family of cell membrane receptors? what do they respond to? what do they mediate?

receptor spans membrane 7 times --> 7TM (seven transmembrane)

what is the structure of GPCRs?

- naturally occurring small molecules: adrenaline (epinephrine), dopamine, prostaglandins, and adenosine - drug-like small molecules: caffeine, morphine, heroin, histamine

what kinds of molecules bind to GPCRs?

aspirin

what med inhibits COX1 & COX2?

- cyclic nucleotides (cAMP, cGMP) - IP3 (inositol triphosphate) and DAG (diacylglycerol) - arachidonic acid & metabolites (eiconsanoids) - intracellular Ca2+

what second messengers are produced downstream of G-proteins?

they carry the message to, and regulate a specific set of target proteins in the plasma membrane including ion channels & membrane-bound enzymes

when G-proteins are activated by a receptor, what do their alpha-GTP and free beta-gamma subunits do?

inactive

when GDP is bound, G-protein is...

active

when GTP is bound, G-protein is...

GPCRs

1/3 of all drugs used today work through...

leukotrienes & some HETE compounds (hydroxyeicosatetraenoic acid)

5-lipoxygenase enzymes produce...

cell opens channel which can be ligand-gated, G protein-linked, or voltage-dependent

how can you raise cytoplasmic Ca2+?

by acting on muscle

how does Ca2+ exert its effect directly?

activated PKA phosphorylates inactive CREB --> active CREB --> CREB-binding protein (CBP) binds to activated target gene --> transcription --> translation --> new protein

how does cAMP affect gene expression?

epinephrine promotes glycogen breakdown by cAMP-PKA in skeletal muscle and liver by 5 steps: 1. activates GPCR 2. increases cAMP 3. activates PKA 4. activates glycogen phosphorylase 5. glycogen breakdown

how does cAMP mediate glycogen breakdown?

uses one or more active transport mechanisms, including Ca-ATPases and Na/Ca exchangers

how does cell remove Ca2+ and terminate Ca2+ signal in cytoplasm?

at least 16 different isoforms in mammalian tissues --> each specific to particular receptors and activate different downstream effectors

how many G-protein alpha subunit isoforms are there?

at least 5 different beta and 11 different gamma subunits in mammalian tissues

how many G-protein beta-gamma subunit isoforms are there?

serotonin, GABA, glycine receptors

ACh receptor superfamily

inhibits adenylyl cyclase activity

G-alpha(i) [G(i)] does what?

- Ach from vagus nerve acts on pacemaker cells --> decreased heart rate - mediated by muscarinic AcH receptors --> 7TM receptors that work thru G proteins - this GPCR activates G-alpha(i) and liberates beta-gamma subunits which bind to a plasma membrane K+ channel, open it, increase K+ efflux from myocytes, hyperpolarize myocytes, and make it harder for them to fire - K+ channel recloses when G-alpha(i) 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

G-alpha(i) and beta-gamma control an ion channel in the heart - how?

activates phospholipase A2 to produce arachidonic acid (AA)

G-alpha(o) [G(o)] activates ____ to produce ____

stimulates phopsholipase C which produces IP3 and DAG

G-alpha(q) [G(q)] stimulates ____ which produces ____

stimulates adenyl cyclase activity which produces cAMP

G-alpha(s) [G(s)] stimulates ____ which produces ____

1. agonist binds to GPCR 2. receptor-G-protein interaction 3. GDP release 4. GTP binding 5. activated alpha (with GTP) dissociates from beta-gamma complex of the G-protein ** alpha and gamma subunits are still attached to the plasma membrane by lipid anchors and move freely laterally to interact with other membrane proteins 6. G-alpha (with GTP) and beta-gamma each activate target proteins 7. hydrolysis of GTP by G-alpha's intrinsic GTPase activity --> inactivates GTP and causes it to dissociate from target protein 8. inactive G-alpha reassociates with beta-gamma to re-form inactive G-protein

G-protein activation

see slides 13 & 14 for graphics

G-protein activation/inactivation

- IP3 receptor on ER is a calcium-specific channel - IP3 binds to IP3 receptors which trigger the opening of the Ca2+ channel - Ca2+ stored inside ER rushes out into cytosol thru open channels - increased Ca2+ signals to other proteins participating in many cellular processes including activation of PKC

IP3 action

Ca2+

IP3 receptor & Ryanodine receptor in ER are _____ channels

- DAG = lipid that remains membrane associated after its produced by PLC-beta - DAG helps recruit and activate protein kinase C --> translocates from cytosol to the plasma membrane - activated PKC phosphorylates other proteins and alters their functional state

action of DAG

see slide 34

arachidonic acid signaling pathway

PLA2

arachidonic acid signaling pathway: direct pathway

DAG and PLC-beta

arachidonic acid signaling pathway: indirect pathway

slow

cAMP affecting gene expression is an example of ____ (slow/quick) response

GPCR --> G(s) --> adenylyl cyclase --> cAMP

cAMP cycle

major response = cortisol secretion

cAMP mediated response: ACTH acting on adrenal gland

acts on heart --> increases heart rate and force of contraction

cAMP mediated response: adrenaline (epinephrine) acting on heart

major response = glycogen breakdown

cAMP mediated response: adrenaline acting on skeletal muscle

major response = fat breakdown

cAMP mediated response: adrenaline, ACTH, and glucagon acting on fat

quick

cAMP mediating glycogen breakdown is an example of ____ (slow/quick) response

cAMP to 5'-AMP

cAMP phosphodiesterase breaks down...

- Ca2+ responsive protein - most widespread and common member of calcium binding proteins that includes troponin C in muscle

calmodulin is what kind of protein?

ADP ribsoylation of G(s) --> abolishes GTPase activity of G(s) by locking it in the active state --> continuously stimulates adenylyl cyclase --> causes excessive outflow of Cl- and H2O into the gut

cholera toxin (bacterial toxin) causes...

prostaglandins, thromboxanes, and prostacyclins

cyclooxygenase enzymes (COX1 & 2) produce...

other HETE and ETE compounds (cis-epoxy-eicosatrienoic acid)

epoxygenase enzymes produce...

- agonists binds a specific GPCR - receptor activates G-protein of the G(o) or G(i)/G(o) family - activated G-protein then activates membrane phospholipase A2 (PLA2) - PLA2 hydrolyzes specific phospholipids to release AA - enzymes in 3 diff pathways catalyze stereospecific insertion of oxygen into AA to produce several different eicosanoids - AA & eicosanoids can act as intracellular second messengers or diffuse out of cell to exert effects like inflammatory & allergic responses, platelet aggregation, vascular smooth muscle and vascular endothelium, and gastric acid secretion

generation & actions of AA and eicosanoids

- act as gates in cell membrane - ligand binding opens gate, which lets in specific ions thru channel in receptor - receptors = protein multimers that form the membrane-spanning pore - cellular response in picture: change in ion concentration triggers cellular response

ion-linked channel receptors

in conjunction with calmodium which is a ubiquitous calcium binding protein

more often, how does Ca2+ work?

GPCRs

muscarinic ACh receptors are...

GPCR genes

mutations in what kind of genes are being increasingly recognized as important in the pathogenesis of various diseases?

autoantibodies to the ACh receptor

myasthenia gravis involves...

an ion-linked channel receptor

nicotinic acetylcholine (nACh) receptor is an example of...

- IP3 receptor in ER (Ca2+ channel) - Ryanodine receptor in ER (Ca2+ channel) - ACh receptor superfamily: serotonin, GABA, glycine receptors

other examples of ion-channel linked receptors

ADP ribosyates G(i) --> inhibits function of alpha(i) in lung airway cells --> leaves it unable to inhibit adenylyl cyclase --> stimulates coughing

pertussis toxin (bacterial toxin)

3 different subunits: alpha (GDP), beta & gamma

subunits of the G-protein

- ion channel-linked receptors --> ligand gated ion channels or "ionotropic receptors" - G-protein linked receptors - enzyme-linked receptors aka "catalytic receptors"

transduction pathways for cell membrane receptors (3)

- G-proteins: GTP/GDP-binding proteins - activated by cell surface receptors and carry signals from receptors to intracellular effector proteins

what are G proteins? what are they activated by? where do they carry signals?

- important as a secondary messenger for blood coagulation, muscle contraction, & nerve function - affects many cellular functions by interacting with various Ca2+ responsive proteins

what are calcium ions important for?

metabolites of AA

what are eicosanoids?

epinephrine & norepi

what are some amino-acid derived hormones that function thru GPCR?

- important: oxytocin, somatostatins, PTH, endothelin-1 (ET-1) - other kinds: glucagon, angiotensin, GnRH, SS, GHRH, FSH, LH, TSH, ACTH

what are some peptide and protein hormones that function thru GPCR?

- 5 different Ca2+/calmodulin-dependent protein kinases (CaM-kinases) - myosin light chain kinases (MLCK)

what can Ca-calmodulin bind to and activate?

when alpha hydrolyzes the GTP and re-associates with beta-gamma to restore the inactive configuration --> this is how G-proteins work like a switch (turned off/on by signal receptor interactions on cell's surface)

when does the G-protein activation cycle end?

outside cell, ER, and in mitochondria within cell (in mM)

where is Ca2+ high?

in cytoplasm --> in nM

where is Ca2+ low?