G-protein coupled receptors

Importance of GPCR

• largest class of receptor

• Mediate wide variety of responses

Basic structure

• 4 extracellular domains

• 7 transmembrane domains

• 4 cytosolic domains

Extracellular domains

• E1 = amino terminal

• E2-E4 =loops

• For ligand binding

Transmembrane domains

• TM1-7

Cytosolic domains

• C1-C3 are loops

• C4 = C terminal , lipid anchor extends into membrane

Ligand binding

• GPCR = barrel in membrane

• TM helices form a cavity

• Often covered by E2 loop, so only binds small ligands

Conformational change in TM helices

• caused by ligand binding

• Helices twist

Activation of receptor

• helices twist and reveals AA in cytosolic domains

• Heterotrimeric G proteins activate

• Bulky ligands, such as large proteins, may bind to extracellular domains

Heterotrimeric G proteins

• alpha, beta and gamma subunits

• Change in shape of receptor leads to nucleotide exchange and activation of G proteins

• When GTP binds, the G alpha subunit activates and the G protein complex dissociates from the receptor

Inactive G protein

• GDP or no guanine nucleotide bound

Active G protein

• GTP bound

G protein after dissociation from receptor

• G alpha - GTP monomer

• G beta gamma dimer

• Both modulate activity of other intracellular proteins

G protein regulation

• G alpha slowly hydrolyses its GTP

• When GTP is hydrolysed, the G alpha subunit is inactive

• G alpha subunit then re associates with the G beta gamma dimer

• Trimmer binds to receptor

Signal desensitisation

• change in shape exposes residues on cytosolic domains which can be phosph

• GPCR kinase phosphorylates the residues

• More phosphorylation = less ability to activate receptor

• More activation of receptor = more phosphorylation

• Therefore, desensitisation

Resensitising

• when all 7 sites are phosphorylated

• Arrestin binds

• Signalling stops

• Over time, arrestin and the P are removed

• Receptor becomes activate again

Heterotrimeric G protein diversity

• lots of different alpha, beta , gamma subunits

• Therefore lots of combos

• Some are tissue specific

Nomenclature of G proteins

• Gs = stimulates adenylate cyclase

• GI = inhibits adenylate cyclase

Types of error (fight or flight)

• type 1 = believing a falsehood

• Type 2 = rejecting a truth

Fight or flight response

• stimulated by the release of cortisol and epinephrine from adrenal glands

• Converts huge amounts of fat and glycogen to substrate for energy production, needed for rapid muscle movement

Cortisol

• long term stress hormone

• Stimulates gluconeogenesis

• Raises blood glucose

Epinephrine

• binds to adrenergic receptors (GPCR)

Alpha adrenergic receptors

• inhibits insulin secretion

• Stimulates glycogenolysis and glycolysis

Beta adrenergic receptors

• triggers glucagon secretion

• Increases lipolysis by adipose tissue

GPCR (adrenergic)

• upon stimulus, epinephrine binds to beta adrenergic receptors

• Activates Gs Heterotrimer

• G alpha s with GTP bound, binds to and stimulates adenylate cyclase, converts ATP to cAMP

• G beta inhibits adenylate cyclase (regulation)

• The G protein complex / GPCR is regenerated

Cyclic AMP

• cAMP

• Second messenger

• Response generated depends on which target proteins are present

Epinephrine response via cAMP

• glycogenolysis in skeletal muscle

• Induces contract reaction in cardiac muscle

• Therefore is very involved in fight or flight response

Protein kinase A

• PKA

• Activated by cAMP

• Phosph target proteins

• Pathways acting through cAMP generally give same response

Target proteins of PKA

• transcription factors

• Ion channels

• Enzymes

Major control of cAMP mediated pathways

• cell-type specific expression of cAMP targets

Epinephrine signalling complexity - beta adrenergic GPCR

• Coupled to Gs Heterotrimeric G protein complex

• G alpha s stim adenylate cyclase

• G beta s inhibit, prevents continual stim

Epinephrine signalling complexity - alpha adrenergic GPCR

• coupled to GI Heterotrimeric G protein complex

• G alpha i , inhibits adenylate cyclase GI

• G beta gamma, activate growth signalling MAPK enzyme cascade

• Cross talk w growth signalling pathway

Epinephrine signalling termination

• adenylate cyclase acts as GAP on G alpha s

• Means short lived response

GAP

• GTPase activating protein

Short term glucagon signalling

• insulin lowers blood sugar via PIP3 second messenger

• Leptin = JAK STAT pathway

Longer term glucagon signalling

• glucagon raises blood sugar via PIP3

• GPCR and cAMP stimulant glycogen breakdown

Cholera toxin

• CTx

• A and B chain

• A chain proteolytically cleaved , catalytic chain CTxA1 disulfide linked to CTxA2

• A2 anchors A1 into B chain pentameter (donut shaped)

CTx transport

• traffics to ER of target cells

• Binds to receptor

• Taken in by endocytosis

• Retrograde trafficking via endosome and Golgi to ER

CTx in ER

• PDI breaks disulfide bridge between A1 and A2

• BiP acts as chaperone to CTxA1, keeps it soluble until it dislocates across ER membrane

• CTxA1 folds in the cytoplasm

PDI

• protein disulfide isomerase

BiP

• binding protein

Toxicity of CTxA1

• ADP-ribosylase transfers ADP-ribose group to G alpha s Means

• No longer hydrolysees GTP, permanently active

• Abnormally high cAMP levels

• Activates cystic fibrosis transmembrane receptors

• Increases efflux of Na+ and water into intestine

• Fatal diarrhoea