what are G-protein coupled receptors?
metabotropic receptors:
- indirectly linked to ion channels through signal transduction mechanisms via G-proteins
what are the structural features of GPCRs?
- 7 TM alpha-helices, linked by extracellular and intracellular loops
- TM3 is centrally located next to binding pocket which is crucial for transduction and ligand binding
- other TMs and extracellular N-terminus contributes to ligand binding
- C-terminus at intracellular side is for G-protein binding
- TM5 and TM6 control intracellular binding pocket for G-protein binding when the GPCR is activated
how are GPCRs activated?
GPCRs exist in an equilibrium:
- no ligand = inactive
- ligand-bound = active
ligand binding induces a conformational change in TM5 and TM6 which opens up the binding pocket on the intracellular side for the G-protein to bind
how are GPCRs distinguished?
- structural features of the extracellular domains which define the ligand binding site
- linked to huge diversity of the stimuli GPCRs can detect
what is an example of a GPCR?
Protease-Activated Receptors (PAR) in platelets:
- Receptor activated by cleavage of the N-terminal which in turn acts as a tethered ligand
- Part of the receptor itself acts as the agonist
- Receptors work together to elicit a response – 3 independent stimuli activate platelets: thrombin, ADP and exposure of the basal lamina
- Results in clot formation by crosslinking of platelets
what are G-proteins?
- Guanine nucleotide-binding proteins – belong to the GTPase family
- Act as molecular switches inside cell to transmit signals from extracellular stimuli
- Regulated by ability to bind and hydrolyse GTP (‘on’) to GDP (‘off’)
- Exist as heterotrimeric complexes made up of alpha, beta and gamma subunits
GTP = on
GDP = off
what is the activation mechanism of GPCRs?
- Resting state – 3 inactive components: alpha, beta, gamma
- Activation by ligand binding (TM5 and TM6 move apart to form ligand binding pocket on intracellular side) and exchange GDP for GTP
- Alpha-subunit binds to this pocket
- Alpha-subunit at rest is bound to GDP
- When bound to the receptor, alpha exchanges GDP for GTP
- Formation of 3 separate active components: alpha-GTP dissociates from membrane and from beta-gamma which can now activate downstream effectors
how is G-protein signalling controlled?
- G-proteins are timers
- Duration of signalling by activated trimeric G-protein is regulated by rate of GTP hydrolysis by the alpha-subunit
- Regulators of G-Protein Signalling (RGS) proteins stimulate GTPase activity in the alpha subunit
how is G-protein action terminated?
- ligand dissociates from GPCR
- alpha hydrolyses GTP to GDP, becoming inactive
- beta-gamma and alpha reassociate to form an inactive G-protein
what are the triggers which may terminate G-protein action?
- Agonist dissociating from the receptor
- GTPase activity of the alpha-subunit
- second messenger breakdown
- inactivation of effector enzymes
how many families of G-proteins exist? how are they specific?
6 families:
- various combinations produce a wide range of responses
- differences in the alpha subunit make G-proteins specific to certain receptors and their effectors
how do GPCRs display specificity?
- In multicellular organisms, selective expression of certain receptors and the molecules involved in signal transduction allow cells to respond specifically to particular stimuli
- It is the specific alpha-subunit of the GPCR and cell type that determine the response using the same signalling as other cell types
how are effectors of GPCRs determined?
by the class of the alpha-subunit:
- effectors include enzymes that create second messengers and ion channels whose gating is regulated either directly (beta-gamma subunits) or indirectly by second messengers and their effectors
what may GPCR effectors be?
- ion channels (ionotropic receptors)
- second messenger systems and enzymes
how do G-proteins directly activate ion channels?
- Active G-protein binds to ionotropic receptor
- Similar mechanism as ligand-gated channels - the G-protein changes the activity of the ionotropic receptor:
- may be slow to open or close
- may stay open or closed for longer - minutes rather than milliseconds - Changes electrical properties of the cell
what are second messengers?
- Second messengers are small molecules that carry signals inside cells
- These include:
- Hydrophobic lipids confined to the membrane in which they are
generated - Small molecules that diffuse through the cytoplasm e.g. cAMP, cGMP
- Calcium ions
- Hydrophobic lipids confined to the membrane in which they are
why do we need second messenger systems?
A single ligand binding to a single GPCR results in the phosphorylation and activation of millions of proteins
how does Vibrio cholera affect GPCRs?
- stimulates the Gs alpha subunit
- greater activation of adenylyl cyclase -> more cAMP -> more PKA activation
- causes increased Cl- secretion and increased Na+ and H2O secretion
- means excess fluid and electrolytes in lumen of small intestine
- leads to diarrhoea and extreme dehydration = cholera
how does Bordatella pertussis affect GPCRs?
- inhibits the Gi alpha subunit
- causes inactivation of the inhibitory G-protein, leading to increased adenylyl cyclase
- causes increase in intracellular cAMP
- leads to erosion of the respiratory epithelium and discharge of lots of mucus
- triggers coughing fits = whooping cough
give examples of mutations in GPCRs:
GOF mutations:
- parathyroid Ca2+ sensor -> hypoparathyroidism
- rhodopsin -> night blindness
- Thyroid hormone receptor -> hyperthyroidism, thyroid cancer
LOF mutations:
- cone cell opsin -> colour blindness
- parathyroid Ca2+ sensor -> hyperparathyroidism, cannot respond to
serum Ca2+
- rhodopsin -> retinitis pigmentosa, retinal degeneration
- thyroid hormone receptor -> hypothyroidism
- vasopressin receptor -> nephrogenic diabetes insipidus, kidneys cannot
reabsorb water
how does a mutation in GPCR lead to uveal melanoma?
GNA1 and GNA11:
- Over 90% of uveal melanoma have mutations in Gq alpha-subunit
- Leads to blocking of GTP hydrolysis so subunits are always active, causing permanent signal transmission
- Constitutively active growth pathways – promote cancer progression
- Thought to occur early on in tumour development
what are the 3 main downstream second messengers of GPCRs?
- second messengers:
- adenylyl cyclase -> cAMP
- guanylate cyclase -> cGMP - Phospholipase C-beta
- produces 2 second messengers -> IP3 and DAG - Calcium (the oldest second messenger)
which G-protein alpha subunits affect adenylyl cyclase?
- Gs-alpha subunit stimulates adenylyl cyclase
- Gi-alpha subunit inhibits adenylyl cyclase
which G-protein alpha subunit affects PLC?
Gq-alpha subunit
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Cell communication27
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Ligand-gated ion channels34
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GPCRs44
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Big Data22
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Cell polarity62
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SNAREs53
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Using yeast to understand membrane trafficking38
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Liz Smythe lectures - protein sorting and vesicular transport126
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Autophagy56
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GTPases35
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DNA structure and repair128
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Mechanobiology68
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Immunology100
