GPCRs Flashcards
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
what is adenylyl cyclase?
- Adenylyl cyclase is a membrane-anchored enzyme and has 10 isoforms
- Carboxyl terminus bind to activate the enzyme
- It is activated by the Gs-alpha-subunit, inhibited by the Gi-alpha subunit
- cAMP signalling increases in the dendrite and cell body when adenylyl cyclase is activated
what are the steps in the cAMP second messenger system?
- Ligand binds to receptor to activate G-protein
- Gs-alpha subunit bound to GTP binds to adenylyl cyclase in the
membrane and activates it - Active adenylyl cyclase catalyses ATP to cAMP
- cAMP (second messenger) activates Protein Kinase A (PKA)
- PKA phosphorylates/activates protein effector
- This activated protein will initiate a response within the cell
what is an example of a Gs-protein cAMP second messenger system?
beta-2-adrenoreceptor regulation of skeletal muscle metabolism:
- Binding of a single adrenaline molecule to the receptor triggers a signalling cascade (Gs-alpha subunit -> adenylyl cyclase -> cAMP -> PKA)
- This results in PKA phosphorylating/activating enzymes that control glycogen metabolism to glucose which is used as energy within the muscles
- Signalling is switched off by:
o Agonist dissociating from the receptor
o GTPase activity of the Gs-alpha-subunit
o cAMP breakdown by phosphodiesterase
o Dephosphorylation of enzymes
what is the cGMP second messenger system?
- Guanylate cyclase is an enzyme that can be receptor-bound or free in the cytoplasm
- It converts guanosine triphosphate (GTP) to 3’,5’-cyclic guanosine monophosphate (cGMP)
- cGMP acts as a second messenger and activates downstream effectors
what determines the local concentration of second messengers?
- Rate of production
- Rate of diffusion from site of production
- Rate of removal
how is cAMP controlled/regulated?
- cAMP production is regulated by adenylyl cyclase
- cAMP is removed by phosphodiesterase as it converts cAMP to 5’-AMP which can no longer bind to enzymes
- phosphodiesterase can be modulated by calcium and calmodulin to speed up breakdown of cAMP
what is the signal that second messengers send?
Message is encoded in the concentration and frequency of changes in concentration of the messenger
what is phospholipase C-beta?
- PLC enzyme cleaves lipids in the phospholipid membrane
- Main target is PIP2: PLC cleaves PIP2 to IP3 and DAG
what second messengers does PLC generate?
Membrane lipids may be targeted by receptor-regulated lipases (PLC) to generate 2 kinds of second messengers:
- IP3: water-soluble and diffuse through cytoplasm
- DAG: a hydrophobic molecule which remains in the membrane
what is the role of lipid kinases in the IP3/DAG second messenger system?
Lipid kinases add phosphate groups to lipids:
- E.g. to DAG to make phosphatidic acid
- E.g. to PI to generate PIP, PIP2, PIP3
how do GPCRs affect PLC?
When ligand is bound to a GPCR, its Gq-alpha subunit activates PLC to generate IP3 and DAG:
- DAG stays in membrane and activates PKC
- IP3 diffuses through cytoplasm and activates calcium channel on the intracellular domain of the ER
how are PLC and PKC so specific?
There are numerous isoforms of PLC and PKC in cells and they all have a conserved X and Y catalytic domain, but differ in their regulatory domains:
- PLC has a PH domain that binds beta-gamma subunit
- PKC (Protein Kinase C) are Ser/Thr kinases, activated by DAG (C1 domain) and Ca2+ (C2 domain)
- PMA phorbol ester is an analogue of DAG used in research to activate PKCs
How does activation of PKC by PLC affect GPCR signalling?
activation of PKC:
- Pseudosubstrate domain self-regulates the PKC to prevent substrates from binding to the substrate-binding domain, but DAG prevents this
- DAG binding causes dissociation of the intramolecular pseudosubstrate domain from active site, once activated PKCs can provide positive/negative feedback in signalling pathway
- Phosphorylation of PLCβ provides negative feedback for GPCR signalling, making the signalling transient
- Phosphorylation of receptors contributes to desensitization
what processes does calcium regulate?
- synaptic transmission
- Hormone secretion & synthesis in some cases
- Fertilization
- Muscle contraction
- Cytokinesis
why is calcium important?
In resting cells cytosolic [Ca2+] is kept low (~100 nM) by ATP-driven Ca2+ pumps – Why?
- Receptors regulate the activity of Ca2+ channels to produce transient rises in Ca2+
- ATP-pumps drive calcium into the extracellular space to keep intracellular calcium low
- Low levels are needed, as if a receptor is activated, the whole signalling pathway is triggered, and calcium influx can occur
what are the 3 ways in which calcium is regulated as a second messenger?
- Calcium influx into the cytosol is regulated by channels in the extracellular membrane and ligand gated channels on the ER
- Moves from high conc in ER (400um) to low conc in cytosol (100nm) - Store-operated channels made up of ORAI and gated by STIM are responsible for store refilling and maintaining ER calcium levels
- Important role in activation of T-lymphocytes – loss of function mutation in Orai1 causes severe combined immunodeficiency (SCID) - Calcium ATP-pumps refill the ER
how can calcium signalling be imaged?
using fluorescent tags such as GFP:
- Used to study signalling in a wide variety of cells in response to many types of stimuli
- To gain insight into disease mechanisms and develop new drugs
- Genetically engineered animal models expressing fluorescent Ca2+ allow in vivo recording of calcium signaling associated with different behaviours
how may GPCRs become desensitised?
by overstimulation of GPCRs
- Tachyphylaxis - e.g. LSD or salbutamol
- Disease e.g. uncontrolled growth in cancer
what are the 2 mechanisms in which GPCRs become desensitised?
- GRK (receptor kinases): stops G-protein from binding to effectors
- Beta-arrestin: phosphorylates the receptor to enable proteosomes to internalise the receptor -> leads to degradation or recycling
why is desensitisation of GPCRs important therapeutically?
- GPCR signalling pathways are important targets for medicine
e.g. in rhodopsin receptors in the retina, GRK may modify the receptor, leading to prolonged photon response and rod apoptosis
