Week 8: GPCR

• G protein couple receptors (GPCR) have a conserved structure/topology which contain

  • 7 transmembrane segments that form an alpha helical barrel structure -> ligand binding occurs on interior of barrel that leads to conformation change
  • an extracellular ligand pocket that connects information to inside at TM regions 5/6
  • its ligands are usually small molecules, 300 ligands are known now, more than 700 GPCR in humans

  
• ligands can have multiple effects on GPCR signalling

  • agonist -> ligand binds to receptor and activates its normal biological response
  • antagonist -> ligand that binds to receptor and blocks activation -> dampening biological response
  • inverse agonist -> ligand that binds to receptor and activates it in an opposite/different response, anything but the normal response
• types of agonists

  • full agonist -> gives normal response, response is concentration dependent, ability can go to full 100% activation
  • partial agonist -> normal response activity is less than 100%
  • neutral antagonist -> keeps activity at 0
  • inverse agonist -> increase in activity but different response

  
• the GPCR is activated when a ligand binds in the pocket which causes TM 5 to be pulled into the cavity (usually sticks out) and comes into contact with TM regions 3, 6, 7 to form a docking site for the G protein alpha subunit to bind

  
• the G protein is a heterotrimeric protein localized and recruited by GPCR with an alpha, beta, gamma subunit

  • alpha -> lipidated, a GTPase (Ras family) which binds GDP/GTP
  • inactive -> GDP bound, bound to beta/gamma
  • active -> GTP bound, released from beta/gamma -> both alpha and beta/gamma are active and have a function downstream when released from each other
  • beta -> bound with gamma
  • gamma -> lipidated, bound with beta

  
• ligand binding to GPCR makes it act as a GEF which allows binding of alpha G protein -> this binding causes AH domain of alpha G protein to open and have affinity for GTP

  
• GTP loading of the alpha subunit has 2 effects:

  • heterotrimer G protein releases from GPCR
  • GTP bound alpha subunit and beta/gamma subunits dissociate, both of which are considered active

  
• the G protein is lipidated (alpha and gamma) to keep it localized to the membrane bound GPCR

  
• the different signal cascades activated by GPCRs are

  • phosphatidylinositol 3 kinase (PI3K)
  • adenylyl cyclase (AC) and protein kinase A (PKA)
  • phospholipase C (PLC)
  • control over a variety of ion channels and transporters

  * also activated by most RTK

  
• GF independent

  Direct activation of PI3K/Akt pathway
• GF dependent

  Indirect activation of PI3K/Akt pathway
• GPCR and PI3K/Akt pathway, activated in 2 ways

  • GF independent
  • agonist binds GPCR and turns on -> G protein binds which opens alpha subunit AH domain -> alpha becomes GTP bound and dissociates from beta/gamma -> beta/gamma DIRECTLY interact with PI3K by allosteric binding which turns it on

  
• GPCR and PI3K/Akt pathway, activated in 2 ways

  • GF dependent
  • agonist binds GPCR and turns on -> G protein binds which opens alpha subunit AH domain -> alpha becomes GTP bound and dissociates from beta/gamma -> GTP alpha activates a downstream TF to increase GF/GF transporter production -> transporter localized, GF transported to extracellular environment so it can bind to its receptor and activate PI3K
  • this is INDIRECT activation

  
• adenylyl cyclase (AC) is a 12 transmembrane protein (close to GPCR) with 2 catalytic domains in the cytosol which is activated BY 1 G protein and synthesizes cAMP at both of its domains

  • catalytic domain takes ATP and cleaves pyrophosphate which causes a cyclic phosphodiester bond to make cAMP
  • 1 G protein activates 1 AC which makes 100-1000 cAMP
  • AC is a molecular amplifier, cAMP is a second messenger

  
• different GPCR can activate or inhibit AC depending on cell and GPCR type

  • Gs -> GTP bound alpha subunit (Gs) can stimulate AC
  • Gi -> GTP bound alpha subunit (Gi) can inhibit AC

  
• fluorescent cAMP reporters are proteins that change fluorescent colour when bound to cAMP -> informs about intracellular cAMP levels

  • NT (serotonin, GPCR agonist) added to neuron -> increase of fluorescent colour change in neuron because cAMP is made and bound

  
• PKA is a heterodimer of dimers (4 subunits) and not membrane bound consisting of

  • 2 regulatory subunits -> keeps kinase subunit off when no cAMP, has 4 allosteric binding sites for cAMP to bind to which allows the release of kinase subunits (regulatory subunits stay dimeric)
  • 2 kinase subunits -> off when bound to regulatory subunits, on when cAMP binds to regulatory subunits and releases kinase subunit where they become monomeric which can enter the nucleus
  • functions to phosphorylate targets like CREB

  
• active PKA phosphorylates CREB on Ser133 which is a TF in the nucleus (monomeric PKA is small enough to fit in NPC)

  
• phosphorylated CREB binds to genes that have a CREB responsive element (CRE) -> CREB binding protein (CBP) binds to the CREB/CRE area to co-activate the complex -> allows for activation/inactivation of specific genes (depending on placement of binding)

  
• CREB plays a role in neuronal dependent long term memory formation in the brain and regulates genes involved in mammalian circadian rhythms

  • decreased CREB -> implicated in Alzheimer's disease and depressive disorders
  • increased CREB -> implicated in schizophrenia
• phospholipase C (PLC) is an enzyme that cleaves PIP2 into their lipid and inositol components, both of which are second messengers

  • diacylglycerol (DAG) -> activates PKC
  • IP3 -> releases Ca from ER

  
• some GPCR activate PLCbeta

  • GPCR binds agonist -> TM 5 pulled into cavity with TM 3, 6, 7 which acts as GEF for alpha G protein -> GTP loads into AH domain of alpha -> G protein subunits separate -> alpha subunit (Gq) activate PLCbeta -> PLCbeta cleaves PIP2 to DAG and IP3 -> IP3 allosterically binds Ca channels at ER to release -> Ca and DAG bind to PKC -> PKC is active now

  
• Ca levels in cytosol is low, in ER is high

  
• there are 3 mechanisms that regulate the IP3 gated Ca channel on the ER:

  • IP3 binding site -> causes Ca channel to open
  • first Ca site -> high affinity Ca binding site that helps the Ca flux, positive feedback, occurs during low Ca levels
  • second Ca site -> low affinity Ca binding site that closes the channel, negative feedback, occurs during high Ca levels

  thus Ca is released in bursts and acts as a second messenger

  
• activation of the PLC causes Ca signal 'waves' in cells, visualized with a fluorescent reporter for Ca that displays a concentration gradient upon opening Ca channels with IP3

  
• PLC activation causes Ca signal oscillations across the ER membrane

  • IP3 binds to receptor to open the Ca channel -> low Ca levels released from ER binds to high affinity Ca binding site on channel to further open channel for Ca flux (positive feedback) -> high Ca levels released bind to low affinity Ca binding site to close the channel (negative feedback) -> cytosolic Ca returns to normal -> if more IP3, process restarts (oscillations)

  
• Ca signal oscillation frequency depends on multiple factors:

  • concentration of ligand present
  • how specific it binds to GPCR
  • how much PLC activated by GPCR
  • how much IP3 is made

  the amount of Ca bursts doesn't change with increasing agonist/drug/hormone, only the frequency will increase because of the positive and negative feedback loop process

  
• Ca signalling is important in membrane traffic, hormone secretion, activation of calmodulin (phosphate binding protein), and activation of the Ca/calmodulin dependent kinase (CAMK)

  
• calmodulin is a universal Ca signal adaptor

  • Ca binding (4 Ca sites) to calmodulin causes a change in conformation which allows it to bind/activate a number of target proteins with a CAM motifs and wraps around them to protect these regions
  • conformation goes from disordered to ordered dumbbell structure

  
• activation of CAM dependent kinases

  • CAMK is a hexameric complex, each protomer has 2 domains (kinase and hub domain) connected by a linker region that contains a CAM motif and phosphorylation site
  • active calmodulin binds to the CAM motif on the linker to keep kinase domain open so it can auto phosphorylate and be 100% active -> can't be dephosphorylated because calmodulin present
  • open kinase domains can also trans phosphorylate other closed kinase domains (because no calmodulin) which activates them 50-80% -> at risk of dephosphorylation because no calmodulin

  
• in the activation of CAMK

  • low levels of GPCR ligand -> low frequency Ca oscillations, insufficient signal to activate CAMK
  • high levels of GPCR ligand -> high frequency Ca oscillations, sufficient signal to activate CAMK, noise threshold passed

  
• GPCR can activate the olfaction sensory system

  • humans have 350 olfactory GPCR (mice have 100) each binding to a different odourant
  • each olfactory neuron produces 1 type of olfactory GPCR
  • ligand binding to olfactory GPCR triggers signalling that initiates an AP in neurons
  • smelling -> your brain interpreting which neurons are activated with AP

  
• GPCR get activated and propagate downstream signals but these signals are not always on, desensitization occurs with prolonged ligand input through negative feedback loops like

  • GPCR phosphorylation
  • beta arrestin binding
  • endocytosis
• GPCR negative feedback loop: phosphorylation of GPCR

  • the ACTIVATED cavity on GPCR (TM 3/5/6/7) can bind alpha G proteins to activate but is also a good substrate for GPCR kinases (GRK) to phosphorylate the cavity at multiple spots
  • phosphorylation alone inhibits alpha G subunit binding moderately
  • phosphorylation can recruit arrestin to this docking site and completely block alpha G subunit from binding

  
• GPCR negative feedback loop: beta arrestin binding induces GPCR down regulation through endocytosis

  • once GPCR cavity is activated by ligand -> GRK phosphorylate cavity -> beta arrestin binds to phosphorylated GPCR -> beta arrestin/GPCR complex undergoes endocytosis by sequestration or degradation
  • beta arrestin recruits adaptor protein 2 (AP2) which is a docking site for clathrin which forces pits to induce endocytosis

  
• beta arrestin can also activate particular signals other than G proteins like

  • Ras/MAPK pathway
  • NFkB pathway (for gene expression)
  • CREB (TF) adding regulation via Ca

  
• a single activated GPCR can bind to G proteins or beta arrestin but not both at the same time
• different ligands of a single GPCR can induce unique structures to bias/prefer G protein or beta arrestin binding

  • if G protein biased -> GPCR activates cAMP and Ca signals
  • if beta arrestin biased -> GPCR activates Ras/MAPK and some transcription to cause GPCR degradation
  • this is to say more ligand/drug may not cause higher response because of this

  
• with high degree of similarity between signalling systems of GPCR and RTK, the cell outcome is determined by the details of

  • signal strength
  • signal duration
  • other unique properties of cells being exposed to GF/hormones