Week 8

• G-protein coupled receptors (GPCRs) are very similar in their structure and topology. But they have a wide range of ligands

  • 7 transmembrane segments form a barrel structure
  • Ligand binding occurs in the interior of the barrel, which leads to a conformational change
  • >700 GPCRs in humans
  • ~300 have known ligands

  
• Agonist - a ligand that binds to the receptor and activates it to cause a biological response.

  
• Antagonist - a ligand that binds to the receptor and blocks its activation, thus dampening a biological response

  
• Inverse agonist - a ligand that binds to the receptor and activates it, but the biological response is the opposite of the natural agonist

  
• Partial antagonist - activates normal biological response but not at its full level

  
• Agonist binding induces a conformational change that activates the GPCR.

  • Ligand binding pulls TM5 into the cavity of the GPCR to create 4 interaction points for G-alpha subunit of a G-protein

  
• Each heterotrimeric G-protein has an alpha, beta, and gamma subunit

  • the alpha subunit is the one that binds to GDP or GTP
  • the alpha subunit bound to GDP and in complex with beta and gamma subunits is inactive

  
• Ligand binding to GPCR leads to a conformational change in the receptor that opens a binding site (AH domain) for the G alpha subunit. The ligand-bound GPCR acts like a Guanyl Exchange Factor (GEF) for the G-alpha subunit by loading in a GTP molecule.

  
• The alpha and gamma subunits of G-proteins are physically tethered to the membrane by a lipid.

  
• GTP loading of the alpha subunit has two effects:

  1. it causes the heteromeric G-protein to be released from the GPCR
  2. The GRP-bound alpha subunit and the Beta-gamma subunit complex dissociate

  Both the alpha and beta-gamma complexes are considered active and have functions to control downstream signal cascades.

  
• GPCRs can activate the PI3K-Akt pathway in two ways:

  1. Growth factor independent
  2. Agonist binds to GPCR and turns it ON --> G-alpha then switches out its GDP for GTP --> G-beta and gamma directly interact with PI3K.
  3. Growth factor dependent
  4. activated G-alpha stimulates a pathway that makes the growth factor needed --> growth factor is then transported out of the cell for GFR to take the stimulus.

  
• Adenylyl cyclase has 2 catalytic domains that produce cAMP from ATP. This process takes off the alpha and beta phosphate. The oxygen of gamma phosphate is linked up to the sugar causing its cyclic nature.
• Adenylyl cyclase acts as a molecular amplifier while cAMP acts as a second messenger.

  
• Both AC domains are found inside the membrane

  
• The alpha-subunit of G-proteins that activate AC is called Gs (stimulatory). The alpha-subunit that inhibits AC is called GI (inhibitory).

  
• Fluorescent cAMP reporter: a protein which changes fluorescence when bout to cAMP -> informs about. the levels of cAMP in the cell
• cAMP binds to and activates protein kinase A (PKA)

  • PKA has two regulatory subunits and two catalytic subunits
  • in the absence of cAMP binding, the regulatory subunits bind to the catalytic subunits, rendering them inactive
  • Binding of cAMP to the regulatory subunits (requires 4 cAMP) of PKA releases the catalytic subunits (monomeric form), which become active
  • when regulatory subunits are released they stay in their dimeric forms.

  
• PKA phosphorylates several targets

  • The activated PKA catalytic subunits have many substrates within the cell
  • CREB (cAMP response element-binding protein) is a TF that is phosphorylated by PKA
  • PKA phosphorylates Ser133 of CREB

  
• The monomeric catalytic site of PKA can enter the nucleus but the full PKA complex can't come in because there's a size-inclusive barrier to get in the nucleus.

  
• PKA phosphorylates the transcription factor CREP

  • phosphorylated CREB binds to genes that contain CREB-responsive element (CRE)
  • CREB-binding protein (CBP) then binds to CREB-CRE to co-activate the complex
  • This allows for the activation or inactivation of specific genes

  
• • CREB plays a role in neuronal-dependent long-term memory formation in the brain
  • CREB down-regulation is implicated in the pathology of Alzheimer's disease
  • Low CREB function is also implicated in depressive disorders
  • CREB hyper-activation is associated wit schizophrenia

  
• CREB regulates genes involved in mammalian circadian rhythms.

  
• Phospholipase C (PLC) is an enzyme that breaks down PIP2 into subunits

  • DAG (activates protein kinase C)
  • IP3 (Releases calcium from the endoplasmic reticulum)

  Both of these units can act as a second messenger

  
• Some GPCRs activate phospholipase C-B (PLCB). Activated alpha g-protein and gamma beta g-protein can turn on PLC.

  
• IP3 acts as an allosteric Calcium opener of Calcium release channels found in the ER.

  
• Protein kinase C is activated by DAG and calcium binding
• The IP3 receptor is an ion-gated channel that is regulated by 3 different mechanisms

  1. IP3 binding (ligand-gated) - opens calcium channels
  2. Low levels of Calcium (positive feedback) - opens Calcium channels (high affinity)
  3. High levels of Calcium (negative feedback) - closes Calcium channels (low affinity)

  
• activation of PLC causes calcium signal waves across the ER membrane:

  1. IP3 binding to its receptor opens the calcium channel
  2. The IP3-receptor is ALSO a calcium-gated calcium channel. Low levels of calcium ions released bind to the IP3-receptor, opening the channel further.
  3. IP3-receptor is also regulated by very high calcium concentration; Multiple calcium ions binding to the receptor closes the channel.
  4. Calcium in cytosol returns to normal levels
  5. If IP3 is still present, the process starts again (producing oscillations)

  
• Calcium signal oscillation frequency depends on multiple factors:

  1. concentration of the ligand there is
  2. How tightly (specific) it binds to the GPCR
  3. How much PLC is activated from the specific GPCR
  4. How much IP3 is produced

  An increase of agonist does not change the amount of burst but it does change the frequency of the burst.

  
• Calcium signalling functions:

  • Changes in membrane traffic and secretion of hormones
  • activation of the phosphatase calmodulin
  • Activation of calcium-dependent kinases: calcium/calmodulin-dependent kinases

  
• Calmodulin: a universal calcium signal adaptor

  • Calmodulin is a "professional" calcium-binding protein
  • Calcium binding to calmodulin causes a change in the conformation of calmodulin
  • calcium bound calmodulin then can bind to several target proteins, causing activation

  
• calmodulin goes from a disordered structure to an ordered structure after binding to calcium

  • requires 4 calcium ions to activate
  • Calcium coordinates protein structure
  • calmodulin looks for Cam motif found in proteins
  • It then tightly binds to it

  
• activation of calcium/calmodulin-dependent protein kinase (CaMK):

  • it is a hexameric complex with each protomer consisting of 2 domains
  • kinase domain
  • Hub domain
  • in between the 2 domains is the linker region
  • Has the cam recognition site
  • Has the phosphorylation site

  
• CaMK: When inactive the linker is buried in the hexameric complex. The hexameric complex is not static as it will close and open repeatedly to capture molecules.

  • CAM looks for the CAM site and wraps around it. This locks the kinase domain to stay ON
  • The kinase domain autophosphorylates with ATP which becomes 100% active
  • Kinase domain can trans phosphorylate a neighbouring protomer. This makes it 50-80% active. Allows calmodulin to come in and bind.

  
• To activate caMK you need high levels of GPCR ligand to produce enough frequency calcium oscillations.

  
• Once calcium concentration in the cytosol return to resting levels (very low), a phosphatase dephosphorylates calmodulin-dependent kinases, causing inactivation.
• Humans have 350 olfactory HPCRs, each one binding to a different odorant.

  • Each olfactory neuron produces only one kind of olfactory GPCR
  • ligand binding to olfactory GPCR triggers signalling that initiates an action potential in that neuron
  • Smelling involves your brain interpreting which neurons are activated with action potentials

  
• GPCR signal transduction can be regulated by 3 types of negative feedback loops:

  1. Phosphorylation of the GPCR
  2. B-Arrestin binding
  3. Edocytosis

  
• Phosphorylation of GPCRs by GPCR kinase (GRK):

  • TM pulls in and makes a complex along with Tm 3,6, and 7.
  • Activated GPCR stimulates GRK to phosphorylate the GPCR on multiple sites
  • Arrestin Binds to Phosphorylated GPCR
  • This stops G-alpha from binding

  
• B-arrestin binding induces GPCR down-regulation through endocytosis.

  1. Phosphorylation of some GPCRs by GPCR kinase
  2. Binding of B-arrestin to phosphorylated GPCR
  3. B-arrestin-GPCR complex undergoes endocytosis
  4. AP2 binds to arrestin which pulls the whole vesicle inside the cell for
  5. sequestration
  6. degradation