Nov 10 - Lecture 29 ~ GPCR

  • alpha and beta gamma protein subunits have distinct functions

    • when bound together, the alpha subunit and beta gamma subunit complex mutually inhibit each other

    • upon separation, alpha and beta gamma can interact with their own target proteins

      • e.g. — the separation of G protein associated with an Ach receptor allows the beta gamma complex to bind and stimulate the opening of a K+ channel

        • the outflow of K ions alters the membrane potential

        • neuron signaling that opens ion channels indirectly via intracellular signaling is metabotrophic

        • the alpha subunit in this pathway has its own interaction partner

          • specifically, it activates or inhibits an enzyme called adenylyl cyclase

  • alpha subunits signal through second messengers

    • alpha subunits signal through their association with enzymes

      • the rxn catalyzed by the enzyme and the effect of the association depends on the alpha subunit itself

    • signal molecules produced (or released) via enzymes are referred to as second messengers

      • examples include: cAMP, IP3, DAG, and Ca2+ 

    • a single activated enzyme can produce many second messenger molecules — more signal amplification

  • Gas and Gai subunits alter cAMP concentrations

    • cyclic AMP is a metabolite of ATP

    • Adenylyl cyclase catalyzes phosphodiester bond formation between a 5’ phosphate and 3’ hydroxyl of the same ribose

    • cyclic AMP phosphodiesterase catalyzes hydrolysis of the 3’ phosphoester bond and conversion of cAMP to AMP

  • cAMP is an activator of protein kinase A (PKA)

    • cAMP has multiple potential target that vary according to cell type

      • cAMP-gated channels open when bound by cAMP

      • cAMP-sensitive channels close when bound by cAM

    • PKA depends on cAMP binding for full activity

      • cAMP binding to a regulatory protein releases active PKA

      • once bound to cAMP, PKA phosphorylates its protein target using ATP as a donor

        • PKA can regulate the function of cytosolic proteins via phosphorylation

  • cholera and pertussis toxins increase cAMP through different mechanisms

    • Cholera and whooping cough are caused by two different bacterial pathogens that colonize different tissues

    • Cholera – Vibrio cholerae in the intestine

    • Whooping cough – Bordetella pertussis in the airways

      • Both toxins covalently modify α subunits

    • Cholera toxin enhances cAMP signaling in enterocytes by locking Gαs in an active state – this leads to increased expression of the chloride channel CFTR on the apical membrane, water secretion, and diarrhea

    • Pertussis toxin enhances cAMP signaling in immune cells (maybe) by preventing activation of Gαi – this is hypothesized to inactivate the immune cells

  • Gq signals through phospholipase C (PLC) and protein kinase C (PKC)

    • Gaq and GBy activate phospholipase C-B (PLC-B), a lipid-hydrolyzing enzyme

    • the main substrate for PLC PI(4,5)P2 which when cleaved yields IP3 and DAG

    • IP3 opens a Ca2+ channel on the ER membrane

      • Ca2+ and DAG activate protein kinase C (PKC) which phosphorylates its own target

  • signaling can result in rapid rises in cytosolic calcium concentration

    • opening of IP3 receptors yields an increase in cytosolic calcium

      • increased calcium conc leads to opening of calcium-dependent ryanodine channels that increase the rate of calcium uptake

  • positive and negative feedback contributes to rapid Ca2+ oscillations

    • opening of ryanodine calcium channels by calcium from IP3 receptors produces a positive feedback process that opens more ryanodine channels

    • as calcium rises, it causes the channels to close

    • ATPases return Ca2+back to the ER causing a drop in calcium concentration, which allows channels to open again

    • this cycle produces oscillations of Ca2+ concentration with frequencies proportional to signal strength

  • calcium-binding regulatory proteins turn calcium mobilization into responses

    • calmodulin is a dumbbell-shaped protein with a pair of calcium binding sites on each head

    • without Ca2+, it has a linear structure; upon Ca2+ binding, the protein jackknifes placing the head groups side-by-side

    • this creates a protein interaction domain that allows calmodulin to bind target proteins

    • calmodulin-dependent protein kinase II is one of many targets of calmodulin

    • the transcription factor NFAT can enter the nucleus only when it isn’t phosphorylated; calcineurin is a calmodulin-dependent phosphatase that dephosphorylates NFAT

  • calmodulin stabilizes CAMK II in an active conformation

  • examples of GPCR signalling — olfactory neurons signal through cAMP

    • when an odorant binds to the olfactory receptors in the olfactory neurons that line the nose, it activates Golf 

    • Golf then activates adenylyl cyclase → cAMP → opens cation channel to initiate action potential

      • Na+ influx causes depolarization of the membrane which sends a nerve impulse that travels to the brain

  • Gaq signaling in endothelial cells leads to nitric oxide production which relaxes smooth muscle

    • vasorelaxation can be initiated via endothelial cells that line the inner surface of the vessel

    • Ach activates Gaq couple receptor in endothelial cells — the increase of Ca2+ activates the enzyme nitric oxide synthase (NOS)

    • NO gas binds and activates guanylyl cyclase which produces cGMP

    • cGMP hyperpolarizes the cell and closes Ca2+ channels

      • this results in the cell relaxing and cGMP broken down by cGMP phosphodiesterase



  • sensory neuron — eyesight 

    • rhodopsin is a GPCR that is activated by light

    • Photodetection is an unusual sense in that the sensing cells are depolarized when they are not stimulated

    • Rhodopsin is a GPCR that associates with a special α subunit called transducin (GαT )

      • GαT activates cGMP phosphodiesterase, which degrades cGMP

      • Falling levels of cGMP cause a cGMP-gated cation channel to close, which hyperpolarizes the cell

      • Because of this, photoreceptors release neurotransmitter in the dark and stop releasing when hit by a photon

    • light inhibits inhibitory nT release

      • nT are inhibitory — they inhibit nerve impulse from travelling to the brain

      • when inhibition is removed, nerve impulse is able to travel to the brain

  • amplification of GPCR signals produce strong responses from few receptors

  • GPCR kinases and arrestins decrease sensitivity of GPCRs

  • Summary / Key Concepts

    • Gαq signaling acts first through phospholipase C (PLC). This enzyme cleaves PI(4,5)P2 to release IP3 & DAG. IP3 increases cytosolic calcium concentration and DAG cooperates with calcium to activate protein kinase C (PKC).

      • Like PKA, PKC has it’s own phosphorylation targets.

    • Calcium can also alter protein function via binding to regulatory proteins like calmodulin. Calcium oscillations are translatable by CAMK II

    • Photoreceptors use GPCRs that signal through transducin, which activates phosphodiesterase that breaks down cyclic GMP – this closes cGMP-gated cation channels and hyperpolarizes the photoreceptor cell.

    • GPCR activation can lead to phosphorylation by GPCR kinases, followed by binding of arrestin and desensitization