Monday, May 16, 2011

Book Notes: Signaling Part 4

  • G proteins do more than play with cAMP and Ca2+
    • the a subunit of G12 activates GEF for the Rho GTPase
    • others bind and open/close ion channels to change membrane potential
    • others phosphorylate channels or change levels of cNMP (e.g. cAMP) that affect channels
  • olfactory receptors utilize cAMP-gated ion channels
    • odor particle binds to olfactory receptor
    • the receptor activates the G protein Golf (G subscript olf for olfactory)
    • Golf activates adenylyl cyclase = lots of cAMP
    • cAMP binds to cation channels that allow Na+ to flood in
    • neuron containing the olfactory receptor is depolarized and sends a potential down the axon to the brain
    • each neuron makes only 1 type of receptor that senses only a small set of odors
  • visual receptors utilize cGMP-gated ion channels
    • rod photoreceptors are cells with a rodlike outer and inner segment, followed by the body, followed by the axon and synapse
    • the outer segment looks like a pancake stack with lots of rhodopsin molecules in them
    • the plasma membrane coating the stacks have lots of cGMP-gated cation channels
    • in the dark, cGMP remains bound to the channels, keeping them open
    • in the light, the cis chromophore in the rhodopsin absorbs a photon and isomerizes into trans
    • the changed chromophore causes the protein opsin to change conformation and activates the G protein transducin (Gt)
    • transducin a subunit activates cGMP PDE, which removes cGMP
  • visual system resets itself VERY QUICKLY so that we can see the change in light in the next instant
    • rhodopsin kinase phosphorylates the tail of activated rhodopsin, to prevent further activation of Gt
    • arrestin binds to Pi-rhodopsin to fully inhibit rhodopsin activity
    • RGS acts as GAP to make Gt hydrolyze the GTP
    • light also causes calcium channels to close, so calcium levels fall, so guanylyl cyclase hurries to make more cGMP, returning to normal "dark" mode
    • negative feedback allows system to adapt to continuous light, and be sensitive to CHANGES in light (seeing camera flash in broad daylight)
  • all the steps of intracellular signaling pathways are potential SIGNAL AMPLIFICATION steps
    • but for rapid sensing, cells must be able to both amplify a signal quickly and destroy the response just as quickly
    • therefore, any and every protein/molecule in the pathway can be a target for regulation
  • GPCRs are usually desensitized in 3 ways
    • receptor inactivation: receptor interaction with G protein is blocked
      • e.g. GRK (GPCR kinase) like RK that phosphorylates GPCR after the receptor has been activated by ligand-binding
      • then (as with rhodopsin) an inhibitor like arrestin binds and blocks the G protein interaction
    • receptor sequestration: move receptor inside the cell to block access to ligand signal
      • arrestin can also couple the receptor to endocytosis machinery and clathrin
      • later dephosphorylation returns the receptor to the surface
    • receptor down-regulation: receptor is destroyed post-activation
      • endocytosis of receptor in some cases lead to the receptor ubiquitylation and lysosomal degradation
  • EZCR (enzyme-coupled receptor): transmembrane receptor with a cytosolic domain that is an enzyme or associates with another enzyme (note, the abbreviation is my own invention)
    • exist 6 classes of EZCRs
      1. receptor Tyr kinase: phosphorylate Tyr residues on itself and some signaling proteins
      2. Tyr kinase associated receptor: recruit cytoplasmic Tyr kinase
      3. receptor Ser/Thr kinase: phosphorylate Ser/Thr on itself and some gene regulatory proteins
      4. His kinase associated receptor: phosphorylate itself on His and transfers the Pi to a signaling protein
      5. receptor guanylyl cyclase: catalyzes production of cGMP
      6. receptorlike Tyr phosphatase: remove Pi from Tyr of signaling proteins (ligands are unknown)
  • RTK (receptor Tyr kinase): bind hormones like insulin, growth factors, and ephrins (cell-surface ligands as opposed to free floating signals)
    • ephrins and EPHrin receptors both make responses in the receptor cell and the ligand cell: "bidirectional signaling"
    • ligand binding causes RTKs to dimerize and cross-phosphorylate each other
    • the phosphorylated Tyr residues serve as docking sites for specific intracellular signaling proteins
    • these signaling proteins are either activated by docking or by receiving phosphorylation from the RTK
    • proteins dock by modular interaction domains (remember SH2, SH3, etc etc), sites of protein protein binding that don't affect function
  • examples of docked proteins are PI3-kinase and PLCgamma (PLCbeta associates with GPCRs)
  • adaptor proteins with lots of modular domains connect docked proteins to other proteins that DON'T have these awesome domains
    • example being RAS
  • Ras and Rho families are the only monomeric GTPase families to relay signals from surface receptors
    • as a family, they can coordinate one signal to many pathways
    • Ras has lipid groups to keep it anchored to the cytoplasmic leaflet of the membrane
  • RTKs activate Ras through adaptors and Ras-GEF
    • case study: fly eye is composed of ommatidia, which are sets of 8 photoreceptor cells and 12 accessory cells
    • the Sevenless (Sev) mutant gene causes flys to be blind in UV light, because mutant Sev prevents normal development of the R7 photoreceptor
    • BUT it turns out for some blind flies, the Sev gene is kept normal: it's the protein Bride of Sevenless (BOSS) who's mutant
    • Boss is a 7pass transmembrane ligand on the R8 cell, and binds to the R7 Sev RTK (see, Sevenless is an RTK, a receptor.  BOSS is the ligand.  That's why it's "bride of Sevenless," because it binds to the Sevenless receptor)
    • when they bind, the R7 precursor cell is induced to develop into R7
    • even if other cells express Sev (which they do), only R7 touches R8, so only the Sev on R7 receives the ligand
    • Steps to signal transduction
      1. Boss binds Sev-RTK
      2. Sev-RTK is phosphorylated and an adaptor DRK with an SH2 domain docks
      3. a special Ras-GEF called Son of Sevenless (SOS) is also bound by DRK's 2 SH3 domains
      4. SOS makes membrane-bound Ras get a new GTP and the activated Ras continues the signal with cytoplasmic free proteins
        • see, SOS is Son of Sevenless because it's activated when Bride of Sevenless binds to Sevenless receptor = love child (haha, biologists are both bored and lewd)
  • Now that Ras is active, where do we go next?
    • Ras-MAP-kinase signaling pathway: Ras activates MAPKKK (Raf), who activates MAPKK (Mek), who activates MAPK (Erk), who activates gene regulatory proteins in the nucleus
    • these gene regulatory proteins quickly turn on "early genes," who are part of the primary response
    • early genes produce more gene regulatory proteins that go to activate later genes for the secondary response
    • in this way, there is a rapid response and a more delayed response
    • this pathway is how the signal goes from surface (where RTKs and Ras await) to the nucleus (where DNA and genes hide)
    • lots of positive and negative feedback: MAP kinases activate both downstream targets and its own activators, BUT they also increases transcription of phosphatases and deactivate its own activators (Erk blocks Raf)
  • different sets of 3 MAP kinases form MAP modules in different pathways
    • however, many modules share one or more of the same kinases
    • scaffolds prevent cross-talk by associating with the sensor, so that activated kinases that bind to the scaffold are automatically next to its downstream targets also docked to the scaffold protein
      • this makes sure the activated kinase doesn't wander off and find other targets of other pathways
  • Rho GTPases regulate the cytoskeleton (actin and microtubules) in response to special guidance receptors
    • inactive Rho is bound to GDI, which prevents association with the GEF
    • Eph RTKs are the receptors that activate surface-bound GEFs that activate Rho
    • ephrins are the guidance ligands that bind Eph RTK
      • e.g. most commonly used for directing axonal growth
      • target cells express the appropriate ephrin on its surface
      • the axon's growth cone has Eph RTK that binds ephrin
      • Rho changes the cytoskeleton to grow in the direction of the bound ephrin to shift the axon thataway
  • PI3-kinase is another protein that docks to RTK
    • PI3 phosphorylates PIs, usually to make PIP3
    • PIP3 floats around until the PTEN phosphatase turns it into PIP2
    • PIP2 is nabbed by PLCbeta or PLCgamma and cleaved into IP3 and DAG for signaling
    • RTKs activate PI3-kinase to make PIP3 available to make PIP2 available for signaling
    • PIP3 have special PH modular domains (pleckstrin homology) that are super specific (because PIP3 and PI3 tend to be involved in growth and related pathways that are vulnerable to become cancerous)
  • PI3-kinase-Akt pathway receives signals from IGF, the "SURVIVE AND GROW" signal
    • RTK activates PI3 kinase, which makes PIP3, which recruits Akt and PDK1 (phosphoinositide-dependent protein kinase 1) to the plasma membrane
    • Akt is activated there and phosphorylates many targets, producing a growth response
      • e.g. one target is called Bad, which causes apoptosis, but phosphorylation of Bad makes it dock onto a scaffold protein, preventing its function
    • a special TOR protein promotes ribosome production, protein synthesis, and nutrient uptake/metabolism, so it is activated by the Akt pathway for growth purposes


pg. 916 - 935

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