Receptors moda

  1. Competitive antagonist for nAChR, used experimentaly becaise of its high affinity
  2. Used to trap NAChR on gel beads
  3. 3 NAChR agonist with different efficacies
    • ACh: 1ms open time
    • Carbachol: 0.5ms
    • Suberyldicholine: 1.7ms
  4. 3 types of ligand gated ion channels, with examples
    • pentameric: AChR, GABAA, Glycine, 5-HT
    • tetrameric: AMPA, NMDA
    • trimeric: P2X for ATP
  5. Which parts of GPCRs interact with G proteins
    • 3rd intracellular loop (PKA)
    • C-terminal tail (PKA, betaARK)
  6. 3 types of G proteins
    • Gs: stimulates AC -> cAMP (beta)
    • Gi: inhibits AC -| cAMP (alpha2)
    • Gq/11: stimulates PLC (alpha1)
  7. Cholera toxin effect
    • Ribosylates ADP in alpha-s and inhibits GTPase activity
    • -> sustained activation of AC
    • -> high cAMP
  8. Pertussis toxin
    • Ribosylates ADP in alpha-i and prevents its activation
    • -> sustained activation of AC (no inhibition
    • -> high cAMP
  9. AIF4-
    • Mimics gamma-phosphate of GTP
    • -> sustained activation of G proteins (and AC)
  10. Agonists for phosphoinositide pathway
    • alpha 1 adrenoceptor
    • muscarinic AChR
    • substance P
  11. Lithium importance in phosphoinositide pathway
    used for?
    • Uncompetitively inhibits phosphatase that converts IP3 to inositol
    • -> blocks recycling of inositol, important in the brain as inositol cannot cross BBB
    • Used in treatment of schizophrenia
  12. How to label calcium?
    Fura-2: fluorescent
  13. Investigating IP3 experimentally
    • recording calcium levels in permeabilised cells:
    • 1) add ATP: Ca sequestered by ER
    • 2) Add Ca
    • 3) Add IP3 : Ca levels increase again, as Ca is released from ER
  14. 3 main ways of desensitising beta-adrenoceptor
    • 1) Uncoupling GPCR and G proteinĀ  (phosphorylation at GPCR)
    • 2) Sequestration of receptors (endocytosis -> destroyed in lysosomes/shuttled back to membrane)
    • 3) Downregulation of receptor production (maybe PKA alters mRNA stability?)
  15. Describe heterologous desensitisation
    • Beta2 stimulation produces PKA
    • PKA phosphorylates beta 2 receptor at 3rd intracellular loop and C terminal tail (and other similar receptors)
    • happens with low [ligand], as is proportional to response
  16. Describe homologous desensitisation
    • ligand bound beta2R phosphorylated by betaARK (adrenoceptor kinase) at C terminal tail
    • this increases the affinity of beta-arrestin
    • beta-arrestin binding uncouples the receptor
    • happens with high [ligand] conc, as it is proportional to occupancy
  17. 4 viral oncogenes thata affect RTKs
    useful drug?
    • v-erbB: causes a truncated, constitutively active EGFR, found in avian erythroblastosis virus
    • c-erbB: overexpressed in squamous cell carcinomas
    • v-sis: PDGF (platelet derived growth factor) chain, found in simian sarcoma virus
    • c-sis: overexpression is tumorigenic

    tyrphostins: inhibit tyrosine kinases, may be useful for controlling neoplastic growth
  18. Imatinib (Glivec)
    Inhibits kinase bcr-abl in chronic myeloid leukaemia
  19. hsp90
    other similar?
    • complexes with inactive steroid receptors: needed for glucocorticoids to bind
    • hsp70: another essential chaperone
  20. Different types of sodium channel
    • nerves: Nav1.1-1.3
    • skeletal muscle: Nav1.4
    • cardiac muscle: Nav1.5
  21. 3 reasons why nAChR has been so widely studied
    • alpha-bungarotoxin is a high affinity ligand that is used as a competitive antagonist
    • receptor structure preserved when solubilised in triton-x 100
    • nAChR available from Torpedo electroplaques
  22. nAChR structure and binding
    • 5 subunits: alpha1, alpha2, beta, gamma, delta
    • ACh binds between alpha and delta, and alpha and gamma
    • each subunit has 4 hydrophobic stretches (M1-M4)
    • M2 may form an alpha helix in the membrane, and the 5 M2 regions line the pore of the channel
  23. Structure of GPCR
    • Single subunit
    • 7 membrane spanning alpha helices
    • Extracellular N terminal tail
    • Intracellular C terminal tail
    • Ligand binding site within TM helices
    • Interact with G proteins with 3rd intracellular loop and C terminal tail
  24. What experiments found the difference between homologous and heterologous desensitisation?
    • Site-directed mutagenesis
    • Type A: could not be phosphorylated at PKA site - desensitisation only at high levels of stimulation with isoprenaline
    • B: could not be phosphorylated at betaARK site - desensitisation at low (nm) and high (um) levels
    • C: could not be phosphorylated at all - no desensitisation
  25. Structure of receptor tyrosine kinase
    • 4 domains: ligand binding, TM, catalytic, autophosphorylation
    • Insulin: 2 chains linked as a dimer
    • EGF and PDGF: single polypeptide chain
  26. Structure of steroid receptor
    • 3 domains:
    • N terminal: transcription activating domain
    • DNA binding domain: zinc finger
    • Hinge region: nuclear localisation sequence exposed on binding
Card Set
Receptors moda