Lecture 14: Cellular Signaling Pathways (CBAD)

Lecture 14 Tuesday 19 November 2024 14:08 1. What are the 4 main classes of receptors? Ligand gated ion channel G-protein coupled receptors Enzyme-linked receptors Nuclear receptors 2. What does the activated GPCR do to the G protein? The GPCR causes the excha...

Lecture 14 Tuesday 19 November 2024 14:08 1. What are the 4 main classes of receptors? Ligand gated ion channel G-protein coupled receptors Enzyme-linked receptors Nuclear receptors 2. What does the activated GPCR do to the G protein? The GPCR causes the exchange of GDP for GTP on the a-subunit of the G protein. 3. What happens to the G protein after GDP is exhcnaged for GTP? The Ga-GTP subunit dissociates from the ßy complex and activates adenylyl cyclase (AC). 4. What does adenylyl cyclase do? Converts ATP into cAMP (a second messenger). 5. How does cAMP activate protein kinase A (PKA)? CAMP binds to the regulatory subunit of PKA, causing them to dissociate from the catalytic subunits, activating PKA. 6. What happens after PKA is activated? The catalytic subunits of PKA phosphorylate target proteins, triggering cellular responses. 7. What did the first G protein to be discovered contain? Contained an alpha subunit that could activate adenylyl cyclise (AC). 8. What did the discovery of the first G protein lead to? The realisation that some hormones could inhibit adenylyl cyclase. this response was also mediated via a G protein. 9. What is the difference between the Ga subunits? Gas: stimulates AC Gai: inhibits AC 10. What couples to Gs? ß-adrenoceptors Vasopressin receptor A2A/B adenosine receptors Inhibits AC 11. What couples to Gi? Alpha2 adrenoceptors Mu and delta opioid receptors A1/3 adenosine receptors. Inhibits AC 12. What does signalling via G proteins depend on? Exchange of GDP for GTP 13. Describe how G proteins act act targets for bacterial toxins (e.g. cholera). Active alpha subunit has GTP bound. Hydrolysis of GTP leads to inactivation. 14. What does cholera toxin (CTx) do to the Ga subunit? Cholera toxin (CTx) acts on alphas subunit and causes ADP- ribosylation. This prevents hydrolysis of GTP. Causes persistent activation of alpha subunit. In colon causes activation of PKA-dependent Cl- channels and secretory diarrhoea. AC -> cAMP -> CFTR -> diarrhoea. 15. How does the cholera toxin signalling pathway lead to diarrhoea? Adenylyl cyclase (AC) is activated, leading to increased cAMP production. This activates the CFTR chloride channel, causing diarrhoea. 16. How do G proteins act as a target for pertusiss toxin? Pertussis toxin acts on alphai subunit Locks subunit into inactive configuration 17. What effect does pertussis toxin have on the ai subunit? Prevents activation by receptors Prevents its inhibitory control over AC/PKA 18. What is the result of pertussis toxin acting on G proteins? increased levels of cAMP and PKA In airways: leads to whooping cough. 19. What is the role of G proteins containing alphaq11 subunits? Allow hormones/ neurotransmitters to activate amplifier enzyme phospholipids C (PLC) 20. How does the activation of aq11-containing G proteins affect acetylcholine signalling? underlies autonomic effects of Actetylcholine (e.g. salivary secretion, bronchial smooth muscle contraction). 21. What role do G proteins containing aq11 subunits play in histamine H1 receptor responses? Mediate responses: Smooth muscle contraction, allergies etc. Responses due to increased internal Ca2+. 22. What is the role of G proteins containing the aq11 subunit in calcium signalling? G proteins with the aq11 subunit activate phospholipids C. PLC cleaves PIP2 into IP3 and DAG. IP3 binds to receptors on the endoplasmic reticulum causing the release of Ca2+ into the cytoplasm 23. What does activation of M3 receptors by aq11-containing G proteins cause? Causes bronchospasm 24. What type of receptors are muscarinic receptors? Metabotropic receptors Coupled to Gq/Gi Ach activates muscarinic receptor (metabotropic receptor) 5 sub types 25. Which muscarinic receptor subtypes are Gq-coupled stimulatory receptors? M1, M3, M5 26. Which muscarinic receptor subtypes are Gi-coupled inhibitory receptors? M2 and M4 27. What autonomic effects are mediated by muscarinic receptors? Salivary secretion, bronchial smooth muscle contraction. Through acetylcholine 28. What do Gq proteins stimulate? Phospholipase C (PLC) 29. What does PLC do? Cleaves PIP2 (a membrane phospholipid) into IP3 and DAG. ○ cleaves the polar head group of the phosphate. 30. What is IP3? What does it do? The water soluble part of PIP2 Travels through the cytosol to stimulate calcium release from ER. ○ IP3 binds to IP3 receptors on the ER, leads to release of calcium. ○ Calcium acts as a second messenger that triggers responses (muscle contraction, gene expression). 31. How is IP3 inactivated? By the conversion of IP3 into IP2, halting the signal. 32. Where does DAG go after being produced? Remains in the hydrophobic part of the membrane. Where it recruits protein kinase C (PKC). 33. What is the role of phospholipase Cß? Hydrolyses PIP2 into IP3 and DAG. 34. What does the hydrolysis of PIP2 lead to? IP3 triggers Ca2+ release from the ER. DAG activates PKC in the membrane. 35. What activates phospholipase Cß? Binding of a hormone to a cell surface G protein-coupled receptor. 36. What allows the release of Ca2+ into the cytosol? IP3 interacts with its receptor (IP3R) in the membrane of the ER. Allows the release of Ca2+ into the cytosol. 37. What transports the Ca2+ back into the ER? The SERCA Ca2+ pump Uses ATP 38. What is IP3? What does it do? A second messenger. Stimulates calcium release from ER. Hydrophilic - enters the cytoplasm. Binds to receptors on ER and promotes release of stored Ca2+. Also promotes Ca2+ influx from extracellular fluid. The increase in intracellular free Ca2+ promotes cellular responses. 39. What is the most important calcium binding protein that mediates intracellular responses? Calmodullin (CaM) 40. What are the calmodullin (CaM) modulated intracellular responses? Each CaM binds 4 Ca2+ ions - Ca2-CaM complex. 41. What does the Ca2+ - CaM complex activate? PDE (the enzyme that degrades cAMP). CaM kinases (CaMKs). 42. What do calmodulin kinases (CaMKs) do? CaMKs phosphorylate serine and threonine residues on a number of substrate proteins. CaMKs are involved in smooth muscle contraction 43. How does the alpha1 adrenergic receptor mediate vascular smooth muscle contraction? Alpha1 adrenal-receptor is a Gq coupled protein receptor. Increases intracellular free Ca2+. Activating CAMKs Leads to vasoconstriction. 44. What are the effects of DAG? DAG is a second messenger that affects cellular signalling. Evokes a cellular response by phosphorylating other proteins. ○ the most important one is protein kinase c (PKC) Plays a role in receptor desensitisation (similar to ß-ARK in GPCR signalling). 45. Why does DAG remain in the plasma membrane? DAG is hydrophobic - remains in the plasma membrane (hydrophobic part). 46. How does DAG affect protein kinase activity? Presence of DAG increases the activity of Ca2+ dependent protein kinase. 47. How does PKC interact with IP3 signalling? PKCs can potentiate the effects of IP3. Enhancing downstream signalling. 48. What functions does PKC regulate? Regulates cell shape. cell proliferation. transcription factor activity. 49. What is the role of an alpha1-adrenoceptor? Causes vasoconstriction. Via Gq-PLC-IP3-CaMK. Increases blood pressure. 50. What is the role of a ß2-adrenoceptor? Causes relaxation of vascular smooth muscle (vasodilation). Via Gs-cAMP-PKA Blood pressure decreases 51. What are the enzyme linked receptors? Receptor guanylyl cylclases Receptor serine/ threonine kinase Receptor tyrosine- kinase Tyrosine kinase- associated receptors. Receptor tyrosine phosphatase. 52. Describe receptor guanylyl cyclase Contains 2 guanylyl cyclase domains which convert GTP to cGMP. CGMP activates downstream kinases. 53. Describe the signalling mechanism of receptor guanylyl cyclase. Binding of ANP induces a conformational change in the receptor that causes receptor dimerisation and activation The guanylyl cyclase activity of the receptor generates cGMP. Increased concentrations of cGMP activates other signalling molecules determining the response. E.g. atrial natriuretic peptide (ANP) relax vascular smooth muscle and dilate blood vessels (vasodilation). 54. What does the guanylyl cyclase activity of the receptor generate? CGMP. ○ Increased cGMP activate other signalling molecules, determining the response. 55. What is the effect of ANP signalling via guanylyl cyclase? ANP signalling via guanylyl cyclase leads to vasodilation, causing relaxation of vascular smooth muscle and dilation of blood vessels. 56. Describe receptor serine/ threonine kinases Contain serine-threonine kinase domains which phosphorylate target proteins (similar to PKA). 57. Describe the signalling mechanism of receptor serine/threonine kinases. First messenger binds to receptor type II. Receptor type I then binds forming a ternary complex with type II and first messenger. Type II receptor phosphorylates type I, activating the Ser-Thr kinase activity of type I. Type I then phosphorylates target proteins (e.g. SMAD proteins). E.g. TGFß mediated cell proliferation. 58. Describe receptor tyrosine kinases (RTKs). Contain tyrosine kinase domains which phosphorylate themselves/ other proteins. 59. Describe the mechanism of signalling for receptor tyrosine kinases. Binding of 2 molecules of insulin causes the receptor to dimerise. Receptors then use their cytoplasmic tryosine kinase activity to phosphorylate eachother at multiple tyrosine residues creating “phosphotyrosine motifs”. 60. What is the role of “phosphotyrosine motifs”? Recruit intracellular signalling leading to the response. 61. What is an example of a cellular response mediated by receptor tyrosine kinases? Insulin mediated glucose uptake and storage in liver and muscles. 62. What initiates RAS activation in the MAP kinase signalling pathway? A signal molecule binds to and activates a receptor tyrosine kinase (RTK). The activated RTK undergoes dimerisation and autophosphorylation at specific tyrosine residues. 63. What role does the adaptor protein play in RAS activation? An adaptor protein docks on a phosphotyrosine residue of the activated RTK. It recruits RAS-GEF. 64. How does RAS-GEF activate Ras? Ras-GEF stimulates Ras to exchange its bound GDP for GTP, activating Ras. 65. What happens to the activated Ras protein? The Ras protein, bound to GTP, is anchored to the plasma membrane via a covalently attached lipid group. It initiates the transmission of the signal to downstream pathways. 66. Describe MAPKKK Mitigation-activated protein kinase kinase kinase First kinase in the cascade (e.g. RAF). Activated by Ras-GTP and phosphorylates MAPKK. ATP->ADP. 67. Describe MAPKK. Second kinase in the cascade (e.g. MEK) Activated by MAPKKK Phosphorylates MAPK ATP->ADP 68. Describe MAPK. Third kinase in the cascade (e.g. ERK) Activated by MAPKK Translocates to the nucleus to phosphorylate transcription factors, altering gene expression. 69. Describe tyrosine kinase-associated receptors. The receptors do not contain kinase domains. Associated non-covalently with the cytoplasmic domains. 70. Describe the mechanism of signalling for tyrosine kinase- associated receptors. Binding of first messenger to the receptor induces conformational change. Causes dimerisation of the receptor. The dimerisation causes activation of the associated Tyr kinases (e.g. JAK2). These kinases then phosphorylate tyrosine residues on both themselves and the receptor, creating “phosphotyrosine motifs”. (Like tyr-kinase receptors). These motifs recruit intracellular signalling molecules leading to the response. (Re STAT proteins). E.g: cytokines signalling pathway (IL-6 acute phase response). 71. How are tyrosine kinases activated in tyrosine kinase-associated receptors? Dimerisation of the receptor activates the associated tyrosine kinases (e.g. JAK2). 72. What do tyrosine kinases do once activated in this signalling mechanism? Phosphorylate tyrosine residues on both themselves and the receptor. Creating phosphotyrosine motifs. 73. What is the role of phosphotyrosine motifs in tyrosine kinase associated receptor signalling? Recruit intracellular signalling molecules. E.g. STAT proteins Leading to cellular responses. 74. What is an example of a signalling pathway mediated by tyrosine kinase-associated receptors? The cytokine signalling pathway. such as the IL-6 acute phase response. 75. Describe receptor tyrosine phosphatases Receptors contain tyrosine phosphatase domains and remove Tyr residues to dephosphorylate target proteins. E.g. Tyr residues created by Tyr kinase receptors signalling. 76. Describe the mechanism of signalling for receptor tyrosine phosphatase. 1st messenger binding to the receptor induces a conformational change that activates the Tyr phosphatase activity of the receptor. Target proteins are dephosphorylated by Tyr phosphatase activity. This causes phosphorylation of downstream proteins (e.g. Lck and Fyn). E.g. CD45 Induces the maturation of lymphocytes via binding to this receptor. 77. What does the tyrosine phosphatase activity of the receptor do? Dephosphorylates target proteins, modulating their activity. 78. How does receptor tyrosine phosphatase signalling affect downstream proteins? Dephosphorylation of targets leads to the phosphorylation of downstream proteins: e.g., Lck and Fyn. 79. What is an example of receptor tyrosine phosphatase signalling? CD45 induces the maturation of lymphocytes by binding to this receptor. 80. What are the differences in the structure of GPCRs and RTKs? GPCRs: have 7 transmembrane helices. RTKs: have 1 transmembrane helix. 81. What enzymatic activities are associated with GPCRs and RTKs? GPCRs: no enzymatic activity; activate G proteins to relay signal. RTKs: the receptor itself has catalytic activity that triggers phosphorylation. 82. Do GPCRs and RTKs require receptor dimerisation? GPCRs: do not require dimerisation. RTKs: ligand binding can lead to dimerisation of neighbouring receptors. 83. How do GPCRs and RTKs relay their signals? GPCRs: via secondary messengers (e.g. cAMP or IP3/DAG). RTKs: relay signals without secondary messengers, using phosphorylation cascades. 84. What is the duration of pathway activation in GPCRs and RTKs? GPCRs: seconds RTKs: hours 85. What downstream effects do GPCRs and RTKs trigger? GPCRs: can lead to protein phosphorylation or ion channel opening. RTKs: primarily lead to phosphorylation cascades and activation of signalling pathways like Ras-GEF and PI 3-kinase. 86. What are the signalling mechanisms for GPCRs? Activated GPCR: G protein->adenylyl cyclase->cAMP->PKA. G protein->phospholipase C->IP3->Ca2+->calmodulin->CaM kinase. G protein->phospholipase C->diacylglycerol->PKC 87. What are the signalling mechanisms for RTK? Activated RTK: PI 3-Kinase->phosphorylated inositol phospholipid->protein kinase 1->Akt kinase Ras-GEF->Ras->MAPKKK->MAPKK->MAPK 2*.The GPCR stimulation of adenylyl cyclise, cAMP and protein kinase A Enzyme linked receptors Receptor guanylyl cyclase: Receptor serine/ threonine kinases: Receptor tyrosine kinases (RTKs) 71*. Tyrosine-kinase associated receptors: 76*. Receptor tyrosine phosphate: 87*. GPCR vs RTK: