Cellular aspects of molecular pharmacology


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Cellular aspects of molecular pharmacology

  2. 2. Receptors G-protein coupled receptors  Ion Channel Receptors Receptor-enzymes Cytosolic-nuclear receptors
  3. 3. G-protein coupled receptors
  4. 4. G-protein coupled receptor: GPCR Examples: Adrenergic receptors Muscarinic ACh receptors GABAB receptors Metabotropic Glutamate receptors Dopamine receptors Metabotropic Serotonin receptors
  5. 5. G-protein coupled receptor: GPCR More examples—hormones: Angiotensin receptor Bradykinin receptor Thrombin receptor FSH receptor LH receptor TSH receptor ACTH receptor
  6. 6. GPCR structure A single subunit with 7 transmembrane segments out in
  7. 7. A depiction of how GPCRs activate signaling
  8. 8. Ligand + GPCR GPCR* GPCR* abg a + bg signaling abg Heterotrimeric GTP-binding protein (G protein) GPCR G-protein coupled receptor signalin g Another depiction of how GPCRs activate signaling
  9. 9. Heterotrimeric GTP-binding proteins cycle between GTP- and GDP-bound state Binding of ligand to G-protein coupled receptor facilitates exchange of GTP for GDP, a and bg dissociate a b g GDP a b g GTP INACTIVE ACTIVE
  10. 10. Heterotrimeric G proteins •Activated by binding of ligand to 7-transmembrane receptor (G-protein coupled receptor) •Ligand binding causes dissociation of a and bg subunits •Dissociation allows GDP to exchange for GTP •GTP binding causes conformational change, a subunit can now interact with effector (e.g. AC) •GTP is hydrolyzed to GDP, a subunit dissociates from effector •Signal is terminated
  11. 11. Adenylate cyclase Membrane protein makes cAMP from ATP ATP cAMP
  12. 12. Three major families of G-proteins Gs couples to Adenylate Cyclase stimulates AC activity increases cAMP activates Protein Kinase A Gi couples to Adenylate Cyclase inhibits AC activity decreases cAMP inhibits Protein Kinase A Gq couples to Phospholipase C increases diacylgyclerol (DAG) increases IP3 increases intracellular Ca2+
  13. 13. Gs Gi Gq b-adrenergic receptor ACTH receptor FSH receptor a2-adrenergic receptor M2 muscarinic receptor a1-adrenergic receptor M1, M3 muscarinic receptors Angiotensin receptor cAMP PKA activity cAMP PKA activity PLC activity DAG, IP3 Ca2+ PKC activity G-protein Receptor examples Signaling pathway
  14. 14. cAMP-dependent stimulation of glucose liberation from glycogen
  15. 15. bAR a b g GTP AC PKA cAMP-dependent stimulation of cardiac muscle contraction Ca2+ Ca2+ Ryanodine Receptor Sarcoplasmic reticulum bAR = b-adrenergic receptor
  16. 16. cAMP is constantly inactivated by phosphodiesterase (active) (inactive) Theophylline blocks the phosphodiesterase
  17. 17. Phospholipase C activation produces Diacylglycerol and IP3 PI 4,5 biphosphate (PIP2) Plasma membrane lipid plasma membrane Diacylglycerol (DAG) Inositol 1,4,5-triphosphate (IP3) cytosolic messenger plasma membrane
  18. 18. a1-adrenergic receptors acting on vascular smooth muscle: PLC IP3 Ca2+ calmodulin myosin light chain kinase Myosin-P contraction An example of the Gq signaling pathway
  19. 19. IP3 receptors: intracellular calcium channels
  20. 20. Controlling intracellular calcium levels IP3 receptors Ca2+ Intracellular Ca2+ levels are << micromolar
  21. 21. Guanylate cyclase receptors
  22. 22. Guanylate Cyclase Receptors Two Types: 1. Transmembrane Guanylate Cyclase Receptor activated by peptide hormones 2. Soluble Guanylate Cyclase Receptor activated by nitric oxide (NO) target for anti-angina drugs nitroglycerin Atrial natriuretic peptide/factor ANP
  23. 23. Transmembrane Guanylate Cyclase Receptor 3 domains Hormone binding Transmembrane Intracellular GC domain with catalytic activity Single transmembrane receptor—similar to tyrosine kinase receptor (binds ANP)
  24. 24. Signal transduction initiated by binding of ANP to Transmembrane Guanylate Cyclase Receptor 1. Atrial natriuretic peptide binds to receptor, causes conformational change 2. Guanylate cyclase is activated 3. cGMP is generated 4. cGMP dependent protein kinase is activated 5. proteins are phosphorylated
  25. 25. Smooth muscle cell contraction cGMP-dependent protein kinase (PKG) phosphorylates many proteins that modulate muscle contraction Ca2+ channels K+ channels IP3 receptors Myosin light chain phosphatase NPR-A ANP receptor
  26. 26. Soluble Guanylate Cyclase Receptor (binds NO) NO, a gas, can cross cell membranes Interacts with heme group on GC inside the cell
  27. 27. Mechanism of activation of Guanylate Cyclase by NO NOS nitric oxide synthase catalyzes oxidation of arginine to produce citrilline + NO gas
  28. 28. Signal transduction initiated by generation of NO 1. Nitric oxide synthase (NOS) is activated in generator cell 2. Nitric oxide is formed by NOS from arginine 3. Gaseous nitric oxide diffuses out of generator cell and into target cell 4. Nitric oxide reacts with iron-containing heme group on Guanylate Cyclase 5. Activated Guanylate Cyclase converts GTP to cyclic GMP 6. cGMP binds to cGMP-dependent protein kinase (PKG) 7. proteins are phosphorylated by PKG
  29. 29. Mechanisms by which NO induces relaxation of smooth muscle Smooth muscle contractionNO activates guanylate cyclase cGMP is produced cGMP dependent protein kinase is activated Proteins that regulate contraction are phosphorylated IP3 R MLCP Ca2+ chann Ch K+ K+ chann PKG
  30. 30. Mechanisms by which NO induces relaxation of smooth muscle (review) NO Guanylate Cyclase cGMP PKG IP3R K+ Chann Ca2+ chann Myosin light chain phosphatase
  31. 31. CELL GROWTH P ras MAPK MAPK P phosphorlyates multiple target proteins PDGF dimerize & phosphorylate each other Mechanism of signal transduction
  32. 32. Enzyme-linked Cell Surface Receptors  Receptor Tyrosine kinases: phosphorylate specific tyrosines  Tyrosine kinase associated receptors: associate with intracellular proteins that have tyrosine kinase activity.  Receptorlike tyrosine phosphatases: remove phosphate group  Receptor Serine/ Threonine kinases: phosphorylate specific Serine/ Threonine  Receptor guanylyl cyclases: directly catalyzes the production of cGMP  Histidine kinase associated receptors: kinase phoshorylates itself on histidine and then transfers the phosphate to a second intracellular signaling protein.
  33. 33. Receptor Tyrosine Kinases (RTKs)  Intrinsic tyrosine kinase activity  Soluble or membrane-bound ligands:  Nerve growth factor, NGF  Platelet-derived growth factor, PDGF  Fibroblast growth factor, EGF  Epidermal growt factor, EGF  Insulin  Downstream pathway activation:  Ras-MAP kinase pathway
  34. 34. TYROSINE KINASE RECEPTORS • these receptors traverse the membrane only once • respond exclusively to protein stimuli – cytokines – mitogenic growth factors: • platelet derived growth factor • epidermal growth factor
  35. 35.  Functions include:  Cell proliferation, differentiation  Cell survival  Cellular metabolism  Some RTKs have been discovered in cancer research  Her2, constitutively active form in breast cancer  EGF-R overexpression in breast cancer  Other RTKs have been uncovered in studies of developmental mutations that block differentiation
  36. 36. Outline  Activated RTKs transmit signal to Ras protein  Ras transduces signal to downstream serine-threonine kinases  Ultimate activation of MAP kinase  Activation of transcription factors
  37. 37. Ligand binding to RTKs  Most RTKs are monomeric  ligand binding to EC domain induces dimerization  FGF binds to heparan sulfate enhancing its binding to receptor: dimeric receptor-ligand complex  Some ligands are dimeric: direct dimerization of receptors  Insulin receptors occur naturally as a dimer  Activation is due to the conformational change of the receptor upon ligand binding
  38. 38. Substrate + ATP Substrate-P + ADP Protein Tyrosine Kinase Protein Tyrosine Phosphatase (PTP)
  39. 39. Tyrosine Protein Phosphorylation • Eukaryotic cells coordinate functions through environmental signals - soluble factors, extracellular matrix, neighboring cells. • Membrane receptors receive these cues and transduce signals into the cell for appropriate response. • Tyrosine kinase signalling is the major mechanism for receptor signal transduction. • Tyrosine protein phosphorylation is rare (1%) relative to serine/threonine phosphorylation. • TK pathways mediate cell growth, differentiation, host defense, and metabolic regulation. • Protein tyrosine phosphorylation is the net effect of protein tyrosine kinases (TKs) and protein tyrosine phosphatases (PTPs).
  40. 40. Protein Tyrosine Kinases (TKs) Receptor tyrosine kinases (RTK) – insulin receptor – EGF receptor – PDGF receptor – TrkA Non-receptor tyrosine kinases (NRTK) – c-Src – Janus kinases (Jak) – Csk (C-terminal src kinase) – Focal adhesion kinase (FAK)
  41. 41. TABLE 15–4 Some Signaling Proteins That Act Via Receptor Tyrosine Kinases SIGNALING LIGAND RECEPTORS SOME RESPONSES Epidermal growth factor (EGF) EGF receptor stimulates proliferation of various cell types Insulin insulin receptor stimulates carbohydrate utilization and protein synthesis Insulin-like growth factors IGF receptor-1 stimulate cell growth and survival (IGF-1 and IGF-2) Nerve growth factor (NGF) Trk A stimulates survival and growth of some neurons Platelet-derived growth factors PDGF receptors stimulate survival, growth, and proliferation of various cell types Macrophage-colony-stimulating M-CSF receptor stimulates monocyte/macrophage factor (M-CSF) proliferation and differentiation Fibroblast growth factors FGF receptors stimulate proliferation of various cell (FGF- (FGF1 to FGF-24) (FGF-R1–FGFR4) types; inhibit differentiation of some precursor cells; inductive signals in development Vascular endothelial growth VEGF receptor stimulates angiogenesis factor (VEGF) Ephrins (A and B types) Eph receptors (A and B) stimulate angiogenesis; guide cell and axon migration
  42. 42. Signaling from tyrosine kinase receptors • Ligand induced dimerization • Autophosphorylation • Phosphorylation in the catalytic domain increase the kinase activity • Phosphorylation outside the catalytic domain creates specific binding for other proteins. • Autophosphorylated receptors bind to signaling proteins that have SH2 (phosphotyrosine residues) domains
  43. 43. Consequences of receptor dimerization  Kinase in one subunit P* one or more tyrosine residues on the other  Binding of ATP (insulin-R) or protein substrates (FGF-R)  Enhanced kinase activity: P* of other sites on the receptor  P*-tyrosine residues become docking sites for adapter proteins  Small proteins with SH2, PTB and SH3 domains, but without intrinsic enzymatic or signaling activities  Coupling activated RTKs to components of signaling pathways such as Ras
  44. 44. Channel Families  Voltage-gated  Extracellular ligand-gated  Intracellular ligand-gated  Inward rectifier  Intercellular  Other
  45. 45. Typical Ion Channels with Known Structure: K+ channel (KCSA) Types of ion channels:  Simple pores (GA, GAP junctions)  Substrate gated channels (Nicotinic receptor)  Voltage-gated channels (K-channels)  Pumps (ATP-synthase, K+,Na+-ATPase)
  46. 46. What are the Biochemical Changes that Lead to Channel Gating (Opening or Closing)? Gating involves some type of conformational change in the protein, but other than that there are few definitive answers to the question. However, there are several general proposed models for gating.
  47. 47. Types of Biochemical Mechanisms that Open and Close Channels  Conformational change occurs in a discrete area of the channel, leading to it opening.  The entire channel changes conformation (e.g., electrical synapses).  Ball-and-chain – type mechanism.  Nt or hormone binding causes the channel to open.
  48. 48. Types of Biochemical Mechanisms that Open and Close Channels (Cont’d)  Nt or hormone binding to receptor causes a 2nd messenger to activate a protein kinase that phosphorylates a channel and thus opens it.  Changes in membrane potential.  Membrane deformation (e.g., mechanical pressure).  Selectivity by charge (i.e., positively lined pore allows anions through; negatively lined pore allows cations through).
  49. 49. Extracellular ligand-gated  nicotinic ACh (muscle): a2bg (embryonic), a2b (adult)  nicotinic ACh (neuronal): a(2-10), b(2-4)  glutamate: NMDA, kainate, AMPA  P2X (ATP)  5-HT3  GABAA: a(1-6), b(1-4), g (1-4), , , (1-3)  Glycine
  50. 50. Intracellular ligand-gated leukotriene C4-gated Ca2+  ryanodine receptor Ca2+  IP3-gated Ca2+  IP4-gated Ca2+  Ca2+-gated K+  Ca2+-gated non- selective cation • Ca2+-gated Cl– • cAMP cation • cGMP cation • cAMP chloride • ATP Cl– • volume-regulated Cl– • arachidonic acid- activated K+ • Na+-gated K+
  51. 51. Gated Ion Channels  Another type of membrane transport  Pores in the membrane that open and close in a regulated manner and allow passage of ions -“Dispose” of the gradients  Passive transporters -Ions flow from high to low concentration -No energy is used -If there is no gradient ions will not flow
  52. 52. Gated Ion Channels  Small highly selective pores in the cell membrane  Move ions or H2O  Fast rate of transport 107 ions x s-1  Transport is always down the gradient  Cannot be coupled to an energy source
  53. 53. Ion channels are everywhere  Channels are present in almost every cell  Functions -Transport of ions and H2O -Regulation of electrical potential across the membrane -Signaling
  54. 54. Gating mechanisms  Two discrete states ;open (conducting) closed (nonconducting)  Some channels have also inactivated state (open but nonconducting)  Part of the channel structure or external particle blocks otherwise open channel
  55. 55. What gates ion channels?  Non gated - always open  Gated 􀁺 Voltage across the cell membrane 􀁺 Ligand 􀁺 Mechanical stimulus, heat (thermal fluctuations)
  56. 56. Gating mechanisms  Conformational changes in channel protein are responsible for opening and closing of the pore -3D conformational shape is determined by atomic, electric, and hydrophobic forces  Energy to switch the channel protein from one conformational shape to another comes from the gating source
  57. 57. Ligand gated channels  Glutamate receptors  Nicotinic acetylcholine receptor  Vanilloid receptor family (TRPV) = Neurotransmitter Ion Flow = Current
  58. 58. Ligand gated ion channels  Gated by ligands present outside of the cell  In fact they are receptors  All of them are nonselective cation channels  Mediate effects of neurotransmitters
  59. 59. Nuclear Receptors 1. Proteins interact with steroids and other hormones that diffuse through the cell membrane. 2. Form hormone-receptor complexes that function as activators by binding to enhancers  hormone response elements. 3. Sex hormones: estrogens and androgens; glucocorticoids, cortisol, vitamin D  Ca2+ metabolism; thyroid hormone, retinoic acid  developmental factors.
  60. 60. 1. The majority of nuclear receptors bind to respective enhancer elements and repress transcription. - In the presence of hormone, they form R-H complexes in the nucleus and function as activators by binding to the same enhancers. - Act as repressor or enhancer, depending on the physiological signals. - thus, the response element serves as either enhancer or silencer.
  61. 61. Responses to hydrophobic hormones are mediated by intracellular receptors Transcription Translation Cytoplasm Nucleus Nuclear envelope Plasma membrane Lipophilic hormone carried in blood Hormone binds intracellular receptor inducing receptor dimerization and activation Complex is imported into nucleus Binds to “hormone response element” to regulate gene expression Intracellular receptor Promoter Target gene“Hormone response element” Target cell Lipophillic Hormone
  62. 62. 2. The glucocorticoid (nuclear) receptor is found in the cytoplasm
  63. 63. Glucocorticoid Action 1. GR exists in an inactive form in the cytoplasm  complexed with heat shock protein 90 (hsp90). 2. Glucocorticoid (G) diffuses across cell membrane and enters cytoplasm 3. G binds to GR  changes conformation  dissociates from hsp90 4.  exposes a nuclear localization signal (stretch of aas) on GR. 5. G-GR (hormone-receptor complex, HR) enters nucleus, dimerizes with another HR.
  64. 64. 6. HR dimer binds to enhancer/hormone-response element upstream of hormone activated gene. 7. Binding of HR dimer to enhancer  activates transcription. 8. Most contain 2 zinc fingers (1) controls DNA binding, (2) controls dimerization Critical residues for discriminating between GRE and ERE lie at the base of the first finger -GRE = glucocorticoid responsive element /enhancer (sequence); ERE = estrogen
  65. 65. Specificity of DNA binding dimerization