Molecular pharmacology of cell signling


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molecular biology and pharmacology G protein, receptor, transport of drug ankoring protein, adapter protein, cell signal

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Molecular pharmacology of cell signling

  1. 1. Molecular Pharmacology of Cell Signaling Mohanad AlBayati Mohanad AbdulSattar Ali Al-Bayati, BVM&S, MSc. Physiol., PhD. Assistant Professor of Pharmacology and Toxicology Department of Physiology and Pharmacology College of Veterinary Medicine University of Baghdad Al Ameria, Baghdad Phone: 0964 7802120391 E. Mail:
  2. 2. Previous soundness of drug cell signal • What is cell signaling? • Signal transduction • Receptors • Types • Functions • Steps for signaling
  3. 3. - Guanlyl cyclase activity
  4. 4. Receptors Receptors are the sites at which biomolecules such as hormones, neurotransmitters and the molecules responsible for taste and odour are recognised. A drug that binds to a receptor can either: • Trigger the same events as the native ligand - an agonist. Or • Stop the binding of the native agent without eliciting a response - an antagonist. There are four ‘superfamilies’ of receptors.
  5. 5. . These have 4 or 5 membrane-spanning helical subunits. Their N- and C-terminii are found in the extracellular fluid. This family includes ion channels. . These have 7 helical transmembrane regions. Their N- terminal is extracellular and the C-terminal in intracellular. This family is coupled to the action of G-proteins: They are known as the G- protein coupled receptors. . These are tyrosine kinase-linked receptors with a single transmembrane helix. The insulin and growth factor receptors fall within this family. . These receptors are found in the cell nucleus and are transcription factors. They have looped regions held together by a group of four cysteine residues coordinating to a zinc ion. These motifs are called zinc fingers. The receptor ligands include steroids and thyroid hormones. Receptors
  6. 6. Today soundness of drug cell signal  What is G protein coupled receptor  Regulation  What is G protein  Regulation  Mode of action
  7. 7. G-protein-Coupled Receptors may dimerize or form oligomeric complexes within the membrane. Ligand binding may promote oligomerization, which may in turn affect activity of the receptor. Various GPCR-interacting proteins (GIPs) modulate receptor function. Effects of GIPs may include:  altered ligand affinity  receptor dimerization or oligomerization  control of receptor localization, including transfer to or removal from the plasma membrane  promoting close association with other signal proteins
  8. 8. Neurotransmitter receptors Ligand – gated channels: • Nicotinic acetylcholine receptor • NMDA-type glutamate receptor • Glycine receptor • GABAA receptor • Serotonin receptor (5-HT3) G protein-coupled receptors: • Muscarinic acetylcholine receptor (several types) • Catecholamine receptors • Histamine receptors (H1, H2) • 5-HT receptors other than 5-HT3 • GABAB receptors • ‘Metabotropic’ glutamate receptors • Peptide receptors (Endorphin, cholecystokinin..)
  9. 9. `
  10. 10. Into nucleus
  11. 11. Thanks
  12. 12. The G Protein-Coupled Receptor (GPCR) Superfamily • Largest known receptor family – Constitutes > 1% of the genome. • Comprises receptors for a diverse array of molecules: neurotransmitters, odorants, lipids, neuropeptides, large glycoprotein hormones. • Odorant receptor family alone contains hundreds of genes. • Mammalian GPCRs: nearly 300 different kinds – grouped into 3 main subfamilies:
  13. 13. • Each GPCR family contains some orphan receptors, which have been identified as members of the GPCR superfamily by homology cloning but whose activating ligand is unknown. • But high throughput screening has recently added to the advances in being able to identify the ligand.
  14. 14. • GPCRs Interact guanine nucleotide-binding proteins (aka G-proteins) • Largest family of membrane proteins in the human genome • Eukaryotic trans membrane receptors • Seven helices spanning the membrane
  15. 15.  Roles:  - Light and smell processing - Behavior and mood - Immune response - Autonomic nervous system transmission - Blood pressure - Heart rate - Digestive processes - CRITICAL FACTOR IN MANY DISEASES!
  16. 16.  Five different classes (based on sequence and function):  - Class A: Rhodopsin-like receptors - Class B: Secretin receptor family - Class C: Metabotropic glutamate/pheromone - Class D: Fungal pheromone receptors - Class E: Cyclyic AMP receptors
  17. 17. Almost all Receptors Comprise a Number of Subtypes • Dopamine receptors - 5 subtypes • 5-HT receptors – 13 subtypes • mGlu receptors - 8 subtypes • Acetylcholine receptors – 5 subtypes • Identified by their pharmacological and functional characteristics, rather than by strict sequence homology: - Some receptors for the same ligand show remarkably little homology (e.g., histamine H3 and H4 have the lowest recorded homology (~ 20 %) to other histamine receptors H1 and H2).
  18. 18. Regulation of G protein-coupled receptor function Desensitization/resensitization– a decrease in responsiveness during continuous drug application or a right-shift in a drug dose-response curve. After removal of the drug, receptor activity recovers, although the speed and extent of this resensitization can depend on the duration of agonist activation. Rapid desensitization (sec-min) results from receptor phos, arrestin binding, and receptor internalization. Long-term desensitization (down-regulation) involve changes in receptor and/or G protein levels, and their mRNA stability and expression. Long-term changes in [GPCR]s and [accessory proteins]s known to be induced by chronic drug treatment and involved in several pathologies.
  19. 19. Phosphorylation 2nd messenger kinase G protein receptor kinase (GRK) Arrestin β-arrestin binding to phosphorylated GPCR is required to decrease GTPase activity prior to desensitization. Receptor trafficking, internalization, and recycling (covered earlier; see Protein trafficking and LGIC slides).
  20. 20. Mechanisms of long-term down regulation Long-term (> 1 hr) treatment with agonist induces the loss of total cellular receptor number in addition to the decr in surface receptor number. e.g., antidepressants (e.g., fluoxetine) incr [5HT]synapse decr 5HT receptor density. Receptor endocytosis: C-terminal domain determines whether they enter the recycle pathway or the lysosomal pathway: - 2 distinct motifs: 1. PDZ-domain interats with NHERF in a phos-dependent manner. 2. A short sequence that interacts with NSF (N-ethylmaleimide sensitive factor). Arrestin has also been shown to be important for recycling: e.g., V2 vasopressin receptor, which continues to bind arrestin while in endosomes, does not recycle back to plasma membrane.
  21. 21. D D D D α α β α γ (1) Agonist binding and G protein activation (2) Phosphorylation P P (3) Arrestin binding Arrestin P P Arrestin P P Clathrin(4) Clustering in clathrin-coated pits (5) EndocytosisEndosomes Arrestin P P D (7) Recycling (6) Dissociation of agonist: • Dephosphorylation • Sorting between cycling and lysosomal pathways (8) Traffic to lysosomes Lysosomes Mechanisms of Receptor Regulation
  22. 22. Another Receptor – G Protein Cycle
  23. 23. Structure, function and mechanisms of G-Proteins
  24. 24. What are G-proteins? • G proteins bind GTP: guanosine triphosphate. Control and amplify intracellular signaling pathways Exist in two states 1) bound GTP: active 2) bound GDP: inactive Fig. 15.1 Examples of GTPase proteins Ras, Cdc-42 (hormone, GF, drug)
  25. 25. 1994 Nobel Prize in Medicine, Alfred Gilman and Martin Rodbell, for their „discovery of G-Proteins and the role of these proteins in signal transduction in cells.“
  26. 26. G-Protein = Guanine-nucleotide binding protein (GNBD) 1 2 5 4 3 Guanine Ribose Phosphates α 1 3 42 6 5 7 89 Guanosine EsterAnhydride Guanosine-triphosphate - GTP
  27. 27. G-Protein families • Heterotrimeric G-Proteins (Transducin, Gi, Gq …), in 7-TM receptor signalling • Initiation, elongation, termination factors in protein synthesis (IF1, EF-Tu, EF-TS) • Signal recognition particle (SRP) and its receptor, translocation of nascent polypeptide chains in the ER • Ras-like GTPases (Ras, Rap, Rho, Ran, Rab, Arf, Arl, Sar), molecular switches in signal transduction • Dynamin superfamily of GTPases, remodelling of membranes + 60 further distinct families Leipe et al., JMB (2002)
  28. 28. GTPases and disease. • Damage to these small GTPase switches can have catastrophic consequences for the cell and the organism. • Several small GTPases of the Rac/Rho subfamily are direct targets for clostridial cytotoxins. • Further, Ras proteins are mutated to a constitutively- active (GTP-bound) form in approximately 20% of human cancers.
  29. 29. G-proteins are tightly regulated 3 types of accessory proteins that modulate cycling of G-proteins between GTP/GDP 1. GAPs: GTPase-activating proteins. Stimulate GTP hydrolysis. Inactivate G-protein. Example of a GAP: PLC. 2. GEFs: Guanine nucleotide-exchange factors: G-protein-coupled receptors (GPCR). Stimulate dissociation of GDP (inactive) from G-protein so GTP can bind (active). 3. GDIs: Guanine nucleotide-dissociation inhibitors. Inhibit release of bound GDP (maintain G-protein in inactive state).
  30. 30. The heterotrimeric G proteins transmit signals from a variety of cell surface receptors to enzymes and channels • Stimulated by receptors • Act on effectors • Regulated by nucleotide exchange and hydrolysis
  31. 31. The signal is usually passed from a 7-helix receptor to an intracellular G-protein.  Seven-helix receptors are thus called GPCR, or G- Protein-Coupled Receptors.  Approx. 800 different GPCRs are encoded in the human genome.
  32. 32.  G-proteins are heterotrimeric, with 3 subunits , , .  A G-protein that activates cyclic-AMP formation within a cell is called a stimulatory G-protein, designated Gs with alpha subunit Gs.  Gs is activated, e.g., by receptors for the hormones epinephrine and glucagon. The -adrenergic receptor is the GPCR for epinephrine.
  33. 33. These domains include residues adjacent to the terminal phosphate of GTP and/or the Mg++ associated with the two terminal phosphates. Inhibitory G GTPS PDB 1GIAStructure of G proteins: The nucleotide binding site in G consists of loops that extend out from the edge of a 6-stranded -sheet. Three switch domains have been identified, that change position when GTP substitutes for GDP on G.
  34. 34. GTP hydrolysis occurs by nucleophilic attack of a water molecule on the terminal phosphate of GTP. Switch domain II of G includes a conserved glutamine residue that helps to position the attacking water molecule adjacent to GTP at the active site. O OHOH HH H CH2 H OPOPOP O O O O O O O NH2 NH NN N O H O H GTP hydrolysis
  35. 35. The  subunit of the heterotrimeric G Protein has a -propeller structure, formed from multiple repeats of a sequence called the WD-repeat. The -propeller provides a stable structural support for residues that bind G. It is a common structural motif for protein domains involved in protein-protein interaction. G - side view of -propeller PDB 1GP2 G – face view of -propeller PDB 1GP2
  36. 36. The family of heterotrimeric G proteins includes also:  transducin, involved in sensing of light in the retina.  G-proteins involved in odorant sensing in olfactory neurons. There is a larger family of small GTP-binding switch proteins, related to G.
  37. 37. Small GTP-binding proteins include (roles indicated):  initiation & elongation factors (protein synthesis).  Ras (growth factor signal cascades).  Rab (vesicle targeting and fusion).  ARF (forming vesicle coatomer coats).  Ran (transport of proteins into & out of the nucleus).  Rho (regulation of actin cytoskeleton) All GTP-binding proteins differ in conformation depending on whether GDP or GTP is present at their nucleotide binding site. Generally, GTP binding induces the active state.
  38. 38. A GAP may provide an essential active site residue, while promoting the correct positioning of the glutamine residue of the switch II domain. Frequently a (+) charged arginine residue of a GAP inserts into the active site and helps to stabilize the transition state by interacting with () charged O atoms of the terminal phosphate of GTP during hydrolysis. Most GTP-binding proteins depend on helper proteins: GAPs, GTPase Activating Proteins, promote GTP hydrolysis. protein-GTP (active) GDP GEF GAP GTP Pi protein-GDP (inactive)
  39. 39.  G of a heterotrimeric G protein has innate capability for GTP hydrolysis. It has the essential arginine residue normally provided by a GAP for small GTP-binding proteins.  However, RGS proteins, which are negative regulators of G protein signaling, stimulate GTP hydrolysis by G. protein-GTP (active) GDP GEF GAP GTP Pi protein-GDP (inactive)
  40. 40.  An activated receptor (GPCR) normally serves as GEF for a heterotrimeric G-protein.  Alternatively, AGS (Activator of G-protein Signaling) proteins may activate some heterotrimeric G-proteins, independent of a receptor. Some AGS proteins have GEF activity. protein-GTP (active) GDP GEF GAP GTP Pi protein-GDP (inactive) GEFs, Guanine Nucleotide Exchange Factors, promote GDP/GTP exchange.
  41. 41.  &  subunits have covalently attached lipid anchors that bind a G-protein to the plasma membrane cytosolic surface. Adenylate Cyclase (AC) is a transmembrane protein, with cytosolic domains forming the catalytic site. AC hormone signal outside GPCR plasma membrane GTP GDP ATP cAMP + PPi  cytosol GDP GTP The  subunit of a G-protein (G) binds GTP, & can hydrolyze it to GDP + Pi.
  42. 42. The sequence of events by which a hormone activates cAMP signaling: 1. Initially G has bound GDP, and ,, &  subunits are complexed together. G,, the complex of  &  subunits, inhibits G. AC hormone signal outside GPCR plasma membrane GTP GDP ATP cAMP + PPi  cytosol GDP GTP
  43. 43. 2. Hormone binding, usually to an extracellular domain of a 7-helix receptor (GPCR), causes a conformational change in the receptor that is transmitted to a G-protein on the cytosolic side of the membrane. The nucleotide-binding site on G becomes more accessible to the cytosol, where [GTP] > [GDP]. G releases GDP & binds GTP (GDP-GTP exchange). AC hormone signal outside GPCR plasma membrane GTP GDP ATP cAMP + PPi  cytosol GDP GTP
  44. 44. 3. Substitution of GTP for GDP causes another conformational change in G. G-GTP dissociates from the inhibitory  complex & can now bind to and activate Adenylate Cyclase. AC hormone signal outside GPCR plasma membrane GTP GDP ATP cAMP + PPi  cytosol GDP GTP
  45. 45. Fig 15.3 The G Protein Cycle
  46. 46. GProtein-LinkedReceptors
  47. 47. GProtein-LinkedReceptors
  48. 48. GProtein-LinkedReceptors
  49. 49. GProtein-LinkedReceptors note how activation is reversible
  50. 50. GProtein-LinkedReceptors the more ligand binding, the more K+ in cytoplasm
  51. 51. Regulation at the G protein level Regulator of G protein signaling (RGS = GAPs = GTPase activating proteins) family of proteins (> 20 members) regulate the rate of GTP hydrolysis in the Gα subunit. Can also attenuate G protein actions that are mediated by βγ subunits, because they can alter the number of βγ available by enhancing the affinity of Gα subunits for the βγ after GTP hydrolysis  incr rate of reformation of the heterotimer.
  52. 52. Regulation at the G protein level (cont’d) RGS proteins also important in regulating the temporal characteristics of G protein actions. E.g., RGS proteins accelerate the decay of agonist- induced activation of GIRK (G protein regulated inward rectifying K channels). E.g., RGS proteins accelerate desensitization of adrenergic receptor-induced N-type Ca2+ channel currents.
  53. 53. • ADH - Promotes water retention by the kidneys (V2 Cells of Posterior Pituitary) • GHRH - Stimulates the synthesis and release of GH (Somatotroph Cells of Anterior Pituitary) • GHIH - Inhibits the synthesis and release of GH (Somatotroph Cells of Anterior Pituitary) • CRH - Stimulates the synthesis and release of ACTH (Anterior Pituitary)
  54. 54. • ACTH - Stimulates the synthesis and release of Cortisol (zona fasiculata of adrenal cortex in kidneys) • TSH - Stimulates the synthesis and release of a majority of T4 (Thyroid Gland) • LH - Stimulates follicular maturation and ovulation in women; Stimulates testosterone production and spermatogenesis in men
  55. 55. • FSH - Stimulates follicular development in women; Stimulates spermatogenesis in men • PTH - Increases blood calcium levels (PTH1 Receptor: Kidneys and Bone; PTH2 Receptor: Central Nervous system, Bones, Kidneys, Brain) • Calcitonin - Decreases blood calcium levels (Calcitonin Receptor: Intestines, Bones, Kidneys, Brain) • Glucagon - Stimulates glycogen breakdown (liver) • hCG - Promotes cellular differentiation; Potentially involved in apoptosis
  56. 56.  How G-protein-coupled receptors work (1)  extracellular space cytosol  heterotrimeric G-protein ‘7TM’ - receptor GDP GDP N GTP Ligand
  57. 57. How G-protein-coupled receptors work (2) inactive  N GDP  GTP P  N active
  58. 58. How G-protein-coupled receptors work (3) ATP inactive inactive active cAMP cAMP Protein kinase A Phosphorylation of multiple target proteins   GTP active Adenylate cyclase
  59. 59. Some G-proteins are inhibitory -Adrenoceptor 2-Adrenoceptor s GTP AC active AC inactive i GTP
  60. 60. -Subunits of G proteins may have regulatory activity, too K+ Muscarinic (M2) acetylcholine receptor Kir  AC inactive i GTP
  61. 61. G-proteins regulate diverse effector systems s adenylate cyclase  protein kinase AcAMP  i1 adenylate cyclase  protein kinase AcAMP  q phospholipase C  PIP2 IP3 + DAG protein kinase C phosphorylation of multiple proteins Ca++ ER t cGMP phosphodiesterase  cGMP 
  62. 62. Many transmitters have multiple GPCR with different downstream signaling mechanisms Norepinephrine, 1 IP3 + DAG  epinephrine2 cAMP  1,2 cAMP  Dopamine D2 - D4 cAMP  D1, D5 cAMP  Acetylcholine M1,,M4,M5IP3 + DAG  M2, M3 cAMP 
  63. 63. Thanks