Cell signalling 2


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communication and signalling and transduction by Khuram aziz

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  • D3 is somewhat mysterious – some references say it drops cAMP. Need to find out more about that.
  • Cell signalling 2

    1. 1. Cell signalling <ul><li>By: </li></ul><ul><li>Khuram Aziz </li></ul><ul><li>M.phill biochemiatry </li></ul>
    2. 2. Previous discussion <ul><li>What is cell signaling? </li></ul><ul><li>Signal transduction </li></ul><ul><li>Receptors </li></ul><ul><li>Types </li></ul><ul><li>Functions </li></ul><ul><li>Steps for signaling </li></ul>
    3. 3. Today <ul><li>What is g protein coupled receptor </li></ul><ul><li>Regulation </li></ul><ul><li>What is g protein </li></ul><ul><li>Regulatioon </li></ul><ul><li>Mode of action </li></ul>
    4. 4. Signal Reception: G Protein-Coupled Receptors
    5. 5. <ul><li>G-protein-Coupled Receptors may dimerize or form oligomeric complexes within the membrane. </li></ul><ul><li>Ligand binding may promote oligomerization, which may in turn affect activity of the receptor. </li></ul><ul><li>Various GPCR-interacting proteins ( GIPs ) modulate receptor function. Effects of GIPs may include: </li></ul><ul><ul><li>altered ligand affinity </li></ul></ul><ul><ul><li>receptor dimerization or oligomerization </li></ul></ul><ul><ul><li>control of receptor localization , including transfer to or removal from the plasma membrane </li></ul></ul><ul><ul><li>promoting close association with other signal proteins </li></ul></ul>
    6. 6. Neurotransmitter receptors <ul><li>Ligand – gated channels: </li></ul><ul><ul><li>Nicotinic acetylcholine receptor </li></ul></ul><ul><ul><li>NMDA-type glutamate receptor </li></ul></ul><ul><ul><li>Glycine receptor </li></ul></ul><ul><ul><li>GABA A receptor </li></ul></ul><ul><ul><li>Serotonin receptor (5-HT 3 ) </li></ul></ul><ul><li>G protein-coupled receptors: </li></ul><ul><ul><li>Muscarinic acetylcholine receptor (several types) </li></ul></ul><ul><ul><li>Catecholamine receptors </li></ul></ul><ul><ul><li>Histamine receptors (H 1 , H 2 ) </li></ul></ul><ul><ul><li>5-HT receptors other than 5-HT 3 </li></ul></ul><ul><ul><li>GABA B receptors </li></ul></ul><ul><ul><li>‘ Metabotropic’ glutamate receptors </li></ul></ul><ul><ul><li>Peptide receptors (Endorphin, cholecystokinin..) </li></ul></ul>
    7. 7. The G Protein-Coupled Receptor (GPCR) Superfamily <ul><li>Largest known receptor family – </li></ul><ul><li>Constitutes > 1% of the human genome. </li></ul><ul><li>Comprises receptors for a diverse array of molecules: neurotransmitters, odorants, lipids, neuropeptides, large glycoprotein hormones. </li></ul><ul><li>Odorant receptor family alone contains hundreds of genes. </li></ul><ul><li>Mammalian GPCRs: nearly 300 different kinds – grouped into 3 main subfamilies: </li></ul>
    8. 8. <ul><li>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. </li></ul><ul><li>But high throughput screening has recently added to the advances in being able to identify the ligand. </li></ul>
    9. 9. <ul><li>GPCRs Interact guanine nucleotide-binding proteins (aka G-proteins) </li></ul><ul><li>Largest family of membrane proteins in the human genome </li></ul><ul><li>Eukaryotic trans membrane receptors </li></ul><ul><li>Seven helices spanning the membrane  </li></ul>
    10. 10. <ul><li>Roles: </li></ul><ul><li>                - Light and smell processing                 - Behavior and mood                 - Immune response                 - Autonomic nervous system transmission                 - Blood pressure                 - Heart rate                  - Digestive processes                 - CRITICAL FACTOR IN MANY DISEASES! </li></ul>
    11. 11. <ul><li>Five different classes (based on sequence and function): </li></ul><ul><li>                - 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 </li></ul>
    12. 12. Almost all Receptors Comprise a Number of Subtypes <ul><li>Dopamine receptors - 5 subtypes </li></ul><ul><li>5-HT receptors – 13 subtypes </li></ul><ul><li>mGlu receptors - 8 subtypes </li></ul><ul><li>Acetylcholine receptors – 5 subtypes </li></ul><ul><li>Identified by their pharmacological and functional characteristics, rather than by strict sequence homology: </li></ul><ul><li>- 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). </li></ul>
    13. 14. Regulation of G protein-coupled receptor function <ul><li>Desensitization/resensitization – a decrease in responsiveness during continuous drug application or a right-shift in a drug dose-response curve. </li></ul><ul><li>After removal of the drug, receptor activity recovers, although the speed and extent of this resensitization can depend on the duration of agonist activation. </li></ul><ul><li>Rapid desensitization (sec-min) results from receptor phos, arrestin binding, and receptor internalization. </li></ul><ul><li>Long-term desensitization (down-regulation) involve changes in receptor and/or G protein levels, and their mRNA stability and expression. </li></ul><ul><li>Long-term changes in [GPCR]s and [accessory proteins]s known to be induced by chronic drug treatment and involved in several pathologies. </li></ul>
    14. 15. <ul><li>Phosphorylation </li></ul><ul><li>2 nd messenger kinase </li></ul><ul><li>G protein receptor kinase (GRK) </li></ul><ul><li>Arrestin </li></ul><ul><li>β -arrestin binding to phosphorylated GPCR is required to decrease GTPase activity prior to desensitization. </li></ul><ul><li>Receptor trafficking, internalization, and recycling (covered earlier; see Protein trafficking and LGIC slides). </li></ul>
    15. 16. <ul><li>Mechanisms of long-term down regulation </li></ul><ul><li>Long-term (> 1 hr) treatment with agonist induces the loss of total cellular receptor number in addition to the decr in surface receptor number. </li></ul><ul><li>e.g ., antidepressants ( e.g ., fluoxetine) incr [5HT] synapse  decr 5HT receptor density. </li></ul><ul><li>Receptor endocytosis: C-terminal domain determines whether they enter the recycle pathway or the lysosomal pathway: </li></ul><ul><li>- 2 distinct motifs: </li></ul><ul><li>1. PDZ-domain interats with NHERF in a phos-dependent manner. </li></ul><ul><li>2. A short sequence that interacts with NSF ( N -ethylmaleimide sensitive factor). </li></ul><ul><li>Arrestin has also been shown to be important for recycling: </li></ul><ul><li>e.g., V2 vasopressin receptor, which continues to bind arrestin while in endosomes, does not recycle back to plasma membrane. </li></ul>
    16. 17. D D D D α α β α γ <ul><li>Agonist binding </li></ul><ul><li>and G protein </li></ul><ul><li>activation </li></ul>(2) Phosphorylation P P (3) Arrestin binding Arrestin P P Arrestin P P Clathrin <ul><li>Clustering in </li></ul><ul><li>clathrin-coated </li></ul><ul><li>pits </li></ul>(5) Endocytosis Endosomes Arrestin P P D (7) Recycling <ul><li>Dissociation of agonist: </li></ul><ul><li>Dephosphorylation </li></ul><ul><li>Sorting between cycling </li></ul><ul><li>and lysosomal pathways </li></ul>(8 ) Traffic to lysosomes Lysosomes Mechanisms of Receptor Regulation
    17. 18. Another Receptor – G Protein Cycle
    18. 19. Structure, function and mechanisms of G-Proteins
    19. 20. What are G-proteins? <ul><li>G proteins bind GTP: guanosine triphosphate. Control and amplify intracellular signaling pathways </li></ul><ul><li>Exist in two states 1) bound GTP: active </li></ul><ul><li>2) bound GDP: inactive </li></ul>Fig. 15.1 Examples of GTPase proteins Ras, Cdc-42 (hormone, GF, drug)
    20. 21. 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.“
    21. 22. G-Protein = Guanine-nucleotide binding protein (GNBD) Guanine Ribose Phosphates 1 2 5 4 3 α   1 3 4 2 6 5 7 8 9 Guanosine Ester Anhydride Guanosine-triphosphate - GTP
    22. 23. G-Protein families <ul><li>Heterotrimeric G-Proteins (Transducin, G  i , G  q …), in 7-TM receptor signalling </li></ul><ul><li>Initiation, elongation, termination factors in protein synthesis (IF1, EF-Tu, EF-TS) </li></ul><ul><li>Signal recognition particle (SRP) and its receptor , translocation of nascent polypeptide chains in the ER </li></ul><ul><li>Ras-like GTPases (Ras, Rap, Rho, Ran, Rab, Arf, Arl, Sar), molecular switches in signal transduction </li></ul><ul><li>Dynamin superfamily of GTPases , remodelling of membranes </li></ul><ul><li>+ 60 further distinct families </li></ul><ul><li> Leipe et al., JMB (2002) </li></ul>
    23. 24. GTPases and disease. <ul><li>Damage to these small GTPase switches can have catastrophic consequences for the cell and the organism. </li></ul><ul><li>Several small GTPases of the Rac/Rho subfamily are direct targets for clostridial cytotoxins. </li></ul><ul><li>Further, Ras proteins are mutated to a constitutively-active (GTP-bound) form in approximately 20% of human cancers. </li></ul>
    24. 25. G-proteins are tightly regulated <ul><li>3 types of accessory proteins that modulate cycling of G-proteins between GTP/GDP </li></ul><ul><li>1. GAPs : GTPase-activating proteins. Stimulate GTP hydrolysis. Inactivate G-protein. Example of a GAP: PLC  </li></ul><ul><li>2. GEFs : Guanine nucleotide-exchange factors: G-protein-coupled receptors (GPCR). Stimulate dissociation of GDP (inactive) from G-protein so GTP can bind (active). </li></ul><ul><li>3. GDIs : Guanine nucleotide-dissociation inhibitors. Inhibit release of bound GDP (maintain G-protein in inactive state). </li></ul>
    25. 26. The heterotrimeric G proteins transmit signals from a variety of cell surface receptors to enzymes and channels <ul><li>Stimulated by receptors </li></ul><ul><li>Act on effectors </li></ul><ul><li>Regulated by nucleotide </li></ul><ul><li>exchange and hydrolysis </li></ul>
    26. 27. <ul><li>The signal is usually passed from a 7-helix receptor to an intracellular G-protein . </li></ul><ul><ul><li>Seven-helix receptors are thus called GPCR , or G - P rotein- C oupled R eceptors. </li></ul></ul><ul><ul><li>Approx. 800 different GPCRs are encoded in the human genome. </li></ul></ul>
    27. 28. <ul><li>G-proteins are heterotrimeric , with 3 subunits  ,  ,  . </li></ul><ul><li>A G-protein that activates cyclic-AMP formation within a cell is called a stimulatory G-protein , designated G s with alpha subunit G s  . </li></ul><ul><li>G s is activated, e.g., by receptors for the hormones epinephrine and glucagon . </li></ul><ul><li>The  -adrenergic receptor is the GPCR for epinephrine. </li></ul>
    28. 29. <ul><li>These domains include residues adjacent to the terminal phosphate of GTP and/or the Mg ++ associated with the two terminal phosphates. </li></ul>Structure 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  .
    29. 30. <ul><li>GTP hydrolysis occurs by nucleophilic attack of a water molecule on the terminal phosphate of GTP. </li></ul><ul><li>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. </li></ul>
    30. 31. <ul><li>The  subunit of the heterotrimeric G Protein has a  -propeller structure, formed from multiple repeats of a sequence called the WD-repeat . </li></ul><ul><li>The  -propeller provides a stable structural support for residues that bind G  . </li></ul><ul><li>It is a common structural motif for protein domains involved in protein-protein interaction. </li></ul>
    31. 32. <ul><li>The family of heterotrimeric G proteins includes also: </li></ul><ul><ul><li>transducin , involved in sensing of light in the retina. </li></ul></ul><ul><ul><li>G-proteins involved in odorant sensing in olfactory neurons. </li></ul></ul><ul><li>There is a larger family of small GTP-binding switch proteins , related to G  . </li></ul>
    32. 33. <ul><li>Small GTP-binding proteins include (roles indicated): </li></ul><ul><ul><li>initiation & elongation factors (protein synthesis). </li></ul></ul><ul><ul><li>Ras (growth factor signal cascades). </li></ul></ul><ul><ul><li>Rab (vesicle targeting and fusion). </li></ul></ul><ul><ul><li>ARF (forming vesicle coatomer coats). </li></ul></ul><ul><ul><li>Ran (transport of proteins into & out of the nucleus). </li></ul></ul><ul><ul><li>Rho (regulation of actin cytoskeleton ) </li></ul></ul><ul><li>A ll GTP-binding proteins differ in conformation depending on whether GDP or GTP is present at their nucleotide binding site. </li></ul><ul><li>Generally, GTP binding induces the active state. </li></ul>
    33. 34. <ul><li>A GAP may provide an essential active site residue, while promoting the correct positioning of the glutamine residue of the switch II domain. </li></ul><ul><li>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. </li></ul>Most GTP-binding proteins depend on helper proteins : GAPs , G TPase A ctivating P roteins, promote GTP hydrolysis.
    34. 35. <ul><li>G  of a heterotrimeric G protein has innate capability for GTP hydrolysis. </li></ul><ul><li>It has the essential arginine residue normally provided by a GAP for small GTP-binding proteins. </li></ul><ul><li>However, RGS proteins , which are negative regulators of G protein signaling, stimulate GTP hydrolysis by G  . </li></ul>
    35. 36. <ul><ul><li>An activated receptor (GPCR) normally serves as GEF for a heterotrimeric G-protein. </li></ul></ul><ul><ul><li>Alternatively, AGS (Activator of G-protein Signaling) proteins may activate some heterotrimeric G-proteins, independent of a receptor. </li></ul></ul><ul><ul><li>Some AGS proteins have GEF activity. </li></ul></ul>GEFs , G uanine N ucleotide E xchange Factors, promote GDP/GTP exchange.
    36. 37.  &  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. The   subunit of a G-protein ( G  ) binds GTP , & can hydrolyze it to GDP + P i .
    37. 39. <ul><li>The sequence of events by which a hormone activates cAMP signaling: </li></ul><ul><li>1. Initially G  has bound GDP , and     &   subunits are complexed together. </li></ul><ul><li>G  ,  , the complex of  &  subunits, inhibits G  . </li></ul>
    38. 40. <ul><li>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. </li></ul><ul><li>The nucleotide-binding site on G  becomes more accessible to the cytosol, where [GTP] > [GDP]. </li></ul><ul><li>G  releases GDP & binds GTP ( GDP-GTP exchange ). </li></ul>
    39. 41. <ul><li>3. Substitution of GTP for GDP causes another conformational change in G  . </li></ul><ul><li>G  -GTP dissociates from the inhibitory  complex & can now bind to and activate Adenylate Cyclase. </li></ul>
    40. 42. Fig 15.3 The G Protein Cycle
    41. 43. G Protein-Linked Receptors
    42. 44. G Protein-Linked Receptors
    43. 45. G Protein-Linked Receptors
    44. 46. G Protein-Linked Receptors note how activation is reversible
    45. 47. G Protein-Linked Receptors the more ligand binding, the more K + in cytoplasm
    46. 48. <ul><li>Regulation at the G protein level </li></ul><ul><li>Regulator of G protein signaling (RGS = GAPs = GTPase activating proteins) family of proteins (> 20 members) regulate the rate of GTP hydrolysis in the G α subunit. </li></ul><ul><li>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 G α subunits for the βγ after GTP hydrolysis  incr rate of reformation of the heterotimer. </li></ul>
    47. 49. <ul><li>Regulation at the G protein level (cont’d) </li></ul><ul><li>RGS proteins also important in regulating the temporal characteristics of G protein actions. </li></ul><ul><li>E.g ., RGS proteins accelerate the decay of agonist-induced activation of GIRK (G protein regulated inward rectifying K channels). </li></ul><ul><li>E.g ., RGS proteins accelerate desensitization of adrenergic receptor-induced N-type Ca 2+ channel currents. </li></ul>
    48. 50. <ul><li>ADH - Promotes water retention by the  kidneys  (V2 Cells of Posterior Pituitary) </li></ul><ul><li>GHRH - Stimulates the synthesis and release of GH (Somatotroph Cells of Anterior Pituitary) </li></ul><ul><li>GHIH - Inhibits the synthesis and release of GH (Somatotroph Cells of Anterior Pituitary) </li></ul><ul><li>CRH - Stimulates the synthesis and release of ACTH (Anterior Pituitary) </li></ul>
    49. 51. <ul><li>ACTH - Stimulates the synthesis and release of Cortisol (zona fasiculata of adrenal cortex in kidneys) </li></ul><ul><li>TSH - Stimulates the synthesis and release of a majority of T4 (Thyroid Gland) </li></ul><ul><li>LH - Stimulates follicular maturation and ovulation in women; Stimulates testosterone production and spermatogenesis in men </li></ul>
    50. 52. <ul><li>FSH - Stimulates follicular development in women; Stimulates spermatogenesis in men </li></ul><ul><li>PTH - Increases blood calcium levels (PTH1 Receptor: Kidneys and Bone; PTH2 Receptor: Central Nervous system, Bones, Kidneys, Brain) </li></ul><ul><li>Calcitonin - Decreases blood calcium levels (Calcitonin Receptor: Intestines, Bones, Kidneys, Brain) </li></ul><ul><li>Glucagon - Stimulates glycogen breakdown (liver) </li></ul><ul><li>hCG - Promotes cellular differentiation; Potentially involved in  apoptosis </li></ul>
    51. 53. How G-protein-coupled receptors work (1) extracellular space cytosol    heterotrimeric G-protein ‘ 7TM’ - receptor GDP GDP GTP Ligand    N
    52. 54. How G-protein-coupled receptors work (2) inactive    GDP P active N  GTP   N
    53. 55. How G-protein-coupled receptors work (3) ATP inactive inactive active cAMP cAMP Protein kinase A Phosphorylation of multiple target proteins active Adenylate cyclase    GTP
    54. 56. Some G-proteins are inhibitory  -Adrenoceptor  2 -Adrenoceptor AC active AC inactive  s GTP  i GTP
    55. 57.  -Subunits of G proteins may have regulatory activity, too Muscarinic (M 2 ) acetylcholine receptor K ir AC inactive K +    i GTP
    56. 58. G  -proteins regulate diverse effector systems  q phospholipase C  PIP 2 IP 3 + DAG protein kinase C  phosphorylation of multiple proteins Ca ++ ER  t cGMP phosphodiesterase  cGMP   s adenylate cyclase  protein kinase A  cAMP   i1 adenylate cyclase  protein kinase A  cAMP 
    57. 59. Many transmitters have multiple GPCR with different downstream signaling mechanisms Norepinephrine,  1 IP 3 + DAG  epinephrine  2 cAMP   1 ,  2 cAMP  Dopamine D 2 - D 4 cAMP  D 1 , D 5 cAMP  Acetylcholine       IP 3 + DAG   2 , M 3 cAMP 
    58. 60. <ul><li>Thanks </li></ul>