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Cell signaling3


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for medical students. how cell communicate woith each other and involve in body functioning

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Cell signaling3

  1. 1. Cell signalling <ul><li>By: </li></ul><ul><li>Khuram Aziz </li></ul><ul><li>M.phill Biochemistry </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. <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. 5. Fig 15.3 The G Protein Cycle
  5. 6. TODAY <ul><li>Overview of G Protein-coupled Receptor (GPCR) Signaling </li></ul><ul><li>GPCRs That Regulate Ion Channels </li></ul><ul><li>GPCRs That Regulate Adenylyl Cyclase </li></ul><ul><li>GPCRs That Regulate Phospholipase C </li></ul>
  6. 7. <ul><ul><li>Different isoforms of G  have different signal roles. E.g.: </li></ul></ul><ul><ul><ul><li>The stimulatory G s  , when it binds GTP, activates Adenylate cyclase. </li></ul></ul></ul><ul><ul><ul><li>An inhibitory G i  , when it binds GTP, inhibits Adenylate cyclase. </li></ul></ul></ul><ul><ul><ul><li>Different effectors & their receptors induce G i  to exchange GDP for GTP than those that activate G s  . </li></ul></ul></ul><ul><ul><li>The complex of G  that is released when G  binds GTP is itself an effector that binds to and activates or inhibits several other proteins. </li></ul></ul><ul><ul><li>E.g., G  inhibits one of several isoforms of Adenylate Cyclase , contributing to rapid signal turnoff in cells that express that enzyme. </li></ul></ul>
  7. 8. <ul><li>Cholera toxin catalyzes covalent modification of G s  . </li></ul><ul><ul><li>ADP-ribose is transferred from NAD + to an arginine residue at the GTPase active site of G s  . </li></ul></ul><ul><ul><li>ADP-ribosylation prevents GTP hydrolysis by G s   . </li></ul></ul><ul><ul><li>The stimulatory G-protein is permanently activated . </li></ul></ul><ul><li>Pertussis toxin (whooping cough disease) catalyzes ADP-ribosylation at a cysteine residue of the inhibitory G i  , making it incapable of exchanging GDP for GTP . </li></ul><ul><ul><li>The inhibitory pathway is blocked . </li></ul></ul><ul><li>ADP-ribosylation is a general mechanism by which activity of many proteins is regulated, in eukaryotes (including mammals) as well as in prokaryotes. </li></ul>
  8. 9. G Protein-Linked Receptors
  9. 10. Signal Transduction Components: Kinases/Phosphatases <ul><li>Kinases use ATP to phosphorylate amino acid side-chains in target proteins. Kinases typically are specific for tyrosine or serine/threonine sites. Phosphatases hydrolyze phosphates off of these residues. There are about 600 kinases and 100 phosphatase encoded in the human genome. Activation of most cell-surface receptors leads directly or indirectly to changes in kinase or phosphatase activity. Note that some receptors are themselves kinases (e.g., the insulin receptor). </li></ul>
  10. 11. Signal Trans. Components: 2nd Messengers <ul><li>While there are a large number of extracellular receptor ligands (&quot; first messengers &quot;), there are relatively few small molecules used in intracellular signal transduction (&quot; second messengers &quot;). In fact, only 6 second messengers occur in animal cells. </li></ul>
  11. 12. <ul><li>cAMP , </li></ul><ul><li>cGMP , </li></ul><ul><li>1,2-diacylglycerol ( DAG ), </li></ul><ul><li>inositol 1,4,5-trisphosphate ( IP 3 ) </li></ul><ul><li>calcium </li></ul><ul><li>phosphoinositides </li></ul>
  12. 14. GPCRs That Bind Epinephrine <ul><li>Epinephrine is a hormone that signals the &quot; fight-or-flight &quot; response. It elevates heart rate , dilates the airway , and mobilizes carbohydrate and lipid stores of energy in liver and adipose tissue. In the heart, liver, and adipose tissue, these effects are mediated via binding to ß 1 - & ß 2 - adrenergic GPCRs . Both ß-adrenergic GPCRs signal via G  s , which activates adenylyl cyclase and raises intracellular [ cAMP ]. The  2 - adrenergic GPCR signals via G  i , decreasing adenylyl cyclase activity and intracellular [cAMP]. </li></ul>
  13. 15. <ul><li>The  1 - adrenergic GPCR is coupled to G  q , which activates phospholipase C ( PLC ) and signaling via the IP 3 / DAG pathway .  1 -adrenergic GPCRs are present in the liver and blood vessels in peripheral organs. Binding to  1 -adrenergic GPCRs stimulates glycogen breakdown in the liver, while blood flow to peripheral organs is decreased. </li></ul>
  14. 16. GPCRs that Regulate Ion Channels: Muscarinic Acetylcholine Receptor <ul><li>The neurotransmitter, acetylcholine (ACH) binds to two types of receptors known as the nicotinic and muscarinic acetylcholine receptors . The nicotinic receptor is itself a ligand-gated ion channel that opens on ACH binding. This receptor is located in the neuromuscular junctions of striated muscle. The muscarinic ACH receptor, is a GPCR found in cardiac muscle cells that is coupled to an inhibitory G protein . </li></ul>
  15. 18. <ul><li>The binding of ACH to this receptor triggers dissociation of G i  -GTP from G ß  , which in this case, directly binds to and opens a K + channel. The movement of K + down its concentration gradient to the outside of the cell, increases the positive charge outside the membrane, hyperpolarizing the cell. This results in the slowing of heart rate. </li></ul>
  16. 19. Mechanism of Rhodopsin Signaling <ul><li>. Light absorption by rhodopsin triggers GTP/GDP exchange on the transducin G  t subunit, and dissociation of this trimeric G protein. G  t -GTP binds to and activates a cGMP phosphodiesterase , reducing intracellular cGMP level. This indirectly results in the closing of non-selective Na + /Ca 2+ ion channels in the cytoplasmic membrane and hyperpolarization of the membrane potential. This results in decreased release of neurotransmitter from the cells. Thus, light </li></ul>
  17. 20. <ul><li>is perceived by the brain due to a decrease in nerve impulses coming from rod cells. Studies have shown that only 5 photons must be absorbed per rod cell to transmit a signal. A single activated molecule of rhodopsin activates ~500 transducin molecules in a classic example of signal amplification. However, GTP is rapidly hydrolyzed by G  t , allowing for visual perception at ~1000 frames per second. </li></ul>
  18. 21. Visual Adaptation & Rhodopsin Signaling <ul><li>Rod cell signaling actually is reduced after prolonged exposure to high light intensity . This is apparent as a time delay during which vision is compromised when we move from bright light to a dark room. The change in sensitivity of our eyes to high and low light levels is known as visual adaptation . The biochemical mechanism by which adaptation primarily occurs Similar to what occurs in desensitization of ß-adrenergic receptors, activated rhodopsin is deactivated by rhodopsin kinase phosphorylation which increases at high light intensities. </li></ul>
  19. 23. <ul><li>Tri-phosphorylated rhodopsin is bound by arrestin , which completely inhibits transducin activation. Another mechanism of adaptation involves transport of transducin from the outer to the inner segments of rod cells during prolonged exposure to high light intensity. Visual images are formed due to differences in light intensity sensed by high- or low-light-intensity adapted rhodopsin molecules, and not due to absolute levels of illumination. </li></ul>
  20. 25. GPCRs that Regulate Adenylyl Cyclase <ul><li>Adenylyl cyclase is an effector enzyme that synthesizes cAMP. G  -GTP subunits bind to the catalytic domains of the cyclase, regulating their activity. G  s - GTP activates the catalytic domains, whereas G  i - GTP inhibits them. A given cell type can express multiple types of GPCRs that all couple to adenylyl cyclase. The net activity of adenylyl cyclase thus depends on the combined level of G protein signaling via the multiple GPCRs. In liver, GPCRs for epinephrine and glucagon both activate the cyclase. In adipose tissue (Fig. 15.21), epinephrine, glucagon, and ACTH activate the cyclase via G  s -GTP, while PGE 1 and adenosine inactivate the cyclase via G  i -GTP. </li></ul>
  21. 27. cAMP activates one or more kinases. What are phosphatases?
  22. 28. Adenylyl Cyclase & Protein Kinase A <ul><li>Adenylyl cyclase is an integral membrane protein that contains 12 transmembrane segments (Fig. 15.22). It also has 2 cytoplasmic domains that together form the catalytic site for synthesis of cAMP from ATP . One of the primary targets of cAMP is a regulatory kinase called protein kinase A ( PKA ), or cAMP-dependent protein </li></ul>
  23. 29. <ul><li>kinase. PKA exists in two different states inside cells (Fig. 15.23). In the absence of cAMP, the enzyme forms a inactive tetrameric complex in which 2 PKA catalytic subunits are non-covalently associated with 2 regulatory subunits . When cAMP concentration rises, cAMP binds to the regulatory subunits which undergo a conformational change, releasing the active catalytic subunits. </li></ul>
  24. 33. <ul><li>myocyte  ( skeletal muscle ) muscular system epinephrine  -->  β- adrenergic receptor </li></ul><ul><li>produce  glucose </li></ul><ul><ul><li>stimulate  glycogenolysis </li></ul></ul><ul><ul><ul><li>phosphorylate  glycogen phosphorylase  via phosphorylase kinase  (activating it) </li></ul></ul></ul><ul><ul><ul><li>phosphorylate  Acetyl-CoA carboxylase (inhibiting it) </li></ul></ul></ul><ul><ul><li>inhibit  glycogenesis </li></ul></ul><ul><ul><ul><li>phosphorylate   glycogen synthase  (inhibiting it) </li></ul></ul></ul><ul><ul><li>stimulate  glycolysis </li></ul></ul><ul><ul><ul><li>phosphorylate  phosphofructokinase 2 (stimulating it, cardiomyocytes only) </li></ul></ul></ul>
  25. 34. <ul><li>H epatocyte liver epinephrine  -->  β- adrenergic receptor </li></ul><ul><li>glucagon  -->  Glucagon receptor </li></ul><ul><li>produce  glucose </li></ul><ul><ul><li>stimulate  glycogenolysis </li></ul></ul><ul><ul><ul><li>phosphorylate  glycogen phosphorylase (activating it) [2] </li></ul></ul></ul><ul><ul><ul><li>phosphorylate  Acetyl-CoA carboxylase (inhibiting it) </li></ul></ul></ul><ul><ul><li>inhibit  glycogenesis </li></ul></ul><ul><ul><ul><li>phosphorylate  glycogen synthase  (inhibiting it) [2] </li></ul></ul></ul><ul><ul><li>stimulate  gluconeogenesis </li></ul></ul><ul><ul><ul><li>phosphorylate  fructose 2,6-bisphosphatase (stimulating it) </li></ul></ul></ul><ul><ul><li>inhibit  glycolysis </li></ul></ul><ul><ul><ul><li>phosphorylate  phosphofructokinase -2 phosphatase  (activating it) </li></ul></ul></ul><ul><ul><ul><li>phosphorylate  pyruvate kinase  (inhibiting it) </li></ul></ul></ul>
  26. 35. <ul><li>neurons  in  nucleus accumbens nervous system dopamine  -->  dopamine receptor Activate  reward system principal cells  in kidneykidney Vasopressin  -->  V2 receptor </li></ul><ul><li>theophylline  ( PDE inhibitor ) </li></ul><ul><li>exocytosis  of  aquaporin 2  to  apical membrane .  </li></ul><ul><li>synthesis of aquaporin 2  </li></ul><ul><li>phosphorylation of aquaporin 2 (stimulating it) </li></ul>
  27. 36. GPCRs That Activate Phospholipase C <ul><li>Another common GPCR signaling pathway involves the activation of phospholipase C ( PLC ). This enzyme cleaves the membrane lipid, phosphatidylinositol 4,5-bisphosphate ( PIP 2 ) to the second messengers, inositol 1,4,5-trisphosphate ( IP 3 ) and diacylglycerol ( DAG ). In this case, the G  o and G  q G  proteins conduct the signal from the GPCR to PLC. This is the pathway used in  1 -adrenergic GPCR signaling in the liver. </li></ul>
  28. 37. Activation of phospholipase C by protein-tyrosine kinases Phospholipase C-γ (PLC-γ) binds to activated receptor protein-tyrosine kinases via its SH2 domains. Tyrosine phosphorylation increases PLC-γ activity, stimulating the hydrolysis of PIP2.
  29. 38. The Formation of Inositol Triphosphate and Diacylglycerol PIP2 – phosphatidyl inositol-4,5-bisphophate IP3 – inositol-1,4,5-triphosphate PLC – phospholipase C DAG - diacylglycerol
  30. 40. IP 3 /DAG Signaling Pathway <ul><li>The steps downstream of PLC that make up the IP 3 /DAG signaling pathway. IP 3 diffuses from the cytoplasmic membrane to the ER where it binds to and triggers the opening of IP 3 - gated Ca 2+ channels . Another kinase, protein kinase C ( PKC ) binds to DAG in the cytoplasmic membrane and is activated. In liver, the rise in cytoplasmic [Ca 2+ ] activates enzymes such as glycogen phosphorylase kinase, which phosphorylates and activates glycogen phosphorylase. Glycogen phosphorylase kinase is activated by Ca 2+ - calmodulin . In addition, PKC phosphorylates and inactivates glycogen synthase. </li></ul>
  31. 41. DAG
  32. 42. Inositol Triphosphate 2 nd Messenger
  33. 43. The Structure and Function of the Calcium-Calmodulin Complex Kinases Phosphatases
  34. 44. Nitric Oxide (NO)/cGMP Signaling <ul><li>A related signaling pathway involving phospholipase C operates in vascular endothelial cells and causes adjacent smooth muscle cells to relax in response to circulating acetylcholine . In the NO/cGMP signaling pathway , the downstream target of Ca 2+ /calmodulin is nitric oxide synthase , which synthesizes the gas NO from arginine. NO diffuses into smooth muscle cells and causes relaxation by activating guanylyl cyclase and increasing [ cGMP ]. As a result arteries in tissues such as the heart dilate, increasing blood supply to the tissue. NO also is produced from the drug nitroglycerin which is given to heart attack patients and patients being treated for angina . </li></ul>
  35. 46. Responding to the Signal: Effector Proteins Cytoskeletal Rearrangement Changes in gene expression Cell Cycle Arrest Effector Protein <ul><li>The final step in cell signaling is activation of the effector proteins </li></ul><ul><li>The effector proteins carry out the cellular response to the signal </li></ul><ul><li>Often the cellular response involves expression of previously inactive genes which requires effector proteins called transcriptional activators or transcription factors </li></ul><ul><li>Transcription factors are proteins that bind to specific DNA sequences called promoters that are upstream of the genes that are turned on </li></ul><ul><li>Promoters that are upstream of genes that are only activated during specific cellular responses are called response elements </li></ul><ul><li>Effector proteins can also directly act on proteins that regulate cell shape to induce changes in morphology by rearranging the cytoskeleton </li></ul><ul><li>Other types of effector proteins directly regulate cell growth by arresting the cell cycle or altering cellular metabolism </li></ul>Effector protein
  36. 47. <ul><li>Thanks </li></ul>