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3.17.2010 – Cell Signaling Continued<br /><ul><li>Preface:
Smooth muscle dialates in response to the peripheral NS relesasing neurotransmitter acetylcholine
Acetylcholine receptor is on endothelial cell, and Ach binds to it.  That endothelial cell synthesizes NO which diffuses a...
Signaling Pathway for smooth muscle relaxation
Peripheral NS sends signals to cells to allow vessel dialtion
Endothelial cells line interior of blood vessels
Smooth muscle cells relax for dilation
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  1. 1. 3.17.2010 – Cell Signaling Continued<br /><ul><li>Preface:
  2. 2. Smooth muscle dialates in response to the peripheral NS relesasing neurotransmitter acetylcholine
  3. 3. Acetylcholine receptor is on endothelial cell, and Ach binds to it. That endothelial cell synthesizes NO which diffuses across membrane into muscle which is bound by NO receptor, which then allows smooth muscle relaxation.
  4. 4. Signaling Pathway for smooth muscle relaxation
  5. 5. Peripheral NS sends signals to cells to allow vessel dialtion
  6. 6. Endothelial cells line interior of blood vessels
  7. 7. Smooth muscle cells relax for dilation
  8. 8. Acetylcholine binds to acetylcholine receptor – A G protein linked receptor
  9. 9. What that means is that this receptor has a particular structure to interact with Heterotrimeric G proteins -
  10. 10. G protein linked receptor – active subunit binds GTP when turned on and hydrolyzes to form GDP for inactivity
  11. 11. Bind to and active heterotrimeric G proteins, which is made of three subunits:
  12. 12. Alpha – larger of three subunits
  13. 13. Binds GTP and GDP (reason why it’s called a G protein)
  14. 14. Active when bound to GTP, but GTP will only be in GTP state for so long – androgenous hydrolase activity, hydrolysis takes place and GDP bound state as Pi is removed.
  15. 15. Beta
  16. 16. Gamma
  17. 17. Heterotrimeric proteins are all different and their alpha/beta/gamma subunits have different functions
  18. 18. Can activate or inactivate enzymes
  19. 19. The alpha subunit always binds GTP, but when there is separation and diffusion through the membrane it depends on the type of HG-protein to which protein the alpha or beta/gamma subunit will be doing the next action
  20. 20. In active state, beta/gamma subunit and alpha subunit separate and the beta and gamma act as a dimer
  21. 21. Alpha then – binds to GDP and hydrolyzes to GTP
  22. 22. When bound by Ach, GTP is bound by alpha subunit that makes cascading reactions that leads to hydrolysis of PIP2 in the membrane
  23. 23. PIP2 – phosphoinositol bisphosphate – membrane phospholipid
  24. 24. After receptor is activated, cleaved by enzyme into 2 types of signaling molecules
  25. 25. IP3
  26. 26. Inositol tri-phosphate
  27. 27. Binds to receptors on ER called IP3 receptors
  28. 28. When bound to IP3 receptor, opens channel and releases Ca2+ from ER and increases concentration of Ca2+ in the cell.
  29. 29. Ca2+ is an important signaling molecule
  30. 30. Binds to calmodulin
  31. 31. Calmodulin actives NO synthase
  32. 32. Diacylglycerol
  33. 33. Sits by self in membrane
  34. 34. Nitroglycerine
  35. 35. Inhibits calcium influcx into cells. Taken for heart attacks.
  36. 36. Summary: Signaling Pathway for Smooth Muscle Relaxation
  37. 37. Ach binds to Ach receptor
  38. 38. Binding to the receptor induces conformational change on receptor that allows interaction with heterotrimeric G protein
  39. 39. Interaction of the heterotrimeric G protein activates G protein allowing alpha subunit to bind GTP
  40. 40. Alpha subunit binding to GTP causes a release of the alpha unit from beta/gamma subunit
  41. 41. The beta/gamma subunit activates an enzyme near the plasma membrane of cell, and allows that enzyme to cleave phospholipids in the membrane
  42. 42. PIP2 – Phosphoinositol bisphospahte, main component of phospholipid membrane, is cleaved by an enzyme that breaks it into two important signaling molecules
  43. 43. Diacyl glycerol
  44. 44. IP3
  45. 45. Inositol triphosphate
  46. 46. Diffuses through cytosplasm to bind to IP3 receptors on the smooth ER.
  47. 47. Smooth ER serves as a storage site for calcium
  48. 48. Receptor opens up and allows Ca2+ channel to open and allow it into the cytosol, so intracellular calcium levels increase
  49. 49. The Ca2+ is immediately bound up by the calcium binding proteins, in this case called calmodulin – 4 Ca2+ opens up calmodulin structure so it can interact with other proteins
  50. 50. One result of this is the activation of NO synthase – so now we have increased levels of active NO synthase, and the NO synthase will convert arginine to NO.
  51. 51. NOTE:: All of these steps occur within the cytoplasm of the endothelial cell after Ach binds to the receptor in order to just make NO, which can diffuse across the membrane and lead to relaxation.
  52. 52. Activated heterotrimeric G protein turns on enzyme to cleave PIP2 in membrane
  53. 53. When NO Diffuses across membrane to smooth muscle cell, it also binds to receptor for NO and then from there, the signaling continues to allow the smooth muscle to relax, this is not a direct effect
  54. 54. Binding of NO to receptor activates an enzyme called guananyl cyclase, which converts GTP to cGMP, by removing two Pi.
  55. 55. cGMP, like cAMP, is an important signaling molecule
  56. 56. in this case, 2 bind and activate protein kinase G by binding it
  57. 57. many kinases have specific conformation for catalytic sites for kinase functions (phosphorylation of targets) until regulatory sites are bound and in this case, the reg. sites are bound by cGMP, and when they are bound, changes confo. Of proteins to open to catalytic sites are open and can recognize targets.
  58. 58. Phosphorylates Ca2+ channel in smooth muscle
  59. 59. Phosphorylation of the channel inactivates it.
  60. 60. When inactive, Ca2+ cannot, extracellular Ca2+ channels in the plasma membrane cannot open when phosphorylated, come into EXC space form smooth muscle. Induces relaxaition, Ca2+ is required for muscle contraction, so with low Ca2+ levels, smooth muscle is forced to relax. Muscle relaxation is caused by phosphorylation of Ca2+ channel.
  61. 61. Eventually will be dephosphorylated by phosphatases and the smooth muscle can contract
  62. 62. As long as protein kinase G is active, you have relaxation of smooth muscle.
  63. 63. Controlled by cGMP, which is controlled by levels of NO
  64. 64. Nitroglycerine
  65. 65. Phosphorylates Ca2+ channels.
  66. 66. Inhibits calcium influcx into cells. Taken for heart attacks.
  67. 67. Relaxes blood vessels and allows heart to work less hard to pump blood.
  68. 68. Blood pressure drops to lower levels
  69. 69. Small hydrophobic signaling molecules – Ligand receptor binding example
  70. 70. Move directly through membrane and interact with intracellular receptors
  71. 71. Previous examples regulated channel function and enzyme function and activity in cell, not gene function
  72. 72. Steroid hormones
  73. 73. Bind intracellular receptors and form transcription factors, which regulate levels of gene transcription
  74. 74. Small, hydrophobic, bind
  75. 75. Cortisol
  76. 76. Great example of signaling – regulates a host of activities
  77. 77. Kidney function
  78. 78. Immune system
  79. 79. Number of blood cells (primarily WBC)
  80. 80. Sleep patterns
  81. 81. Stress hormone
  82. 82. Where it begins
  83. 83. Hypothalamus secretes hrmones that activate pituitary
  84. 84. Pituitary releases ACTH – adrenocorticotropin hormone
  85. 85. Circulates through blood system tostimulate adrenal cortex
  86. 86. Adrenal cortex (anterior to kidneys – on top) produces cortisol
  87. 87. Released into blood stream
  88. 88. Intracellular receptor sits in cytoplasm in an inactive state until bound by cortisol that diffuses across membrane
  89. 89. When binding, confo change and exposes a nuclear localization signal that is bound by importin,
  90. 90. Complex carried to nucleus and acts as a transcription factor to regulate gene transcription in those cells with that receptor
  91. 91. Now what types of proteins made is regulated
  92. 92. Cortisol is so important because it regulates so many functions
  93. 93. If levels are abnormal:
  94. 94. Increased levels of cortisol results from too much ACTH production or overactive cortisol production – Cushing Syndrome – (10-15 per million people)
  95. 95. Difficult to regulate because trying to eliminate cortisol is more detrimental than not having enough
  96. 96. Hyperactive immune system, autoimmune, kidney function
  97. 97. Decreased cortisol – Addison’s Disease
  98. 98. Caused as a result of tuberculosis or fungal infection
  99. 99. Immune system tries to deal with either of these and creates granulomas – sites where healthy cells are killed in the process of trying to deal with the infection
  100. 100. Reduces size and function of adrenal cortex. Replaces with immune cell masses
  101. 101. Therefore can’t make as much cortisol
  102. 102. Dehydration due to kidney functions
  103. 103. Lack of immune cell function
  104. 104. Concurrent infection
  105. 105. Synthetic cortisol is taken
  106. 106. Estradiol
  107. 107. Sex hormone
  108. 108. Produced by ovaries and prepares egg/uterine lining for implantation and fertilization
  109. 109. Testosterone
  110. 110. Thyroxine
  111. 111. produced by thyroid and regulates metabolic rates of growth in humans
  112. 112. Signal Transduction Pathways
  113. 113. Movement in step-wise pattern to activate target protein
  114. 114. Beneficial because sometimes intracellular signaling components serve as amplification steps
  115. 115. Beneficial because provides a mechanism of regulation at each successive step
  116. 116. Intracellular signaling proteins are also regulated
  117. 117. At any time an stop or shut down/slow down
  118. 118. End result: changed or will change cellular response
  119. 119. Target proteins
  120. 120. Metabolic enzymes
  121. 121. Skeletal proteins leading to changes in cell structure or motility
  122. 122. Gene transcription, or proteins produced
  123. 123. Two very important components of signaling part of the intracellular signaling protein pathways are molecular switches, called as such because they work just like a light switch.
  124. 124. Kinase activation
  125. 125. Kinases are on when phosphorylated and off when phosphatases dephosphorylate target
  126. 126. Small G protein activation (GTP binding protein)
  127. 127. When bound to GTP – active
  128. 128. Regulated by GEFS – guanine nucleotide exchange factors squeeze out GDP and allow GTP to bind for activation
  129. 129. When bound to GDP – inactive
  130. 130. GAPs – GTPase activating proteins
  131. 131. Enhance hydrolysis and found in cyotplasm to help regulate off state of Small G proteins
  132. 132. will not link up with G protein linked coupled receptors - they are molecular switches within intracellular signaling pathways
  133. 133. Molecular switches will not link up with G protein-linked coupled receptors like heterotrimeric G protens.
  134. 134. Categories of Receptors
  135. 135. Ion-channel-linked receptors
  136. 136. Transmembrane receptors that bind a ligand.
  137. 137. They themselves will then open up to allow ions to move through
  138. 138. IP3 receptors, previously talked about
  139. 139. Receptors bind IP3, and calcium goes out
  140. 140. G-protein-linked receptors
  141. 141. Acetylcholine receptor that binds Ach and activates an enzyme that cleaves PIP2 in membrane.
  142. 142. Family of receptors that are transmembrane,
  143. 143. Have seven transmembrane spanning domains,
  144. 144. A.k.a. serpentine-7-receptors
  145. 145. Interact with heterotrimeric G proteins, the alpha/beta/gamma subunit proteins and NOT the small G proteins that are single subunit proteins that bind GTP or GDP.
  146. 146. Small g proteins are a single subunit protein
  147. 147. Heterotrimeric proteins have three subunit and they interact with the G protein linked serpentine 7 receptors.
  148. 148. When activated, will be bound to GTP by the alpha subunit
  149. 149. Activated subunit of heterotrimeric g protein will then turn on enzyme activity
  150. 150. Ach example: turned on enzyme that cleaved PIP2 in the membrane
  151. 151. Enzyme-linked Receptors
  152. 152. Often requires a dimer, a ligand that exists as a dimer to bind two transmembrane components of the receptor
  153. 153. Receptor isn’t functional until those components are brought together
  154. 154. Usually exist as single transmembrane-spanning G proteins that are brought together by ligand binding
  155. 155. After being brought together, induces conformational change to allow two subunits to communicate with each other.
  156. 156. Usually by cross phosphorylation or autophosphorylation where one half of the receptor is phosphorylated by the other half.
  157. 157. In some cases that will activate kinases or other types of proteins
  158. 158. Small GTP binding proteins are activated downstream of enzyme-linked receptors.
  159. 159. Using the example of g-protein linked receptors to differentiate between the two types of proteins
  160. 160. G-protein linked receptors have two important regions
  161. 161. Extracellular loop between 6 and 7
  162. 162. Binds ligand on extracellular side
  163. 163. Cytoplasmic region between transmembrane regions 5 and 6
  164. 164. Exposed after ligand binding (confo change) that will interact with heterotrimeric g protein in plasma membrane
  165. 165. Sits in an inactive state in the membrane where the alpha subunit is bound to GDP
  166. 166. Difference between heterotrimeric g proteins and small g proteins is that the heterotrimeric g proteins are linked to the membrane and small g proteins are not – they are solube proteins in the cytoplasm.
  167. 167. 3 subunits function as an inactive proteins when alpha is bound to GDP
  168. 168. when loop is exposed on receptor, the heterotrimeric g protein moves as a unit and binds to loop
  169. 169. alpha subunit underges a conformational change exposing a site for GTP binding (GEFs come in)
  170. 170. GTP when bound, leads to activation of heterotrimeric g protein, which separates the alpha beta/gamma subunits and the two separate subunits (alpha and beta/gamma) are activated
  171. 171. Next activation step whatever it is.
  172. 172. Differences Summary
  173. 173. Heterotrimeric G proteins – trimeric protein with three subunits that make a single functional proteins where alpha subunit interacts with GTP and GDP.
  174. 174. Structures
  175. 175. Linked to membrane
  176. 176. Interacts with transmembrane G protein linked serpentine 7 coupled receptors.
  177. 177. Small G proteins
  178. 178. Soluble proteins in cytosol
  179. 179. Single subunit that binds GTP or GDP (active inactive respectively)
  180. 180. Do NOT interact with transmembrane G protein linked serpentine 7 coupled receptors.
  181. 181. Will interact with enzyme linked receptors or within those pathways
  182. 182. Ran/Rab/Rac/Rho/Cdc42 (know)