2. Ran

3. Rac

4. Rho

5. Cdc42

6. Heterotrimeric G Proteins

7. Alpha – Binds GTP, or GDP, and will hydrolyze GTP to GDP

8. Beta

9. Gamma

10. Bind to serpentine 7 receptors, which activate heterotrimeric G proteins

11. Embedded into membrane

12. Cytosolic side of receptor is the binding site for heterotrimeric g proteims, which contains a loop between strands 5 and 6 and ligand binding domain is between 6 and 7. – extracellular

13. When ligand binds to it, it exposes an activation site (binding site) for heterotrimeric g proteins, which is linked to the membrane (meristic acid that links protein to membrane) and when exposed, interacts with HTGP

14. Inactive G Proteins (alpha beta gamma)

15. Once the alpha subunit is removed, the enzyme is inactive. When the alpha subunit is bound to GDP, it no longer associates with the enzyme and it exposes binding sites for B/G subunit, so they can all come together.

16. Signaling Pathways

17. Alpha subunit

18. As long as active subunit is bound, the enzyme will continue intracellular signaling.

19. When bound to GTP, it releases itself from B/G and the activation sites for target proteins/enzymes are now ready to interact

20. Interacts with target proteins and are activated as long as alpha subunit is bound

21. Hydrolysis of GTP

22. GDP makes inactive

23. How activation takes place

24. One ligand binds receptor

25. One h-t-g-p interacts with receptor

26. One alpha subunit acts with proteins

27. Enzymes provide an amplification step

28. Table

29. Gs heterotimeric G proteins aka alpha s

30. Stimulates the function of adenylyl cyclase (a.k.a. adenylate cyclase) – designation for alpha subunit of this g protein.

31. Referred to as alpha-s, when bound to GTP, moves through membrane, attaches to adenylyl cyclase and turns it on. Therefore, that leads to an increase in cAMP in the cell

32. Adenyly cyclase converts ATP to cAMP

33. An Active Gs subunit

34. Alpha subunit is active and bound to GTP

35. Alpha subunit migrates to adenylyl cyclase and turns on its function

36. Adenylyl cyclase removes a pyrophosphate (2) from ATP and make cAMP, a favorable reaction by breaking the phospoanhydride bonds holding the Pi on to the ATP

37. Cyclic form is unstable, being formed to AMP

38. Necessary so it can be turned off rapidly.

39. Golf

40. Olfactory neurons - activates adenylyl cyclase

41. Alpha subunit is activation subunit

42. Different protein because it’s in a different cell type.

43. GI (aka alpha I subunit on Gi protein that carries the function.

44. Inhibitory/inhibiting – also characterize the alpha subunit as being the

45. Inhibits adenylyl cyclase therefore decreasing cAMP

46. In some cells B/G activates K+ channels

47. Go

48. Heterotrimeric (as are all) that does different things in different cells, where in some cases the B/G is only active, but sometimes both A and B/G participate in target activation

49. Beta gamma turns on or opens up K+ channels in the PM allowng K+ to rush out of cell

50. When A and B/G are both active, Phospholipase c-beta is turned on

51. PLCB or PLCG both cleave PIP2 (generating DAG that remains attached to membrane and releases IP3 into the cytosol to bind to IP3 receptors in the ER) in the membrane

52. PLCB activation

53. HTGprotein called G0 that is turned on after ligand binds to g protein linked receptor – g protein linked receptors

54. PLCG activation is downstream of enzyme linked receptor.

55. G1 – transducin

56. Processes visual information

57. Structure and Metabolism of cAMP

58. Can generate kinases

59. Ligand binds to erceptor

60. The Roles of G Proteins and Cyclic AMP in Signal Transduction

61. How cAMP is generated by G proteins

62. Ligand binds

63. Activation of heterotrimeric G protein where alpha is bound to GTP

64. Moves through membrane and interacts with a single molecule of adenylyl cyclase, which is also found in inner leaflet of PM

65. Activates enzymatic activity

66. Converts ATP to cAMP

67. Used for signaling

68. Rapidly degraded by phosphodiesterase to convert it to AMP

69. Release energy

70. When alpha subunit has a GDP attached, the G protein becomes inactive and production of cAMP stops sits in membrane in wait for another signal

71. What does an increased level of cAMP mean?

72. cAMP binds to a kinase, called protein Kinase A, which sits in cytosol in inactive state until bound by cAMP. Similar to cGMP. PKG and PKA both have a structure so they have regulatory sites that can be bound by an intracellular signaling protein

73. when they are bound, open up and release catalytic site that does the phosphorylating.

74. cGMP, activates PKG, synthesized by guanylyl cyclase

75. for PKA, two molecules of cAMP must bind to activate

76. adenylyl cyclase activates cAMP

77. Once PKA or PKG is activated we have catalytic sites that are turned on ready for phosphorylation, and both target serine and threonine as target proteins.

78. Recognize the amino acids on either side of the phosphorylated threonine/serine really.

79. Kinases use particular sequences to indicate a particular site of phosphorylation

80. When PKA active, bound by cAMP, exposes NLS, and be transported to nucleus, and catalytic site

81. When in the nucleus, the active PKA will Pi target.

82. Important target is CREB – cyclic AMP response element binding protein

83. When phosphorylated by active PKA, regulates gene transcprition by binding to regions on DNA called CRE, or cyclic AMP response element.

84. CREB binds to CRE, bind to sequence of DNA and geulate gene transcription

85. In cardiac muscle cells, activation of this pathway increases strength of contraction

86. In smooth muscle cells, decrease strength of contraction

87. Neurons – activation of CREB leads to synthesis of neurotrophins, or molecules that help other neurons survive

88. Liver cells – glycogen synthesis and usage is enhanced by activation of PKA, Pi of CREB and regulation of gene transcription

89. Important: pathway is the same, but different outcomes.

90. Can be regulated and adenylyl cyclase can be turned off by alpha subunit binding.

91. Levels of cAMP by turning on or off adenylyl cyclase

92. Also for cAMP regulation: active phosphodiesterase will break down cyclic AMP to AMP, and inactivation will cause cAMP to stick around to active more PKA. Synthesis and degredation is regulatable.

93. Phospholipid signaling

94. Cleavage of PIP2

95. Cleaves by phospholipase C Beta or Gamma

96. PLCB – through g protein activation

97. PLCG – through enzyme linked receptor

98. Phospholipase enzyme targets bond between phosphate of inositol ring and oxygen releasing inositiol triphosphate and diacyl glycerol.

99. IP3 binds to IP3 receptors to release Ca2+ from intracellular storage

100. DAG and IP3 signaling work together to turn on a kinase in a cell

101. Activation of these leads to regulation of blood platelets

102. Smooth muscles

103. Insulin secretion

104. Amylase secretion

105. Glycogen degradation

106. Antibody production

107. G protein linked receptor that binds ligand will have a heterotrimeric g proteinactive

108. Activated alpha will move through membrane and activate PLCB that exposes cleavage site that can cleave phosphoinositol bisphosphosphate into DAG and IP3 signaling molecule

109. As IP3 moves to receptors on intracellular Ca2+ storage sites, it binds to IP3 receptors and it itself opens up and allow calcium to move to the cytosol. (it is an ion channel, the receptor)

110. DAG and Ca2+ will activate PKC inside the cell.

111. PKC enzyme phosphorylates serine and threonine is activated by DAG in phospholipid bilayer, binding of PS (phosphatidalserine, inserted in PM and flipped to inner leaflet) in the lipid bilayer and the binding of calcium that is required for activation of PKC

112. IP3 signaling pathway is a main way that levels of Ca2+ increase for active PKC

113. Once active, it can Pi its targets

114. Importance of Calcium

115. Uses ATP dependent/independent mechanisms for regulation

116. Sodium/calcium exchanger at PM for Na into cell used to pump Ca out of cell

117. CA2+ pump that pumps out calcium out of the cell at all times or into the ER

118. Ca is in the matrix of mitochondria too

119. Very local signaling molecule, will happen right on the PM etc

120. Calmodulin has a complex carboxy and amino termini. Within globular ends, there are two binding sites for calcium so four calcium molecules are bound by calmodulin in cytoplasm and is then activated

121. Once activated can form kinase, which can wrap itself around a phosphorylating enzyme and stays attached to it. Ca calmodulin complex is PART OF THE KINASE SO IT CANNOT FUNCTION WITHOUT THE ENZYME

122. CAM Kinasae – calcium calmodulin kinase

123. Aka Calcium dependendent calmodulin kinase

124. Can then Pi targets

126. 2 cAMP add to protein kinase A

127. activated in the same way as protein kinase G

128. phosphorylates serine and threonine on target substrates.

129. Active PKA (in nucleus)

130. Phosphorylates CREB (cycle AMP response element binding protein)

131. Interacts with sites on the genome that have been called CRE sites. (cAMP response elements)

132. This interaction drives gene transcription

133. Skeletal and liver cell, increase glycogen, synthesis and usage

134. Smooth muscle cells, decrease strength of contraction

135. Cardiac muscle increases when pathway activated

136. The cell can monitor the concentration of cAMP by the regulation of phosphodiesterase

137. Cleavage of PIP2 in the Membrane

138. Phosphoinositolbisphosphate (PIP2)

139. Platelet Activation (table slide)

140. Once PKC isactive, PKC Pi’s targets, and these targets are also serine and threonine sites on target proteins.

141. Ca2+ is important for intracellular signaling.

142. Exchangers keep Ca2+ low in cell

143. Na+/Ca2+ pump

144. Pumps my hydrolyze ATP to bring Ca2+ out of the cell

145. Calmodulin is activated by 4 Ca2+

146. CaM Kinase only active when calmodulin wraps around it, only when it is bound by Ca2+ forming the Ca2+-CaM Kinase