Constitutive Activity in Receptors and Other Proteins, Part A

1. 1. Introduction 2. 2. Modifying CB1 Activity 3. 3. Behavioral Models 4. Detecting Constitutive Activity and Protean Agonism at Cannabinoid-2 Receptor 5. 1. Introduction 6. 2. General Considerations 7. 3. Evaluation of Constitutive Activity Using GTPγS Assay 8. 4. Evaluation of Constitutive Activity Using cAMP Assay 9. 5. Evaluation of Constitutive Activity Using RT-CES 10. 6. Evaluation of Protean Agonism with cAMP Assay 11. 7. Comparison of the Methods 12. Modulation of the Constitutive Activity of the Ghrelin Receptor by Use of Pharmacological Tools and Mutagenesis 13. 1. Introduction 14. 2. The Ghrelin Receptor and Its Constitutive Activity 15. 3. Structural Basis of Constitutive Activity 16. 4. Residues Responsible for the Inverse Agonism and Efficacy Swap 17. 5. Physiological Relevance of Constitutive Activity 18. 6. Experimental Procedures 19. Assessment of Constitutive Activity and Internalization of GPR54 (KISS1-R) 20. 1. Introduction 21. 2. Materials 22. 3. Methods 23. Acknowledgment 24. Assessment of Constitutive Activity in E-Type Prostanoid Receptors 25. 1. Introduction 26. 2. Assays Used to Assess EP3 Receptor Constitutive Activity 27. 3. Assays Used to Assess EP4 Receptor Constitutive Activity 28. α1D-Adrenergic Receptors 29. 1. Introduction 30. 2. Methods 31. 3. Constitutive Activity 32. 4. Plasma Membrane α1D-Adrenergic Receptors 33. 5. Possible Physiological Implications 34. Acknowledgments 35. Constitutive Activity of the Histamine H1 Receptor 36. 1. Introduction 37. 2. Methods to Study Constitutive H1R Signaling 38. 3. Constitutive Activity as Tool to Elucidate Receptor Activation and Crosstalk 39. 4. Conclusion 40. Acknowledgments 41. Constitutive Activity of Somatostatin Receptor Subtypes 42. 1. Introduction 43. 2. Choosing Cells to be Studied 44. 3. Modifying SSTR Density 45. 4. Summary 46. Assessment of Homologous Internalization of Constitutively Active N111G Mutant of AT1 Receptor 47. 1. Introduction 48. 2. Preparation of Receptor Plasmid and Protein 49. 3. Radioligand Binding Assay 50. 4. Inositol Phosphate Accumulation Assay 51. 5. Internalization Assay 52. 6. Western Blot Analysis 53. 7. Data Analysis 54. 8. Concluding Remark 55. Methods to Detect Cell Surface Expression and Constitutive Activity of GPR6 56. 1. Introduction 57. 2. GPR6 is Expressed in Intracellular Compartments 58. 3. Comparison of the Cell Surface Protein Detection Methods 59. 4. Comparison of the Constitutive Gs-Activity Detection Methods 60. 5. Conclusions 61. β3-Adrenoceptor Agonists and (Antagonists as) Inverse Agonists 62. 1. Introduction 63. 2. β3-Adrenoceptor 64. 3. Methodologies 65. Acknowledgments 66. Constitutive Activity of the Lutropin Receptor and Its Allosteric Modulation by Receptor Heterodimerization 67. 1. Introduction 68. 2. General Principles for Quantifying Receptor Activation 69. 3. Modifying Cell Surface Expression Levels of Recombinant hLHR and Mutants Thereof 70. 4. Quantifying Cell Surface hLHR Expression 71. 5. Quantifying cAMP Production in Cells Expressing the hLHR 72. 6. Experimental Strategies for Characterizing the Attenuating Effects of a Signaling Inactive hLHR on a Coexpressed wt or CAM hLHR 73. Acknowledgment 74. Assessing Constitutive Activity of Extracellular Calcium-Sensing Receptors In Vitro and in Bone 75. 1. Introduction 76. 2. Materials and Methods 77. 3. Results 78. 4. Conclusions 79. Acknowledgments 80. Constitutive Activity of Neural Melanocortin Receptors 81. 1. Introduction 82. 2. Signaling Assay for the Neural Melanocortin Receptors 83. 3. Naturally Occurring Constitutively Active MC4R Mutants 84. 4. Inverse Agonism of AgRP at the MC3R 85. 5. Computational Modeling of the Constitutively Active MC4R Mutants 86. Acknowledgments 87. Measurement of Constitutive Activity of BMP Type I Receptors 88. 1. Introduction 89. 2. Determining ALK2 Constitutive Activity 90. 3. Determining the Effects of ALK2 Constitutive Activity on Osteoblast Differentiation 91. 4. Concluding Remarks 92. Acknowledgments 93. Probing the Constitutive Activity Among Dopamine D1 and D5 Receptors and Their Mutants 94. 1. Introduction 95. 2. Design of Genetically Modified D1-Like Receptor Constructs and Cloning Strategy 96. 3. Transfection of D1R and D5R Expression Constructs in HEK293 Cells 97. 4. Radioligand-Binding Assays 98. 5. Whole Cell cAMP Assays 99. 6. Results Validating Experimental Approaches 100. 7. Concluding Remarks 101. Acknowledgments 102. Identification of Gain-of-Function Variants of the Human Prolactin Receptor 103. 1. Introduction 104. 2. Experimental Procedures 105. 3. Identification of Constitutive Activity: Results and Discussion 106. 4. Conclusions 107. Acknowledgments 108. Investigations of Activated ACVR1/ALK2, a Bone Morphogenetic Protein Type I Receptor, That Causes Fibrodysplasia Ossificans Progressiva 109. 1. Introduction 110. 2. Patient Methodologies 111. 3. Cellular Methodologies 112. 4. Tissue Methodologies 113. 5. In Vivo Methodologies 114. Acknowledgments 115. Identification and Evaluation of Constitutively Active Thyroid Stimulating Hormone Receptor Mutations 116. 1. Introduction 117. 2. TSHR Gene Mutational Screening 118. 3. Determination of TSHR Constitutive Activity In Vitro 119. 4. Measurement of TSHR Expression at Cell Surface by Flow Cytometry Analysis 120. 5. TSH–TSHR Binding Assays 121. 6. TSHR Phosphorylation Analysis 122. Acknowledgments 123. Assessment of Constitutive Activity of a G Protein-Coupled Receptor, Cpr2, in Cryptococcus neoformans by Heterologous and Homologous Methods 124. 1. Introduction of Receptors and Constitutive Receptors 125. 2. Identification of Cpr2 as a Natural Occurring Constitutively Active Receptor 126. 3. Additional Constitutively Active Receptors Identified in Fungi 127. Acknowledgments 128. In Vitro and In Vivo Assessment of Mu Opioid Receptor Constitutive Activity 129. 1. Introduction 130. 2. Measuring Opioid Receptor Constitutive Activity In Vitro 131. 3. cAMP Quantification Assay in CHO Cells Expressing Cloned Opioid Receptors 132. 4. In Vivo Assessment of Antagonist Potency in Opioid Naïve Subjects 133. 5. In Vivo Assessment of Antagonist Potency to Precipitate Withdrawal 134. 6. Summary 135. Acknowledgments 136. Constitutively Active μ-Opioid Receptors 137. 1. Introduction 138. 2. Methods for Measuring Constitutive Activity 139. 3. Conclusions 140. Acknowledgments 141. Protein Kinase CK2 is a Constitutively Active Enzyme that Promotes Cell Survival: Strategies to Identify CK2 Substrates and Manipulate its Activity in Mammalian Cells 142. 1. Introduction 143. 2. Purification of CK2 for in Vitro Studies 144. 3. Assays for CK2 Activity 145. 4. Modulation of CK2 in Mammalian Cells 146. 5. Conclusions 147. Acknowledgments 148. Assessment of CK2 Constitutive Activity in Cancer Cells 149. Abbreviations 150. 1. Introduction 151. 2. Assay of CK2 in Crude Biological Samples 152. 3. In-Cell Assay of Endogenous CK2 Activity 153. 4. Identification/Validation of In Vivo CK2 Targets with Specific Inhibitors 154. Acknowledgments 155. Structural Basis of the Constitutive Activity of Protein Kinase CK2 156. 1. Introduction 157. 2. A Constitutively Active CK2α Structure and Its Stabilizing Elements 158. 3. Analyzing the Constitutive Activity of Protein Kinase CK2 159. Acknowledgments 160. Measuring the Constitutive Activation of c-Jun N-terminal Kinase Isoforms 161. 1. Introduction 162. 2. Important Reagents for Studying JNK Activity 163. 3. Protein Expression and Purification of JNK Proteins and c-JUN 164. 4. Measuring the Autophosphorylation Ability of the JNK Isoforms 165. 5. Determining the Kinase Activity of the JNK Isoforms 166. 6. Monitoring the Formation of JNK Homodimers 167. 7. Measuring Nuclear Translocation of JNK Protein 168. 8. Future Directions 169. Acknowledgments 170. Measurement of Constitutive MAPK and PI3K/AKT Signaling Activity in Human Cancer Cell Lines 171. 1. Introduction 172. 2. Maintaining Melanoma Cell Lines 173. 3. Western Blotting 174. 4. Phospho-Flow Cytometry 175. 5. Immunofluorescence 176. 6. Conclusions 177. Acknowledgments 178. Constitutive Activity of GPR40/FFA1 179. Abbreviations 180. 1. Introduction 181. 2. Measuring FFA1-Mediated Calcium Mobilization 182. 3. Measuring Direct Activation of G Proteins via FFA1 183. Constitutive Activity of TRP Channels 184. 1. Introduction 185. 2. TRP Channels and Cellular Degeneration 186. 3. Constitutive TRP Channel Activity Which Does Not Lead to Cellular Degeneration 187. 4. Constitutive TRP Channel Activity Which Leads to Cellular Degeneration 188. Acknowledgments 189. Measurement of Orexin (Hypocretin) and Substance P Effects on Constitutively Active Inward Rectifier K+ Channels in Brain Neurons 190. 1. Introduction 191. 2. Dissociated Culture of Cholinergic Neurons in the Basal Forebrain 192. 3. Effects of Orexin (Hypocretin) and Substance P on Constitutively Active Inward Rectifier K+ (KirNB) Channels 193. 4. Signal Transduction of Substance P and Orexin Effects on KirNB Channels 194. Acknowledgments 195. Characterization of G Protein-Coupled Receptor Kinase 4 and Measuring Its Constitutive Activity In Vivo 196. 1. Introduction 197. 2. Selection of Cells/Model Systems to Study GRK4 Function 198. 3. Generation of Kinase Dead-GRK4 199. 4. Functional Characterization of GRK4 Constitutive Activity 200. 5. Agonist-Mediated GRK4 Activity 201. 6. Summary 202. Acknowledgments 203. Voltage-Clamp-Based Methods for the Detection of Constitutively Active Acetylcholine-Gated IK,ACh Channels in the Diseased Heart 204. 1. Introduction 205. 2. Recording of Constitutive IK,ACh Using Patch-Clamp Techniques 206. 3. Conclusions and Perspective 207. Acknowledgments 208. Assaying WAVE and WASH Complex Constitutive Activities Toward the Arp2/3 Complex 209. 1. Introduction 210. 2. Establishment of Stable Cell Lines Expressing Tagged WAVE and WASH Complexes 211. 3. Large-Scale Purification of WAVE and WASH Complexes 212. 4. Aggregation Analysis of WAVE and WASH Multiprotein Complexes 213. 5. Activity Measurements using Pyrene Actin Polymerization Assays 214. 6. Detection of an Endogenous Activity of the WAVE Complex 215. 7. Concluding Remarks 216. Acknowledgments