Constitutive Activity in Receptors and Other Proteins, Part A (eBook)

Front Cover 1
Methods Inenzymology: Constitutive Activity inReceptors and OtherProteins, Part A 4
Copyright 5
Contents 6
Contributors 16
Preface 26
Methods in Enzymology 28
Section One: Identification and Measurement of Constitutive Activity 58
Chapter 1: Constitutive Activity at the Cannabinoid CB1 Receptor and Behavioral Responses 60
1. Introduction 61
2. Modifying CB1 Activity 63
3. Behavioral Models 69
References 83
Chapter 2: Detecting Constitutive Activity and Protean Agonism at Cannabinoid-2 Receptor 88
1. Introduction 89
2. General Considerations 91
3. Evaluation of Constitutive Activity Using GTPgammaS Assay 92
4. Evaluation of Constitutive Activity Using cAMP Assay 96
5. Evaluation of Constitutive Activity Using RT-CES 99
6. Evaluation of Protean Agonism with cAMP Assay 103
7. Comparison of the Methods 105
References 106
Chapter 3: Modulation of the Constitutive Activity of the Ghrelin Receptor by Use of Pharmacological Tools and Mutagenesis... 110
1. Introduction 111
2. The Ghrelin Receptor and Its Constitutive Activity 112
3. Structural Basis of Constitutive Activity 114
4. Residues Responsible for the Inverse Agonism and Efficacy Swap 118
5. Physiological Relevance of Constitutive Activity 120
6. Experimental Procedures 122
References 126
Chapter 4: Assessment of Constitutive Activity and Internalization of GPR54 (KISS1-R) 132
1. Introduction 133
2. Materials 134
3. Methods 136
Acknowledgments 148
References 149
Chapter 5: Assessment of Constitutive Activity in E-Type Prostanoid Receptors 152
1. Introduction 153
2. Assays Used to Assess EP3 Receptor Constitutive Activity 154
3. Assays Used to Assess EP4 Receptor Constitutive Activity 162
References 164
Chapter 6: a1D-Adrenergic Receptors: Constitutive Activity and Reduced Expression at the Plasma Membrane 166
1. Introduction 167
2. Methods 168
3. Constitutive Activity 171
4. Plasma Membrane a1D-Adrenergic Receptors 174
5. Possible Physiological Implications 176
Acknowledgments 178
References 178
Chapter 7: Constitutive Activity of the Histamine H1 Receptor 184
1. Introduction 185
2. Methods to Study Constitutive H1R Signaling 190
3. Constitutive Activity as Tool to Elucidate Receptor Activation and Crosstalk 199
4. Conclusion 201
Acknowledgments 201
References 202
Chapter 8: Constitutive Activity of Somatostatin Receptor Subtypes 206
1. Introduction 207
2. Choosing Cells to be Studied 208
3. Modifying SSTR Density 209
4. Summary 220
References 220
Chapter 9: Assessment of Homologous Internalization of Constitutively Active N111G Mutant of AT1 Receptor 222
1. Introduction 223
2. Preparation of Receptor Plasmid and Protein 224
3. Radioligand Binding Assay 225
4. Inositol Phosphate Accumulation Assay 227
5. Internalization Assay 228
6. Western Blot Analysis 230
7. Data Analysis 231
8. Concluding Remark 232
References 232
Chapter 10: Methods to Detect Cell Surface Expression and Constitutive Activity of GPR6 236
1. Introduction 237
2. GPR6 is Expressed in Intracellular Compartments 237
3. Comparison of the Cell Surface Protein Detection Methods 244
4. Comparison of the Constitutive Gs-Activity Detection Methods 249
5. Conclusions 251
References 251
Chapter 11: beta3-Adrenoceptor Agonists and (Antagonists as) Inverse Agonists: History, Perspective, ConstitutiveActivity, and Stereospecific Binding 254
1. Introduction 255
2. beta3-Adrenoceptor 258
3. Methodologies 271
Acknowledgments 281
References 282
Chapter 12: Constitutive Activity of the Lutropin Receptor and Its Allosteric Modulation by Receptor Heterodimerization 288
1. Introduction 289
2. General Principles for Quantifying Receptor Activation 292
3. Modifying Cell Surface Expression Levels of Recombinant hLHR and Mutants Thereof 296
4. Quantifying Cell Surface hLHR Expression 300
5. Quantifying cAMP Production in Cells Expressing the hLHR 302
6. Experimental Strategies for Characterizing the Attenuating Effects of a Signaling Inactive hLHR on a Coexpressed wt or CAM 303
Acknowledgments 307
References 307
Chapter 13: Assessing Constitutive Activity of Extracellular Calcium-Sensing Receptors In Vitro and in Bone 310
1. Introduction 311
2. Materials and Methods 315
3. Results 319
4. Conclusions 320
Acknowledgments 322
References 322
Chapter 14: Constitutive Activity of Neural Melanocortin Receptors 324
1. Introduction 325
2. Signaling Assay for the Neural Melanocortin Receptors 325
3. Naturally Occurring Constitutively Active MC4R Mutants 327
4. Inverse Agonism of AgRP at the MC3R 330
5. Computational Modeling of the Constitutively Active MC4R Mutants 330
Acknowledgments 334
References 334
Chapter 15: Measurement of Constitutive Activity of BMP Type I Receptors 338
1. Introduction 338
2. Determining ALK2 Constitutive Activity 341
3. Determining the Effects of ALK2 Constitutive Activity on Osteoblast Differentiation 346
4. Concluding Remarks 348
Acknowledgments 349
References 349
Chapter 16: Probing the Constitutive Activity Among Dopamine D1 and D5 Receptors and Their Mutants 352
1. Introduction 353
2. Design of Genetically Modified D1-Like Receptor Constructs and Cloning Strategy 356
3. Transfection of D1R and D5R Expression Constructs in HEK293 Cells 358
4. Radioligand-Binding Assays 360
5. Whole Cell cAMP Assays 362
6. Results Validating Experimental Approaches 370
7. Concluding Remarks 381
Acknowledgments 383
References 384
Chapter 17: Identification of Gain-of-Function Variants of the Human Prolactin Receptor 386
1. Introduction 387
2. Experimental Procedures 390
3. Identification of Constitutive Activity: Results and Discussion 402
4. Conclusions 410
Acknowledgments 410
References 410
Chapter 18: Investigations of Activated ACVR1/ALK2, a Bone Morphogenetic Protein Type I Receptor, That Causes Fibrodysplasia 414
1. Introduction 415
2. Patient Methodologies 416
3. Cellular Methodologies 419
4. Tissue Methodologies 423
5. In Vivo Methodologies 426
Acknowledgments 428
References 428
Chapter 19: Identification and Evaluation of Constitutively Active Thyroid Stimulating Hormone Receptor Mutations 432
1. Introduction 433
2. TSHR Gene Mutational Screening 439
3. Determination of TSHR Constitutive Activity In Vitro 443
4. Measurement of TSHR Expression at Cell Surface by Flow Cytometry Analysis 445
5 TSH-TSHR Binding Assays 446
6 TSHR Phosphorylation Analysis 448
Acknowledgments 449
References 449
Chapter 20: Assessment of Constitutive Activity of a G Protein-Coupled Receptor, Cpr2, in Cryptococcus neoformans by Heterolo 454
1. Introduction of Receptors and Constitutive Receptors 455
2. Identification of Cpr2 as a Natural Occurring Constitutively Active Receptor 456
3. Additional Constitutively Active Receptors Identified in Fungi 466
Acknowledgments 466
References 466
Chapter 21: In Vitro and In Vivo Assessment of Mu Opioid Receptor Constitutive Activity 470
1. Introduction 471
2. Measuring Opioid Receptor Constitutive Activity In Vitro 472
3. cAMP Quantification Assay in CHO Cells Expressing Cloned Opioid Receptors 482
4. In Vivo Assessment of Antagonist Potency in Opioid Naïve Subjects 487
5. In Vivo Assessment of Antagonist Potency to Precipitate Withdrawal 492
6. Summary 497
Acknowledgments 498
References 498
Chapter 22: Constitutively Active mu-Opioid Receptors 502
1. Introduction 503
2. Methods for Measuring Constitutive Activity 504
3. Conclusions 522
Acknowledgments 522
References 523
Chapter 23: Protein Kinase CK2 Is a Constitutively Active Enzyme that Promotes Cell Survival: Strategies to Identify CK2 Subs 528
1. Introduction 529
2. Purification of CK2 for In Vitro Studies 531
3. Assays for CK2 Activity 538
4. Modulation of CK2 in Mammalian Cells 542
5. Conclusions 548
Acknowledgments 548
References 549
Chapter 24: Assessment of CK2 Constitutive Activity in Cancer Cells 552
1. Introduction 553
2. Assay of CK2 in Crude Biological Samples 556
3. In-Cell Assay of Endogenous CK2 Activity 561
4. Identification/Validation of In Vivo CK2 Targets with Specific Inhibitors 563
Acknowledgments 568
References 568
Chapter 25: Structural Basis of the Constitutive Activity of Protein Kinase CK2 572
1. Introduction 573
2. A Constitutively Active CK2a Structure and Its Stabilizing Elements 573
3. Analyzing the Constitutive Activity of Protein Kinase CK2 578
Acknowledgments 585
References 585
Chapter 26: Measuring the Constitutive Activation of c-Jun N-terminal Kinase Isoforms 588
1. Introduction 589
2. Important Reagents for Studying JNK Activity 591
3. Protein Expression and Purification of JNK Proteins and c-JUN 592
4. Measuring the Autophosphorylation Ability of the JNK Isoforms 595
5. Determining the Kinase Activity of the JNK Isoforms 597
6. Monitoring the Formation of JNK Homodimers 597
7. Measuring Nuclear Translocation of JNK Protein 598
8. Future Directions 601
Acknowledgments 603
References 603
Chapter 27: Measurement of Constitutive MAPK and PI3K/AKT Signaling Activity in Human Cancer Cell Lines 606
1. Introduction 607
2. Maintaining Melanoma Cell Lines 609
3. Western Blotting 610
4. Phospho-Flow Cytometry 616
5. Immunofluorescence 619
6. Conclusions 622
Acknowledgments 623
References 623
Chapter 28: Constitutive Activity of GPR40/FFA1 626
1. Introduction 627
2. Measuring FFA1-Mediated Calcium Mobilization 632
3. Measuring Direct Activation of G Proteins via FFA1 638
References 644
Chapter 29: Constitutive Activity of TRP Channels 648
1. Introduction 649
2. TRP Channels and Cellular Degeneration 651
3. Constitutive TRP Channel Activity Which Does Not Lead to Cellular Degeneration 652
4. Constitutive TRP Channel Activity Which Leads to Cellular Degeneration 661
Acknowledgments 666
References 666
Chapter 30: Measurement of Orexin (Hypocretin) and Substance P Effects on Constitutively Active Inward Rectifier K+ Channels 670
1. Introduction 671
2. Dissociated Culture of Cholinergic Neurons in the Basal Forebrain 672
3. Effects of Orexin (Hypocretin) and Substance P on Constitutively Active Inward Rectifier K+ (KirNB) Channels... 677
4. Signal Transduction of Substance P and Orexin Effects on KirNB Channels 682
Acknowledgments 685
References 686
Chapter 31: Characterization of G Protein-Coupled Receptor Kinase 4 and Measuring Its Constitutive Activity In Vivo 688
1. Introduction 689
2. Selection of Cells/Model Systems to Study GRK4 Function 691
3. Generation of Kinase Dead-GRK4 694
4. Functional Characterization of GRK4 Constitutive Activity 696
5. Agonist-Mediated GRK4 Activity 705
6. Summary 706
Acknowledgments 706
References 707
Chapter 32: Voltage-Clamp-Based Methods for the Detection of Constitutively Active Acetylcholine-Gated IK,ACh Channels in the 710
1. Introduction 711
2. Recording of Constitutive IK,ACh Using Patch-Clamp Techniques 712
3. Conclusions and Perspective 728
Acknowledgments 730
References 730
Chapter 33: Assaying WAVE and WASH Complex Constitutive Activities Toward the Arp2/3 Complex 734
1. Introduction 735
2. Establishment of Stable Cell Lines Expressing Tagged WAVE and WASH Complexes 737
3. Large-Scale Purification of WAVE and WASH Complexes 739
4. Aggregation Analysis of WAVE and WASH Multiprotein Complexes 741
5. Activity Measurements using Pyrene Actin Polymerization Assays 744
6. Detection of an Endogenous Activity of the WAVE Complex 748
7. Concluding Remarks 750
Acknowledgments 750
References 751
Author Index 754
Subject Index 792
Color Plate 806