Constitutive Activity in Receptors and Other Proteins, Part A

1. Auflage 2010
ISBN: 978-0-12-381299-5
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band Volume 484, 804 Seiten

Reihe: Methods in Enzymology

This volume of Methods in Enzymology covers the current methodology for the detection and assessment of constitutively active proteins. The chapters written by expert authors who are leaders in the field, provide hints and tricks not available in primary research publications.It is extensively referenced, with useful figures and tables throughout the volume. - Expert authors who are leaders in the field - Extensively referenced and useful figures and tables - Provides hints and tricks to facilitate reproduction of methods

Autoren/Hrsg.

Inhaltsverzeichnis

1;Front Cover;1
2;Methods Inenzymology: Constitutive Activity inReceptors and OtherProteins, Part A;4
3;Copyright;5
4;Contents;6
5;Contributors;16
6;Preface;26
7;Methods in Enzymology;28
8;Section One: Identification and Measurement of Constitutive Activity;58
8.1;Chapter 1: Constitutive Activity at the Cannabinoid CB1 Receptor and Behavioral Responses;60
8.1.1;1. Introduction;61
8.1.2;2. Modifying CB1 Activity;63
8.1.3;3. Behavioral Models;69
8.1.4;References;83
9;Chapter 2: Detecting Constitutive Activity and Protean Agonism at Cannabinoid-2 Receptor;88
9.1;1. Introduction;89
9.2;2. General Considerations;91
9.3;3. Evaluation of Constitutive Activity Using GTPgammaS Assay;92
9.4;4. Evaluation of Constitutive Activity Using cAMP Assay;96
9.5;5. Evaluation of Constitutive Activity Using RT-CES;99
9.6;6. Evaluation of Protean Agonism with cAMP Assay;103
9.7;7. Comparison of the Methods;105
9.8;References;106
10;Chapter 3: Modulation of the Constitutive Activity of the Ghrelin Receptor by Use of Pharmacological Tools and Mutagenesis...;110
10.1;1. Introduction;111
10.2;2. The Ghrelin Receptor and Its Constitutive Activity;112
10.3;3. Structural Basis of Constitutive Activity;114
10.4;4. Residues Responsible for the Inverse Agonism and Efficacy Swap;118
10.5;5. Physiological Relevance of Constitutive Activity;120
10.6;6. Experimental Procedures;122
10.7;References;126
11;Chapter 4: Assessment of Constitutive Activity and Internalization of GPR54 (KISS1-R);132
11.1;1. Introduction;133
11.2;2. Materials;134
11.3;3. Methods;136
11.4;Acknowledgments;148
11.5;References;149
12;Chapter 5: Assessment of Constitutive Activity in E-Type Prostanoid Receptors;152
12.1;1. Introduction;153
12.2;2. Assays Used to Assess EP3 Receptor Constitutive Activity;154
12.3;3. Assays Used to Assess EP4 Receptor Constitutive Activity;162
12.4;References;164
13;Chapter 6: a1D-Adrenergic Receptors: Constitutive Activity and Reduced Expression at the Plasma Membrane;166
13.1;1. Introduction;167
13.2;2. Methods;168
13.3;3. Constitutive Activity;171
13.4;4. Plasma Membrane a1D-Adrenergic Receptors;174
13.5;5. Possible Physiological Implications;176
13.6;Acknowledgments;178
13.7;References;178
14;Chapter 7: Constitutive Activity of the Histamine H1 Receptor;184
14.1;1. Introduction;185
14.2;2. Methods to Study Constitutive H1R Signaling;190
14.3;3. Constitutive Activity as Tool to Elucidate Receptor Activation and Crosstalk;199
14.4;4. Conclusion;201
14.5;Acknowledgments;201
14.6;References;202
15;Chapter 8: Constitutive Activity of Somatostatin Receptor Subtypes;206
15.1;1. Introduction;207
15.2;2. Choosing Cells to be Studied;208
15.3;3. Modifying SSTR Density;209
15.4;4. Summary;220
15.5;References;220
16;Chapter 9: Assessment of Homologous Internalization of Constitutively Active N111G Mutant of AT1 Receptor;222
16.1;1. Introduction;223
16.2;2. Preparation of Receptor Plasmid and Protein;224
16.3;3. Radioligand Binding Assay;225
16.4;4. Inositol Phosphate Accumulation Assay;227
16.5;5. Internalization Assay;228
16.6;6. Western Blot Analysis;230
16.7;7. Data Analysis;231
16.8;8. Concluding Remark;232
16.9;References;232
17;Chapter 10: Methods to Detect Cell Surface Expression and Constitutive Activity of GPR6;236
17.1;1. Introduction;237
17.2;2. GPR6 is Expressed in Intracellular Compartments;237
17.3;3. Comparison of the Cell Surface Protein Detection Methods;244
17.4;4. Comparison of the Constitutive Gs-Activity Detection Methods;249
17.5;5. Conclusions;251
17.6;References;251
18;Chapter 11: beta3-Adrenoceptor Agonists and (Antagonists as) Inverse Agonists: History, Perspective, ConstitutiveActivity, and Stereospecific Binding;254
18.1;1. Introduction;255
18.2;2. beta3-Adrenoceptor;258
18.3;3. Methodologies;271
18.4;Acknowledgments;281
18.5;References;282
19;Chapter 12: Constitutive Activity of the Lutropin Receptor and Its Allosteric Modulation by Receptor Heterodimerization;288
19.1;1. Introduction;289
19.2;2. General Principles for Quantifying Receptor Activation;292
19.3;3. Modifying Cell Surface Expression Levels of Recombinant hLHR and Mutants Thereof;296
19.4;4. Quantifying Cell Surface hLHR Expression;300
19.5;5. Quantifying cAMP Production in Cells Expressing the hLHR;302
19.6;6. Experimental Strategies for Characterizing the Attenuating Effects of a Signaling Inactive hLHR on a Coexpressed wt or CAM;303
19.7;Acknowledgments;307
19.8;References;307
20;Chapter 13: Assessing Constitutive Activity of Extracellular Calcium-Sensing Receptors In Vitro and in Bone;310
20.1;1. Introduction;311
20.2;2. Materials and Methods;315
20.3;3. Results;319
20.4;4. Conclusions;320
20.5;Acknowledgments;322
20.6;References;322
21;Chapter 14: Constitutive Activity of Neural Melanocortin Receptors;324
21.1;1. Introduction;325
21.2;2. Signaling Assay for the Neural Melanocortin Receptors;325
21.3;3. Naturally Occurring Constitutively Active MC4R Mutants;327
21.4;4. Inverse Agonism of AgRP at the MC3R;330
21.5;5. Computational Modeling of the Constitutively Active MC4R Mutants;330
21.6;Acknowledgments;334
21.7;References;334
22;Chapter 15: Measurement of Constitutive Activity of BMP Type I Receptors;338
22.1;1. Introduction;338
22.2;2. Determining ALK2 Constitutive Activity;341
22.3;3. Determining the Effects of ALK2 Constitutive Activity on Osteoblast Differentiation;346
22.4;4. Concluding Remarks;348
22.5;Acknowledgments;349
22.6;References;349
23;Chapter 16: Probing the Constitutive Activity Among Dopamine D1 and D5 Receptors and Their Mutants;352
23.1;1. Introduction;353
23.2;2. Design of Genetically Modified D1-Like Receptor Constructs and Cloning Strategy;356
23.3;3. Transfection of D1R and D5R Expression Constructs in HEK293 Cells;358
23.4;4. Radioligand-Binding Assays;360
23.5;5. Whole Cell cAMP Assays;362
23.6;6. Results Validating Experimental Approaches;370
23.7;7. Concluding Remarks;381
23.8;Acknowledgments;383
23.9;References;384
24;Chapter 17: Identification of Gain-of-Function Variants of the Human Prolactin Receptor;386
24.1;1. Introduction;387
24.2;2. Experimental Procedures;390
24.3;3. Identification of Constitutive Activity: Results and Discussion;402
24.4;4. Conclusions;410
24.5;Acknowledgments;410
24.6;References;410
25;Chapter 18: Investigations of Activated ACVR1/ALK2, a Bone Morphogenetic Protein Type I Receptor, That Causes Fibrodysplasia;414
25.1;1. Introduction;415
25.2;2. Patient Methodologies;416
25.3;3. Cellular Methodologies;419
25.4;4. Tissue Methodologies;423
25.5;5. In Vivo Methodologies;426
25.6;Acknowledgments;428
25.7;References;428
26;Chapter 19: Identification and Evaluation of Constitutively Active Thyroid Stimulating Hormone Receptor Mutations;432
26.1;1. Introduction;433
26.2;2. TSHR Gene Mutational Screening;439
26.3;3. Determination of TSHR Constitutive Activity In Vitro;443
26.4;4. Measurement of TSHR Expression at Cell Surface by Flow Cytometry Analysis;445
26.5;5 TSH-TSHR Binding Assays;446
26.6;6 TSHR Phosphorylation Analysis;448
26.7;Acknowledgments;449
26.8;References;449
27;Chapter 20: Assessment of Constitutive Activity of a G Protein-Coupled Receptor, Cpr2, in Cryptococcus neoformans by Heterolo;454
27.1;1. Introduction of Receptors and Constitutive Receptors;455
27.2;2. Identification of Cpr2 as a Natural Occurring Constitutively Active Receptor;456
27.3;3. Additional Constitutively Active Receptors Identified in Fungi;466
27.4;Acknowledgments;466
27.5;References;466
28;Chapter 21: In Vitro and In Vivo Assessment of Mu Opioid Receptor Constitutive Activity;470
28.1;1. Introduction;471
28.2;2. Measuring Opioid Receptor Constitutive Activity In Vitro;472
28.3;3. cAMP Quantification Assay in CHO Cells Expressing Cloned Opioid Receptors;482
28.4;4. In Vivo Assessment of Antagonist Potency in Opioid Naïve Subjects;487
28.5;5. In Vivo Assessment of Antagonist Potency to Precipitate Withdrawal;492
28.6;6. Summary;497
28.7;Acknowledgments;498
28.8;References;498
29;Chapter 22: Constitutively Active mu-Opioid Receptors;502
29.1;1. Introduction;503
29.2;2. Methods for Measuring Constitutive Activity;504
29.3;3. Conclusions;522
29.4;Acknowledgments;522
29.5;References;523
30;Chapter 23: Protein Kinase CK2 Is a Constitutively Active Enzyme that Promotes Cell Survival: Strategies to Identify CK2 Subs;528
30.1;1. Introduction;529
30.2;2. Purification of CK2 for In Vitro Studies;531
30.3;3. Assays for CK2 Activity;538
30.4;4. Modulation of CK2 in Mammalian Cells;542
30.5;5. Conclusions;548
30.6;Acknowledgments;548
30.7;References;549
31;Chapter 24: Assessment of CK2 Constitutive Activity in Cancer Cells;552
31.1;1. Introduction;553
31.2;2. Assay of CK2 in Crude Biological Samples;556
31.3;3. In-Cell Assay of Endogenous CK2 Activity;561
31.4;4. Identification/Validation of In Vivo CK2 Targets with Specific Inhibitors;563
31.5;Acknowledgments;568
31.6;References;568
32;Chapter 25: Structural Basis of the Constitutive Activity of Protein Kinase CK2;572
32.1;1. Introduction;573
32.2;2. A Constitutively Active CK2a Structure and Its Stabilizing Elements;573
32.3;3. Analyzing the Constitutive Activity of Protein Kinase CK2;578
32.4;Acknowledgments;585
32.5;References;585
33;Chapter 26: Measuring the Constitutive Activation of c-Jun N-terminal Kinase Isoforms;588
33.1;1. Introduction;589
33.2;2. Important Reagents for Studying JNK Activity;591
33.3;3. Protein Expression and Purification of JNK Proteins and c-JUN;592
33.4;4. Measuring the Autophosphorylation Ability of the JNK Isoforms;595
33.5;5. Determining the Kinase Activity of the JNK Isoforms;597
33.6;6. Monitoring the Formation of JNK Homodimers;597
33.7;7. Measuring Nuclear Translocation of JNK Protein;598
33.8;8. Future Directions;601
33.9;Acknowledgments;603
33.10;References;603
34;Chapter 27: Measurement of Constitutive MAPK and PI3K/AKT Signaling Activity in Human Cancer Cell Lines;606
34.1;1. Introduction;607
34.2;2. Maintaining Melanoma Cell Lines;609
34.3;3. Western Blotting;610
34.4;4. Phospho-Flow Cytometry;616
34.5;5. Immunofluorescence;619
34.6;6. Conclusions;622
34.7;Acknowledgments;623
34.8;References;623
35;Chapter 28: Constitutive Activity of GPR40/FFA1;626
35.1;1. Introduction;627
35.2;2. Measuring FFA1-Mediated Calcium Mobilization;632
35.3;3. Measuring Direct Activation of G Proteins via FFA1;638
35.4;References;644
36;Chapter 29: Constitutive Activity of TRP Channels;648
36.1;1. Introduction;649
36.2;2. TRP Channels and Cellular Degeneration;651
36.3;3. Constitutive TRP Channel Activity Which Does Not Lead to Cellular Degeneration;652
36.4;4. Constitutive TRP Channel Activity Which Leads to Cellular Degeneration;661
36.5;Acknowledgments;666
36.6;References;666
37;Chapter 30: Measurement of Orexin (Hypocretin) and Substance P Effects on Constitutively Active Inward Rectifier K+ Channels;670
37.1;1. Introduction;671
37.2;2. Dissociated Culture of Cholinergic Neurons in the Basal Forebrain;672
37.3;3. Effects of Orexin (Hypocretin) and Substance P on Constitutively Active Inward Rectifier K+ (KirNB) Channels...;677
37.4;4. Signal Transduction of Substance P and Orexin Effects on KirNB Channels;682
37.5;Acknowledgments;685
37.6;References;686
38;Chapter 31: Characterization of G Protein-Coupled Receptor Kinase 4 and Measuring Its Constitutive Activity In Vivo;688
38.1;1. Introduction;689
38.2;2. Selection of Cells/Model Systems to Study GRK4 Function;691
38.3;3. Generation of Kinase Dead-GRK4;694
38.4;4. Functional Characterization of GRK4 Constitutive Activity;696
38.5;5. Agonist-Mediated GRK4 Activity;705
38.6;6. Summary;706
38.7;Acknowledgments;706
38.8;References;707
39;Chapter 32: Voltage-Clamp-Based Methods for the Detection of Constitutively Active Acetylcholine-Gated IK,ACh Channels in the;710
39.1;1. Introduction;711
39.2;2. Recording of Constitutive IK,ACh Using Patch-Clamp Techniques;712
39.3;3. Conclusions and Perspective;728
39.4;Acknowledgments;730
39.5;References;730
40;Chapter 33: Assaying WAVE and WASH Complex Constitutive Activities Toward the Arp2/3 Complex;734
40.1;1. Introduction;735
40.2;2. Establishment of Stable Cell Lines Expressing Tagged WAVE and WASH Complexes;737
40.3;3. Large-Scale Purification of WAVE and WASH Complexes;739
40.4;4. Aggregation Analysis of WAVE and WASH Multiprotein Complexes;741
40.5;5. Activity Measurements using Pyrene Actin Polymerization Assays;744
40.6;6. Detection of an Endogenous Activity of the WAVE Complex;748
40.7;7. Concluding Remarks;750
40.8;Acknowledgments;750
40.9;References;751
40.10;Author Index;754
40.11;Subject Index;792
40.12;Color Plate;806