Note: Front Cover -- Methods in Enzymology: Constitutive Activity in Receptors and Other Proteins, Part B -- Copyright Page -- Contents -- Contributors -- Preface -- Volume in Series -- Section One: Inverse Agonism and Inverse Agonists -- Chapter 1: Identification and Characterization of Steroidogenic Factor-1 Inverse Agonists -- 1. Introduction -- 2. Characterization of Inverse Agonists of SF-1 by uHTS -- 3. Effect of SF-1 Inverse Agonists on Adrenocortical Tumor Cell Proliferation and Steroid Production -- 4. Conclusion -- Acknowledgments -- References -- Chapter 2: Assessment of Inverse Agonism for the Angiotensin II Type 1 Receptor -- 1. Introduction -- 2. Protocol for Cell Culture and Transfection -- 3. Radioligand Assay for AT1 Receptor -- 4. Protocol for Cell Stretching -- 5. Assessment of AT1 Receptor Activation -- 6. Conclusion -- Acknowledgments -- References -- Chapter 3: Measurement of Inverse Agonism in beta-Adrenoceptors -- 1. Introduction: Basal Spontaneous Receptor Activity. The Rise of the Concept of Inverse Agonism -- 2. beta-Adrenoceptors: Main Features -- 3. Methodological Aspects of the Assessment of Inverse Agonist Properties at betaAR -- 4. Clinical Potential Uses of betaAR Inverse Agonists -- References -- Chapter 4: Techniques for Studying Inverse Agonist Activity of Antidepressants at Recombinant Nonedited 5-HT2C-INI Receptor and Native Neuronal 5-HT2CReceptors -- 1. Introduction -- 2. Constitutive Activity Toward the Gαq-PLC Effector Pathway of 5-HT2C-INI Receptors Transiently Expressed in HEK-293 Cells -- 3. Plasma Membrane Insertion of 5-HT2C-INI Receptors -- 4. Enhanced Responsiveness of Constitutively Active 5-HT2C Receptors Upon Prolonged Treatment with Inverse Agonists -- Acknowledgments -- References -- Chapter 5: Differential Inverse Agonism at the Human Muscarinic M3 Receptor. , 1. Introduction to Muscarinic Receptors -- 2. Role of M3R in Regulating Smooth Muscle Function and Clinical Use of Anticholinergics -- 3. In Vitro Assays to Monitor hM3R Activation and Constitutive Activity -- 4. Constitutive Activity and Receptor Upregulation Studies -- 5. Physiological Relevance of hM3R Constitutive Activity -- References -- Chapter 6: Ghrelin Receptor: High Constitutive Activity and Methods for Developing Inverse Agonists -- 1. Introduction -- 2. Constitutive Activity and Development of Ghrelin Receptor Inverse Agonists -- 3. Synthesis of Ghrelin Receptor Inverse Agonists: Solid-Phase Peptide Synthesis (SPPS) -- 4. Functional Assays for Ghrelin Receptor Inverse Agonists -- References -- Chapter 7: Constitutive Activity and Inverse Agonism at the α1a and α1b Adrenergic Receptor Subtypes -- 1. Introduction -- 2. Combination of Computational Modeling and Site-Directed Mutagenesis of the Receptor to Identify Constitutively Activating Mutations -- 3. Measuring Constitutive Activity of Receptor-Mediated Gq Activation -- 4. Inverse Agonism at the α1-ARs -- 5. Conclusions -- Acknowledgments -- References -- Chapter 8: Measurement of Inverse Agonism of the Cannabinoid Receptors -- 1. Introduction -- 2. Gi-cAMP Assay -- 3. GTPgammaS Binding Assay -- 4. Electrophysiological Assays -- 5. Summary -- References -- Chapter 9: Constitutively Active Thyrotropin and Thyrotropin-Releasing Hormone Receptors and Their Inverse Agonists -- 1. Introduction -- 2. TRH-R2 and Its Inverse Agonist Midazolam -- 3. TSHR and Its Inverse Agonist NCGC00161856 -- Acknowledgments -- References -- Chapter 10: Inverse Agonists and Antagonists of Retinoid Receptors -- 1. Introduction -- 2. Functional Classification of Retinoid Receptor Ligands -- 3. Structural Basis of Retinoid Receptor Action -- 4. Synthetic Routes and Toolbox for Rational Retinoid Design. , 5. Protocols for the Study of Ligand Function -- 6. Chemical Syntheses -- Acknowledgments -- References -- Chapter 11: gamma-Aminobutyric Acid Type A (GABAA) Receptor Subtype Inverse Agonists as Therapeutic Agents in Cognition -- 1. Introduction -- 2. Inverse Agonism: Definition -- 3. Negative Allosteric Regulators of GABAA-R in Cognitive Impairment -- 4. Methods for Evaluating the Affinity and Efficacy at GABAA Receptor Subtypes -- 5. Conclusion -- References -- Chapter 12: Assays for Inverse Agonists in the Visual System -- 1. Introduction -- 2. Opsin Preparation -- 3. Transducin Preparation -- 4. Transducin Activation Assay -- Acknowledgments -- References -- Chapter 13: Receptor-Driven Identification of Novel Human A3 Adenosine Receptor Antagonists as Potential Therapeutic Agents -- 1. Introduction -- 2. Newer Potential Therapeutic Role of A3 Adenosine Receptors -- 3. A3 Adenosine Receptor Antagonists -- 4. Receptor-Based Antagonist Design -- Acknowledgments -- References -- Chapter 14: Inverse Agonist Activity of Steroidogenic Factor SF-1 -- 1. Introduction -- 2. SF-1 Inverse Agonism in the R-SAT Assay of Cellular Proliferation -- 3. SF-1 Inverse Agonism in Luciferase Transcriptional Assay -- 4. SF-1 Inverse Agonism in Adrenocortical Cultures -- Acknowledgments -- References -- Chapter 15: Methods to Measure G-Protein-Coupled Receptor Activity for the Identification of Inverse Agonists -- 1. Introduction -- 2. [35S]GTPgammaS Binding Assay in Membrane Homogenates -- 3. [35S]GTPgammaS Autoradiography in Brain Sections -- Acknowledgments -- References -- Section Two: Novel strategies and techniques for constitutive activity and inverse agonism -- Chapter 16: Use of Pharmacoperones to Reveal GPCR Structural Changes Associated with Constitutive Activation and Trafficking -- 1. Introduction -- 2. Methods for Measuring Receptors and Receptor Activity. , 3. Assessment of Results -- 4. Conclusions -- Acknowledgments -- References -- Chapter 17: Application of Large-Scale Transient Transfection to Cell-Based Functional Assays for Ion Channels and GPCRs -- 1. Introduction -- 2. Large-Scale Transient Transfection -- 3. Cryopreservation of Cells -- 4. Application to Ion Channel Assays -- 5. Application to GPCR Assays -- 6. Conclusion -- Acknowledgment -- References -- Chapter 18: Quantification of RNA Editing of the Serotonin 2C Receptor (5-HT2CR) Ex Vivo -- 1. RNA Editing of the 5-HT2CR -- 2. Functional Properties of 5-HT2CR Edited Isoforms -- 3. Current Methods for Quantification of 5-HT2CR Editing Events Ex Vivo -- 4. Quantification of 5-HT2CR Editing Events Ex Vivo with qRT-PCR -- Acknowledgments -- References -- Chapter 19: Strategies for Isolating Constitutively Active and Dominant-Negative Pheromone Receptor Mutants in Yeast -- 1. Introduction -- 2. Selecting a Yeast Strain and Expression Vector -- 3. Transforming Plasmids into Yeast -- 4. "Gap-Repair" Approach for Targeting Mutagenesis to Genes on Plasmids -- 5. Isolation of Constitutively Active Mutants -- 6. Isolation of Dominant-Negative Mutants -- 7. Further Methods for Analysis of Mutant Receptors -- Acknowledgments -- References -- Chapter 20: Development of a GPR23 Cell-Based beta-Lactamase Reporter Assay -- 1. Introduction -- 2. GPCR Cell-Based Assays -- 3. Development of a Cell-Based beta-Lactamase Reporter Assay for Constitutively Active GPR23 -- 4. Identification of GPR23 Inverse Agonists Using a beta-Lactamase Reporter Screen -- 5. Concluding Remarks -- Acknowledgments -- References -- Chapter 21: Computational Modeling of Constitutively Active Mutants of GPCRs: C5a Receptor -- 1. Introduction -- 2. Modeling CAMs Based on Experimental Data for the Ground and Activated States of GPCRs -- 3. Rotational Sampling of the TM Regions of GPCRs. , 4. Modeling Structural Mechanisms of Constitutive Activity in C5aRs -- 5. Conclusions and Perspectives -- Acknowledgments -- References -- Chapter 22: TSH Receptor Monoclonal Antibodies with Agonist, Antagonist, and Inverse Agonist Activities -- 1. Introduction -- 2. Production of Monoclonal Antibodies to the TSHR with the Characteristics of Patient Serum Autoantibodies -- 3. Characterization of 5C9 a Human Autoantibody with TSH Antagonist and TSHR Inverse Agonist Activity -- 4. Effects of TSHR Mutations on the Activity of MAb 5C9 -- 5. Structure of MAb 5C9 Fab -- 6. Conclusions -- Acknowledgment -- References -- Chapter 23: Current Standards, Variations, and Pitfalls for the Determination of Constitutive TSHR Activity In Vitro -- 1. Introduction -- 2. Detection of Constitutive TSHR Activity -- 3. Methods and Required Materials for LRA -- 4. Conclusion -- Acknowledgment -- References -- Chapter 24: Toward the Rational Design of Constitutively Active KCa3.1 Mutant Channels -- 1. Introduction -- 2. Production of Constitutively Active KCa3.1 Mutant Channels -- 3. Concluding Remarks -- References -- Chapter 25: Fusion Proteins as Model Systems for the Analysis of Constitutive GPCR Activity -- 1. Introduction -- 2. Expression of Fusion Proteins: hH4R-Gαi2 and hH4R-GAIP as Paradigms -- 3. Investigation of GPCR Constitutive Activity with Fusion Proteins: H4R as Paradigm -- 4. Application of the Fusion Protein Approach to other GPCRs -- Acknowledgments -- References -- Chapter 26: Screening for Novel Constitutively Active CXCR2 Mutants and Their Cellular Effects -- 1. Introduction -- 2. Establishment of a Yeast System to Identify CXCR2 CAMs -- 3. Establishment of a Mammalian System to Characterize CXCR2 CAMs -- References. , Chapter 27: A Method for Parallel Solid-Phase Synthesis of Iodinated Analogs of the Cannabinoid Receptor Type I (CB1) Inverse Agonist Rimonabant.

Note: Front Cover -- Methods Inenzymology: Constitutive Activity inReceptors and OtherProteins, Part A -- Copyright -- Contents -- Contributors -- Preface -- Methods in Enzymology -- Section One: Identification and Measurement of Constitutive Activity -- Chapter 1: Constitutive Activity at the Cannabinoid CB1 Receptor and Behavioral Responses -- 1. Introduction -- 2. Modifying CB1 Activity -- 3. Behavioral Models -- References -- Chapter 2: Detecting Constitutive Activity and Protean Agonism at Cannabinoid-2 Receptor -- 1. Introduction -- 2. General Considerations -- 3. Evaluation of Constitutive Activity Using GTPgammaS Assay -- 4. Evaluation of Constitutive Activity Using cAMP Assay -- 5. Evaluation of Constitutive Activity Using RT-CES -- 6. Evaluation of Protean Agonism with cAMP Assay -- 7. Comparison of the Methods -- References -- Chapter 3: Modulation of the Constitutive Activity of the Ghrelin Receptor by Use of Pharmacological Tools and Mutagenesis... -- 1. Introduction -- 2. The Ghrelin Receptor and Its Constitutive Activity -- 3. Structural Basis of Constitutive Activity -- 4. Residues Responsible for the Inverse Agonism and Efficacy Swap -- 5. Physiological Relevance of Constitutive Activity -- 6. Experimental Procedures -- References -- Chapter 4: Assessment of Constitutive Activity and Internalization of GPR54 (KISS1-R) -- 1. Introduction -- 2. Materials -- 3. Methods -- Acknowledgments -- References -- Chapter 5: Assessment of Constitutive Activity in E-Type Prostanoid Receptors -- 1. Introduction -- 2. Assays Used to Assess EP3 Receptor Constitutive Activity -- 3. Assays Used to Assess EP4 Receptor Constitutive Activity -- References -- Chapter 6: α1D-Adrenergic Receptors: Constitutive Activity and Reduced Expression at the Plasma Membrane -- 1. Introduction -- 2. Methods -- 3. Constitutive Activity. , 4. Plasma Membrane α1D-Adrenergic Receptors -- 5. Possible Physiological Implications -- Acknowledgments -- References -- Chapter 7: Constitutive Activity of the Histamine H1 Receptor -- 1. Introduction -- 2. Methods to Study Constitutive H1R Signaling -- 3. Constitutive Activity as Tool to Elucidate Receptor Activation and Crosstalk -- 4. Conclusion -- Acknowledgments -- References -- Chapter 8: Constitutive Activity of Somatostatin Receptor Subtypes -- 1. Introduction -- 2. Choosing Cells to be Studied -- 3. Modifying SSTR Density -- 4. Summary -- References -- Chapter 9: Assessment of Homologous Internalization of Constitutively Active N111G Mutant of AT1 Receptor -- 1. Introduction -- 2. Preparation of Receptor Plasmid and Protein -- 3. Radioligand Binding Assay -- 4. Inositol Phosphate Accumulation Assay -- 5. Internalization Assay -- 6. Western Blot Analysis -- 7. Data Analysis -- 8. Concluding Remark -- References -- Chapter 10: Methods to Detect Cell Surface Expression and Constitutive Activity of GPR6 -- 1. Introduction -- 2. GPR6 is Expressed in Intracellular Compartments -- 3. Comparison of the Cell Surface Protein Detection Methods -- 4. Comparison of the Constitutive Gs-Activity Detection Methods -- 5. Conclusions -- References -- Chapter 11: beta3-Adrenoceptor Agonists and (Antagonists as) Inverse Agonists: History, Perspective, ConstitutiveActivity, and Stereospecific Binding -- 1. Introduction -- 2. beta3-Adrenoceptor -- 3. Methodologies -- Acknowledgments -- References -- Chapter 12: Constitutive Activity of the Lutropin Receptor and Its Allosteric Modulation by Receptor Heterodimerization -- 1. Introduction -- 2. General Principles for Quantifying Receptor Activation -- 3. Modifying Cell Surface Expression Levels of Recombinant hLHR and Mutants Thereof -- 4. Quantifying Cell Surface hLHR Expression. , 5. Quantifying cAMP Production in Cells Expressing the hLHR -- 6. Experimental Strategies for Characterizing the Attenuating Effects of a Signaling Inactive hLHR on a Coexpressed wt or CAM -- Acknowledgments -- References -- Chapter 13: Assessing Constitutive Activity of Extracellular Calcium-Sensing Receptors In Vitro and in Bone -- 1. Introduction -- 2. Materials and Methods -- 3. Results -- 4. Conclusions -- Acknowledgments -- References -- Chapter 14: Constitutive Activity of Neural Melanocortin Receptors -- 1. Introduction -- 2. Signaling Assay for the Neural Melanocortin Receptors -- 3. Naturally Occurring Constitutively Active MC4R Mutants -- 4. Inverse Agonism of AgRP at the MC3R -- 5. Computational Modeling of the Constitutively Active MC4R Mutants -- Acknowledgments -- References -- Chapter 15: Measurement of Constitutive Activity of BMP Type I Receptors -- 1. Introduction -- 2. Determining ALK2 Constitutive Activity -- 3. Determining the Effects of ALK2 Constitutive Activity on Osteoblast Differentiation -- 4. Concluding Remarks -- Acknowledgments -- References -- Chapter 16: Probing the Constitutive Activity Among Dopamine D1 and D5 Receptors and Their Mutants -- 1. Introduction -- 2. Design of Genetically Modified D1-Like Receptor Constructs and Cloning Strategy -- 3. Transfection of D1R and D5R Expression Constructs in HEK293 Cells -- 4. Radioligand-Binding Assays -- 5. Whole Cell cAMP Assays -- 6. Results Validating Experimental Approaches -- 7. Concluding Remarks -- Acknowledgments -- References -- Chapter 17: Identification of Gain-of-Function Variants of the Human Prolactin Receptor -- 1. Introduction -- 2. Experimental Procedures -- 3. Identification of Constitutive Activity: Results and Discussion -- 4. Conclusions -- Acknowledgments -- References. , Chapter 18: Investigations of Activated ACVR1/ALK2, a Bone Morphogenetic Protein Type I Receptor, That Causes Fibrodysplasia -- 1. Introduction -- 2. Patient Methodologies -- 3. Cellular Methodologies -- 4. Tissue Methodologies -- 5. In Vivo Methodologies -- Acknowledgments -- References -- Chapter 19: Identification and Evaluation of Constitutively Active Thyroid Stimulating Hormone Receptor Mutations -- 1. Introduction -- 2. TSHR Gene Mutational Screening -- 3. Determination of TSHR Constitutive Activity In Vitro -- 4. Measurement of TSHR Expression at Cell Surface by Flow Cytometry Analysis -- 5 TSH-TSHR Binding Assays -- 6 TSHR Phosphorylation Analysis -- Acknowledgments -- References -- Chapter 20: Assessment of Constitutive Activity of a G Protein-Coupled Receptor, Cpr2, in Cryptococcus neoformans by Heterolo -- 1. Introduction of Receptors and Constitutive Receptors -- 2. Identification of Cpr2 as a Natural Occurring Constitutively Active Receptor -- 3. Additional Constitutively Active Receptors Identified in Fungi -- Acknowledgments -- References -- Chapter 21: In Vitro and In Vivo Assessment of Mu Opioid Receptor Constitutive Activity -- 1. Introduction -- 2. Measuring Opioid Receptor Constitutive Activity In Vitro -- 3. cAMP Quantification Assay in CHO Cells Expressing Cloned Opioid Receptors -- 4. In Vivo Assessment of Antagonist Potency in Opioid Naïve Subjects -- 5. In Vivo Assessment of Antagonist Potency to Precipitate Withdrawal -- 6. Summary -- Acknowledgments -- References -- Chapter 22: Constitutively Active mu-Opioid Receptors -- 1. Introduction -- 2. Methods for Measuring Constitutive Activity -- 3. Conclusions -- Acknowledgments -- References -- Chapter 23: Protein Kinase CK2 Is a Constitutively Active Enzyme that Promotes Cell Survival: Strategies to Identify CK2 Subs -- 1. Introduction. , 2. Purification of CK2 for In Vitro Studies -- 3. Assays for CK2 Activity -- 4. Modulation of CK2 in Mammalian Cells -- 5. Conclusions -- Acknowledgments -- References -- Chapter 24: Assessment of CK2 Constitutive Activity in Cancer Cells -- 1. Introduction -- 2. Assay of CK2 in Crude Biological Samples -- 3. In-Cell Assay of Endogenous CK2 Activity -- 4. Identification/Validation of In Vivo CK2 Targets with Specific Inhibitors -- Acknowledgments -- References -- Chapter 25: Structural Basis of the Constitutive Activity of Protein Kinase CK2 -- 1. Introduction -- 2. A Constitutively Active CK2α Structure and Its Stabilizing Elements -- 3. Analyzing the Constitutive Activity of Protein Kinase CK2 -- Acknowledgments -- References -- Chapter 26: Measuring the Constitutive Activation of c-Jun N-terminal Kinase Isoforms -- 1. Introduction -- 2. Important Reagents for Studying JNK Activity -- 3. Protein Expression and Purification of JNK Proteins and c-JUN -- 4. Measuring the Autophosphorylation Ability of the JNK Isoforms -- 5. Determining the Kinase Activity of the JNK Isoforms -- 6. Monitoring the Formation of JNK Homodimers -- 7. Measuring Nuclear Translocation of JNK Protein -- 8. Future Directions -- Acknowledgments -- References -- Chapter 27: Measurement of Constitutive MAPK and PI3K/AKT Signaling Activity in Human Cancer Cell Lines -- 1. Introduction -- 2. Maintaining Melanoma Cell Lines -- 3. Western Blotting -- 4. Phospho-Flow Cytometry -- 5. Immunofluorescence -- 6. Conclusions -- Acknowledgments -- References -- Chapter 28: Constitutive Activity of GPR40/FFA1 -- 1. Introduction -- 2. Measuring FFA1-Mediated Calcium Mobilization -- 3. Measuring Direct Activation of G Proteins via FFA1 -- References -- Chapter 29: Constitutive Activity of TRP Channels -- 1. Introduction -- 2. TRP Channels and Cellular Degeneration. , 3. Constitutive TRP Channel Activity Which Does Not Lead to Cellular Degeneration.