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PART I Screening of Ligands for GPCRs |
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Screening of Endogenous Ligands for Orphan GPCRs |
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3 | (34) |
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4 | (1) |
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5 | (2) |
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5 | (1) |
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6 | (1) |
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6 | (1) |
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Particular Examples of Ligand Screening for the Orphan GPCRs |
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7 | (30) |
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Galanin-Like Peptide (GALP) |
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7 | (1) |
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Discovery of a Novel Galanin-Family Peptide, GALP |
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7 | (1) |
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Preparation of Tissue Extract Sample |
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7 | (2) |
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[ 35S] Guanosine 5'O-(γ-Thio)Triphosphate ([ 35S]GTPγS) Binding Assay |
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9 | (1) |
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10 | (1) |
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Physiological Roles of GALP |
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10 | (2) |
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12 | (1) |
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Discovery of Metastin, the Cognate Ligand of OT7T175 (= GPR54) |
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12 | (1) |
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12 | (1) |
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13 | (1) |
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14 | (1) |
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14 | (3) |
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Physiological Roles of Metastin |
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17 | (1) |
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17 | (1) |
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17 | (1) |
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Isolation and Identification of NPW as the Endogenous Ligand for the GPR7 and GPR8 |
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18 | (2) |
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Neuropeptide B (NPB) as a Paralog Peptide of NPW |
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20 | (1) |
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Biological Functions of NPW and NPB |
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20 | (1) |
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21 | (1) |
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21 | (1) |
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Isolation and Identification of UII as the Endogenous Ligand for GPR14 |
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21 | (3) |
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Pathophysiological Significance of UII |
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24 | (1) |
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24 | (1) |
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FFAs Are Signaling Molecules |
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24 | (1) |
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25 | (1) |
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Identification of FFAs as Ligands for GPR40 |
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25 | (2) |
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Role of GPR40 Expressed in βCells |
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27 | (1) |
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Pyroglutamylated RFamide Peptide (QRFP) |
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28 | (1) |
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RFamide Peptides in Mammals |
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28 | (1) |
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Identification of a Novel RFamide Peptide Gene |
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28 | (1) |
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Identification of a Receptor for QRFP |
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29 | (1) |
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Interaction between QRFP and AQ27 |
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30 | (1) |
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Functions of QRFP and Its Receptor |
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30 | (1) |
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30 | (2) |
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32 | (1) |
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32 | (5) |
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Screening of Ligands for Human GPCRs by the Use of Receptor-Gα Fusion Proteins |
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37 | (30) |
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37 | (1) |
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38 | (4) |
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Screening of GPCRs from the Human Genome |
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38 | (2) |
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List of Useful Databases for GPCRs |
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40 | (2) |
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List of Useful Programs for GPCR and Other Protein Research |
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42 | (1) |
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Ligand Screening System for GPCRs Using Receptor-Gα Fusion Proteins |
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42 | (25) |
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Gene Construction for Receptor-Gα Fusion Proteins |
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44 | (5) |
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49 | (7) |
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In vitro [ 35S]GTγS Binding Assay |
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56 | (7) |
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63 | (1) |
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63 | (1) |
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63 | (4) |
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Screening and Identification of Ghrelin, an Endogenous Ligand for GHS-R |
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67 | (18) |
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68 | (1) |
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68 | (1) |
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GHS (Growth-Hormone Secretagogue) and Its Receptor |
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69 | (2) |
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69 | (2) |
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GHS Receptor (GHS-R) Superfamily |
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71 | (1) |
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71 | (8) |
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Construction of GHS-R Expressing Cells |
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71 | (2) |
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Intracellular Calcium Measurement Assay by FLIPR |
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73 | (1) |
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Purification of Rat Ghrelin |
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73 | (3) |
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Structural Determination of Rat Ghrelin |
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76 | (1) |
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Minor Molecular Forms of Human Ghrelin |
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76 | (1) |
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Ghrelin Precursor and Des-Gln14-Ghrelin |
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77 | (2) |
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79 | (6) |
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79 | (1) |
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80 | (1) |
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81 | (1) |
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81 | (4) |
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Ligand Screening of Olfactory Receptors |
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85 | (28) |
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86 | (2) |
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The G Protein-Coupled Olfactory Receptor Superfamily |
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86 | (1) |
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Odorant Signaling Pathways |
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86 | (2) |
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Functional Expression of Olfactory Receptors in Olfactory Neurons |
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88 | (4) |
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Adenovirus-Mediated Gene Transfer |
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88 | (1) |
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Generation of GFP-Tagged Gene-Targeted Mice |
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89 | (1) |
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Ca2+ Imaging and EOG Recording |
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90 | (1) |
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Ca2+ Imaging Assay in Dissociated Olfactory Neurons |
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90 | (2) |
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92 | (1) |
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Methods for Functional Receptor Expression in Heterologous Cells |
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92 | (3) |
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92 | (1) |
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N-Terminal Tagging and Glycosylation |
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93 | (1) |
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Chaperones for Membrane Trafficking |
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94 | (1) |
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Methods for Odorant Response Assays of Olfactory Receptors |
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95 | (9) |
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95 | (2) |
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97 | (2) |
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Luciferase Reporter Gene Assay |
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99 | (1) |
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100 | (1) |
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Assays for Multiple G Protein-Mediated Signals |
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100 | (2) |
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Measurement of Ion Currents with a Voltage Clamp Method |
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102 | (1) |
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Measurement with Depolarization Step Pulses |
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103 | (1) |
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Other Functional Assay Systems |
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104 | (1) |
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Structure-Activity Matrices of Olfactory Receptors |
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105 | (1) |
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106 | (7) |
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106 | (1) |
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107 | (6) |
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PART II Functions and Regulations of GPCRs |
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Generation and Phenotypical Analysis of Muscarinic Acetylcholine Receptor Knockout Mice |
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113 | (24) |
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113 | (1) |
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General Strategy Used to Generate Mice Lacking Specific mAChRs |
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114 | (7) |
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Phenotypical Analysis of mAChR Knockout (KO) Mice |
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121 | (8) |
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Summary of Mutant Phenotypes |
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121 | (5) |
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Potential Compensatory Changes of mAChR Expression Levels in mAChR KO Mice |
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126 | (1) |
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Issues Related to the Mouse Genetic Background of the Analyzed mAChR KO Mice |
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127 | (1) |
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Generation and Analysis of mAChR Double KO Mice |
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128 | (1) |
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Relevance for Drug Therapy |
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129 | (2) |
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129 | (1) |
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129 | (1) |
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130 | (1) |
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130 | (1) |
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130 | (1) |
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Conclusions and Future Outlook |
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131 | (6) |
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131 | (1) |
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131 | (6) |
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Systematic Mutagenesis of M1 Muscarinic Acetylcholine Receptors |
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137 | (42) |
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138 | (2) |
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G Protein-Coupled Receptors |
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138 | (1) |
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The Muscarinic Acetylcholine Receptors |
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139 | (1) |
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140 | (1) |
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The Mechanistic Framework for the Interpretation of Mutagenesis Studies on GPCRs |
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141 | (8) |
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The Ternary Complex Model of Receptor-G Protein Interaction |
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141 | (6) |
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The Extended Ternary Complex Model |
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147 | (1) |
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The Dimeric Receptor Ternary Complex Model |
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148 | (1) |
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149 | (8) |
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Choice of Target Sequences |
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149 | (1) |
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150 | (1) |
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Site-Directed Mutagenesis |
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150 | (1) |
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Preparation of Plasmid DNA |
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151 | (1) |
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151 | (1) |
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Membrane Preparations for Binding Assays |
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152 | (1) |
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Binding Assays on Wild-Type and Mutant M1 mAChRs |
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153 | (2) |
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Functional Assays on Mutant Receptors |
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155 | (1) |
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Immunocytochemistry and Immunofluorescence |
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156 | (1) |
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157 | (1) |
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Key Considerations in Performing Mutagenesis Experiments |
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157 | (12) |
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Measuring the Expression of Functional Receptors |
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157 | (1) |
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How Should Functional Data Be Expressed? The Contribution of Basal Signaling Activity |
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158 | (3) |
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The Effect of Variable Receptor Expression on the Functional Dose-Response Curve: The Effective Receptor:G Protein Ratio |
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161 | (3) |
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Mutants that Give No Measurable Binding of [ 3H]NMS |
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164 | (1) |
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164 | (2) |
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166 | (1) |
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Should We Be Using a Dimeric Receptor Ternary Complex Model to Analyze Binding and Dose-Response Data? |
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166 | (3) |
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The Interpretation of Ala-Scanning Mutagenesis Studies |
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169 | (4) |
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Determining the Functional Phenotype of Mutant Receptors |
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169 | (1) |
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Phenotypes Predicted by the Extended Ternary Complex Model of Agonist-Receptor-G Protein Interaction |
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170 | (1) |
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A Case Study: Transmembrane Helix 7 |
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171 | (2) |
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173 | (6) |
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175 | (1) |
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175 | (4) |
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Analysis of the Regulation of Muscarinic Acetylcholine Receptors |
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179 | (18) |
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179 | (1) |
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Radioligand Binding Assays to Measure mAChR Internalization and Downregulation |
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180 | (3) |
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Analysis of mAChR Internalization in Intact Cells Using [ 3H]NMS Binding Assay |
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180 | (2) |
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Analysis of mAChR Downregulation Using [ 3H]QNB Binding Assay to Cell Membranes |
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182 | (1) |
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[ 35S]GTPγS Assay to Measure Coupling of mAChR to Heterotrimeric G Proteins |
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183 | (3) |
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Analysis of mAChR Signal Transduction Pathways |
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186 | (11) |
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Phospholipase C Stimulation |
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186 | (2) |
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Phospholipase D Activation |
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188 | (3) |
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191 | (1) |
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Inhibition of cAMP Accumulation |
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192 | (3) |
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195 | (1) |
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195 | (2) |
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Single-Molecule Analysis of Chemotactic Signaling Mediated by cAMP Receptor on Living Cells |
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197 | (22) |
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198 | (3) |
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Single-Molecule Detection Techniques |
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198 | (1) |
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Chemotactic Signaling and GPCRs |
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199 | (2) |
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Total Internal Reflection Fluorescence Microscopy (TIRFM) |
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201 | (4) |
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202 | (1) |
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Configuration of Objective-Type TIRFM |
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203 | (1) |
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Measurement of Angle of Incident Laser Beam |
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204 | (1) |
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205 | (8) |
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Preparation of Fluorescent-Labeled cAMP Analogue |
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205 | (1) |
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Single-Molecule Imaging on Living Cells and Crude Membranes |
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206 | (1) |
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206 | (1) |
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Cell Preparation and Observation |
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207 | (1) |
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Crude Membrane Preparation |
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207 | (1) |
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Verification of Single-Molecule Imaging |
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208 | (1) |
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Dissociation Rate Analysis |
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208 | (4) |
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212 | (1) |
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Receptor States on Living Cells |
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212 | (1) |
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Green Fluorescent Protein (GFP) Imaging at Single-Molecule Level |
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213 | (6) |
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216 | (1) |
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216 | (1) |
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216 | (3) |
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Oligomerization of G Protein-Coupled Purinergic Receptors |
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219 | (24) |
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220 | (1) |
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Oligomerization of G Protein-Coupled Receptors |
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220 | (1) |
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220 | (1) |
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Analysis of Oligomerization of Purinergic Receptors |
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221 | (22) |
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221 | (1) |
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Transfection and Expression in HEK293T Cells |
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222 | (1) |
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223 | (2) |
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Adenylyl Cyclase Coupling in Co-Transfected Cells |
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225 | (2) |
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227 | (1) |
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Co-Immunoprecipitation of Epitope-Tagged A1R and P2Y1R in Co-Expressed HEK293T Cells |
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227 | (2) |
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Co-Immunoprecipitation of A1R and P2Y1R from Rat Brain |
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229 | (3) |
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Immunocytochemical Analysis |
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232 | (1) |
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Double Immunofluorescence Microscopy of Co-Expressed Purinergic Receptors |
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232 | (1) |
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233 | (1) |
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BRET of Co-Expressed Purinergic Receptors |
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234 | (1) |
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Plasmid Constructs, Transfection, and BRET2 Assay |
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235 | (3) |
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238 | (1) |
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238 | (1) |
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238 | (5) |
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PART III Tertiary Structure of GPCRs and Their Ligands |
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Methods and Results in X-Ray Crystallography of Bovine Rhodopsin |
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243 | (18) |
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243 | (1) |
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Purification and Crystallization |
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244 | (6) |
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Isolation of Membranes from Retina |
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244 | (2) |
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Purification of Membranes |
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246 | (1) |
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247 | (1) |
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247 | (2) |
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Application to Rhodopsin Analogue |
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249 | (1) |
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Structure Determination and Refinement |
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250 | (1) |
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Characterization of Crystals |
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250 | (1) |
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Summary of Structure Determination |
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251 | (1) |
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Structure Refinements at Higher Resolution |
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251 | (1) |
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251 | (6) |
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251 | (1) |
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252 | (1) |
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252 | (3) |
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255 | (2) |
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257 | (4) |
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258 | (1) |
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258 | (3) |
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Determination of Steric Structure of Muscarinic Ligands Bound to Muscarinic Acetylcholine Receptors: Approaches by TRNOE (Transferred Nuclear Overhauser Effect) |
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261 | (22) |
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262 | (1) |
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Expression and Purification of the M2 Receptors |
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263 | (6) |
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Methods for Expressing the M2 Mutant Using Sf9/Baculovirus |
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264 | (2) |
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Methods for Membrane Preparation and Solubilization |
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266 | (2) |
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Methods for Purification of the M2 Mutant |
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268 | (1) |
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Synthesis and Characterization of (S)-Methacholine |
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269 | (5) |
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Methods for Synthesis of (S)-Methacholine |
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271 | (1) |
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Methods for Activity Assays |
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271 | (3) |
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Determination of the (S)-Methacholine Conformation by NOESY and TRNOESY |
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274 | (4) |
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Methods for Determination of the Free (S)-Methacholine Conformation |
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275 | (2) |
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Methods for Determination of the Receptor-Bound (S)-Methacholine Conformation |
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277 | (1) |
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Assessment of (S)-Methacholine Conformation by Docking Studies |
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278 | (5) |
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280 | (1) |
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280 | (3) |
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Modeling of G Protein-Coupled Receptors for Drug Design |
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283 | (20) |
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283 | (2) |
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Structural Models of the Photointermediates, Batho, Lumi, and Meta I |
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285 | (4) |
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Structural Models of Photointermediates, Meta Ib and I380 |
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289 | (1) |
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Structural Model of Fully Activated Photointermediate, Meta II |
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290 | (1) |
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Functional Structures of GPCRs |
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291 | (2) |
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Receptor-Ligand Complex Models for Muscarinic Acetylcholine Receptor |
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293 | (3) |
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The Ligand-Binding Modes of Adrenergic b2 Receptor |
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296 | (2) |
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298 | (5) |
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299 | (1) |
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299 | (4) |
| Index |
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303 | |