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E-raamat: G Protein-Coupled Receptors: Structure, Function, and Ligand Screening

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  • Ilmumisaeg: 04-Aug-2005
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  • ISBN-13: 9781040202487
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  • Kirjastus: CRC Press Inc
  • Keel: eng
  • ISBN-13: 9781040202487
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In an extension rather than update of the 1999 volume in the series G Protein-Coupled Receptors , mostly Japanese scientists describe techniques used to investigate and manipulate the receptors that either were not treated in the earlier volume or have been developed since it appeared. They cover the screening of ligands for the receptors, receptor functions and regulation, and the tertiary structure of the receptors and their ligands. Among specific topics are the ligand screening of olfactory receptors, the analysis of the regulation of muscarinic acetylcholine receptors, and modeling G protein-coupled receptors for drug design. Annotation ©2005 Book News, Inc., Portland, OR (booknews.com)

This book provides a broad base of knowledge of G-protein-coupled receptors. Useful at both the university and industrial levels, this book is of particular interest to those who are developing therapeutic approaches to diseases using drugs that influence receptor activation.
PART I Screening of Ligands for GPCRs
Screening of Endogenous Ligands for Orphan GPCRs
3(34)
Masaaki Mori
Shuji Hinuma
Tetsuya Ohtaki
Masahiko Fujino
Introduction
4(1)
Ligand Sources
5(2)
Tissue Extract
5(1)
Known Molecules
6(1)
Database Search
6(1)
Particular Examples of Ligand Screening for the Orphan GPCRs
7(30)
Galanin-Like Peptide (GALP)
7(1)
Discovery of a Novel Galanin-Family Peptide, GALP
7(1)
Preparation of Tissue Extract Sample
7(2)
[ 35S] Guanosine 5'O-(γ-Thio)Triphosphate ([ 35S]GTPγS) Binding Assay
9(1)
Isolation of GALP
10(1)
Physiological Roles of GALP
10(2)
Metastin
12(1)
Discovery of Metastin, the Cognate Ligand of OT7T175 (= GPR54)
12(1)
FLIPR Assay
12(1)
Isolation of Metastin
13(1)
Structure of Metastin
14(1)
Receptor Interaction
14(3)
Physiological Roles of Metastin
17(1)
Neuropeptide W (NPW)
17(1)
GPR7 and GPR8
17(1)
Isolation and Identification of NPW as the Endogenous Ligand for the GPR7 and GPR8
18(2)
Neuropeptide B (NPB) as a Paralog Peptide of NPW
20(1)
Biological Functions of NPW and NPB
20(1)
Urotensin II (UII)
21(1)
GPR14
21(1)
Isolation and Identification of UII as the Endogenous Ligand for GPR14
21(3)
Pathophysiological Significance of UII
24(1)
Free Fatty Acids (FFAs)
24(1)
FFAs Are Signaling Molecules
24(1)
GPR40
25(1)
Identification of FFAs as Ligands for GPR40
25(2)
Role of GPR40 Expressed in βCells
27(1)
Pyroglutamylated RFamide Peptide (QRFP)
28(1)
RFamide Peptides in Mammals
28(1)
Identification of a Novel RFamide Peptide Gene
28(1)
Identification of a Receptor for QRFP
29(1)
Interaction between QRFP and AQ27
30(1)
Functions of QRFP and Its Receptor
30(1)
Acknowledgments
30(2)
Abbreviations
32(1)
References
32(5)
Screening of Ligands for Human GPCRs by the Use of Receptor-Gα Fusion Proteins
37(30)
Shigeki Takeda
Hinako Suga
Tatsuya Haga
Introduction
37(1)
List of Human GPCR Genes
38(4)
Screening of GPCRs from the Human Genome
38(2)
List of Useful Databases for GPCRs
40(2)
List of Useful Programs for GPCR and Other Protein Research
42(1)
Ligand Screening System for GPCRs Using Receptor-Gα Fusion Proteins
42(25)
Gene Construction for Receptor-Gα Fusion Proteins
44(5)
In vivo Cell-Based Assay
49(7)
In vitro [ 35S]GTγS Binding Assay
56(7)
Acknowledgments
63(1)
Abbreviations
63(1)
References
63(4)
Screening and Identification of Ghrelin, an Endogenous Ligand for GHS-R
67(18)
Masayasu Kojima
Kenji Kangawa
Introduction
68(1)
Reverse Pharmacology
68(1)
GHS (Growth-Hormone Secretagogue) and Its Receptor
69(2)
History
69(2)
GHS Receptor (GHS-R) Superfamily
71(1)
Purification of Ghrelin
71(8)
Construction of GHS-R Expressing Cells
71(2)
Intracellular Calcium Measurement Assay by FLIPR
73(1)
Purification of Rat Ghrelin
73(3)
Structural Determination of Rat Ghrelin
76(1)
Minor Molecular Forms of Human Ghrelin
76(1)
Ghrelin Precursor and Des-Gln14-Ghrelin
77(2)
Nonmammalian Ghrelin
79(6)
Bullfrog Ghrelin
79(1)
Chicken Ghrelin
80(1)
Fish Ghrelins
81(1)
References
81(4)
Ligand Screening of Olfactory Receptors
85(28)
Kazushige Touhara
Sayako Katada
Takao Nakagawa
Yuki Oka
Introduction
86(2)
The G Protein-Coupled Olfactory Receptor Superfamily
86(1)
Odorant Signaling Pathways
86(2)
Functional Expression of Olfactory Receptors in Olfactory Neurons
88(4)
Adenovirus-Mediated Gene Transfer
88(1)
Generation of GFP-Tagged Gene-Targeted Mice
89(1)
Ca2+ Imaging and EOG Recording
90(1)
Ca2+ Imaging Assay in Dissociated Olfactory Neurons
90(2)
EOG Recording
92(1)
Methods for Functional Receptor Expression in Heterologous Cells
92(3)
General Considerations
92(1)
N-Terminal Tagging and Glycosylation
93(1)
Chaperones for Membrane Trafficking
94(1)
Methods for Odorant Response Assays of Olfactory Receptors
95(9)
Ca2+ Imaging Assay
95(2)
cAMP Assay
97(2)
Luciferase Reporter Gene Assay
99(1)
Xenopus Oocyte Assay
100(1)
Assays for Multiple G Protein-Mediated Signals
100(2)
Measurement of Ion Currents with a Voltage Clamp Method
102(1)
Measurement with Depolarization Step Pulses
103(1)
Other Functional Assay Systems
104(1)
Structure-Activity Matrices of Olfactory Receptors
105(1)
Conclusions
106(7)
Acknowledgments
106(1)
References
107(6)
PART II Functions and Regulations of GPCRs
Generation and Phenotypical Analysis of Muscarinic Acetylcholine Receptor Knockout Mice
113(24)
Jurgen Wess
Dinesh Gautam
Sung-Jun Han
Jongrye Jeon
Cuiling Li
Chuxia Deng
Introduction
113(1)
General Strategy Used to Generate Mice Lacking Specific mAChRs
114(7)
Phenotypical Analysis of mAChR Knockout (KO) Mice
121(8)
Summary of Mutant Phenotypes
121(5)
Potential Compensatory Changes of mAChR Expression Levels in mAChR KO Mice
126(1)
Issues Related to the Mouse Genetic Background of the Analyzed mAChR KO Mice
127(1)
Generation and Analysis of mAChR Double KO Mice
128(1)
Relevance for Drug Therapy
129(2)
M1 Receptors
129(1)
M2 Receptors
129(1)
M3 Receptors
130(1)
M4 Receptors
130(1)
M5 Receptors
130(1)
Conclusions and Future Outlook
131(6)
Acknowledgments
131(1)
References
131(6)
Systematic Mutagenesis of M1 Muscarinic Acetylcholine Receptors
137(42)
Edward C. Hulme
Introduction
138(2)
G Protein-Coupled Receptors
138(1)
The Muscarinic Acetylcholine Receptors
139(1)
Mutagenesis Studies
140(1)
The Mechanistic Framework for the Interpretation of Mutagenesis Studies on GPCRs
141(8)
The Ternary Complex Model of Receptor-G Protein Interaction
141(6)
The Extended Ternary Complex Model
147(1)
The Dimeric Receptor Ternary Complex Model
148(1)
Experimental Techniques
149(8)
Choice of Target Sequences
149(1)
Expression Vector
150(1)
Site-Directed Mutagenesis
150(1)
Preparation of Plasmid DNA
151(1)
Transfection of Cells
151(1)
Membrane Preparations for Binding Assays
152(1)
Binding Assays on Wild-Type and Mutant M1 mAChRs
153(2)
Functional Assays on Mutant Receptors
155(1)
Immunocytochemistry and Immunofluorescence
156(1)
Data Analysis
157(1)
Key Considerations in Performing Mutagenesis Experiments
157(12)
Measuring the Expression of Functional Receptors
157(1)
How Should Functional Data Be Expressed? The Contribution of Basal Signaling Activity
158(3)
The Effect of Variable Receptor Expression on the Functional Dose-Response Curve: The Effective Receptor:G Protein Ratio
161(3)
Mutants that Give No Measurable Binding of [ 3H]NMS
164(1)
Reduced Expression
164(2)
Reduced Ligand Affinity
166(1)
Should We Be Using a Dimeric Receptor Ternary Complex Model to Analyze Binding and Dose-Response Data?
166(3)
The Interpretation of Ala-Scanning Mutagenesis Studies
169(4)
Determining the Functional Phenotype of Mutant Receptors
169(1)
Phenotypes Predicted by the Extended Ternary Complex Model of Agonist-Receptor-G Protein Interaction
170(1)
A Case Study: Transmembrane Helix 7
171(2)
Conclusions
173(6)
Acknowledgments
175(1)
References
175(4)
Analysis of the Regulation of Muscarinic Acetylcholine Receptors
179(18)
Chris J. van Koppen
Introduction
179(1)
Radioligand Binding Assays to Measure mAChR Internalization and Downregulation
180(3)
Analysis of mAChR Internalization in Intact Cells Using [ 3H]NMS Binding Assay
180(2)
Analysis of mAChR Downregulation Using [ 3H]QNB Binding Assay to Cell Membranes
182(1)
[ 35S]GTPγS Assay to Measure Coupling of mAChR to Heterotrimeric G Proteins
183(3)
Analysis of mAChR Signal Transduction Pathways
186(11)
Phospholipase C Stimulation
186(2)
Phospholipase D Activation
188(3)
Elevation of [ Ca2+]i
191(1)
Inhibition of cAMP Accumulation
192(3)
Acknowledgments
195(1)
References
195(2)
Single-Molecule Analysis of Chemotactic Signaling Mediated by cAMP Receptor on Living Cells
197(22)
Masahiro Ueda
Yukihiro Miyanaga
Toshio Yanagida
Introduction
198(3)
Single-Molecule Detection Techniques
198(1)
Chemotactic Signaling and GPCRs
199(2)
Total Internal Reflection Fluorescence Microscopy (TIRFM)
201(4)
Theory: Evanescent Field
202(1)
Configuration of Objective-Type TIRFM
203(1)
Measurement of Angle of Incident Laser Beam
204(1)
Ligand-Binding Analysis
205(8)
Preparation of Fluorescent-Labeled cAMP Analogue
205(1)
Single-Molecule Imaging on Living Cells and Crude Membranes
206(1)
Coverslip Preparation
206(1)
Cell Preparation and Observation
207(1)
Crude Membrane Preparation
207(1)
Verification of Single-Molecule Imaging
208(1)
Dissociation Rate Analysis
208(4)
GTP Sensitivity
212(1)
Receptor States on Living Cells
212(1)
Green Fluorescent Protein (GFP) Imaging at Single-Molecule Level
213(6)
Acknowledgments
216(1)
Abbreviations
216(1)
References
216(3)
Oligomerization of G Protein-Coupled Purinergic Receptors
219(24)
Hiroyasu Nakata
Kazuaki Yoshioka
Toshio Kamiya
Introduction
220(1)
Oligomerization of G Protein-Coupled Receptors
220(1)
Purinergic Receptors
220(1)
Analysis of Oligomerization of Purinergic Receptors
221(22)
Receptor Pharmacology
221(1)
Transfection and Expression in HEK293T Cells
222(1)
Ligand Binding
223(2)
Adenylyl Cyclase Coupling in Co-Transfected Cells
225(2)
Immunoprecipitation
227(1)
Co-Immunoprecipitation of Epitope-Tagged A1R and P2Y1R in Co-Expressed HEK293T Cells
227(2)
Co-Immunoprecipitation of A1R and P2Y1R from Rat Brain
229(3)
Immunocytochemical Analysis
232(1)
Double Immunofluorescence Microscopy of Co-Expressed Purinergic Receptors
232(1)
BRET Analysis
233(1)
BRET of Co-Expressed Purinergic Receptors
234(1)
Plasmid Constructs, Transfection, and BRET2 Assay
235(3)
Acknowledgments
238(1)
Abbreviations
238(1)
References
238(5)
PART III Tertiary Structure of GPCRs and Their Ligands
Methods and Results in X-Ray Crystallography of Bovine Rhodopsin
243(18)
Tetsuji Okada
Rumi Tsujimoto
Miho Muraoka
Chihiro Funamoto
Introduction
243(1)
Purification and Crystallization
244(6)
Isolation of Membranes from Retina
244(2)
Purification of Membranes
246(1)
Selective Solubilization
247(1)
Crystallization
247(2)
Application to Rhodopsin Analogue
249(1)
Structure Determination and Refinement
250(1)
Characterization of Crystals
250(1)
Summary of Structure Determination
251(1)
Structure Refinements at Higher Resolution
251(1)
Crystal Structure
251(6)
Crystal Lattice
251(1)
Overall Structure
252(1)
Transmembrane Region
252(3)
Constitutive Activity
255(2)
Remarks
257(4)
Acknowledgments
258(1)
References
258(3)
Determination of Steric Structure of Muscarinic Ligands Bound to Muscarinic Acetylcholine Receptors: Approaches by TRNOE (Transferred Nuclear Overhauser Effect)
261(22)
Hiroyasu Furukawa
Toshiyuki Hamada
Hiroshi Hirota
Masaji Ishiguro
Tatsuya Haga
Introduction
262(1)
Expression and Purification of the M2 Receptors
263(6)
Methods for Expressing the M2 Mutant Using Sf9/Baculovirus
264(2)
Methods for Membrane Preparation and Solubilization
266(2)
Methods for Purification of the M2 Mutant
268(1)
Synthesis and Characterization of (S)-Methacholine
269(5)
Methods for Synthesis of (S)-Methacholine
271(1)
Methods for Activity Assays
271(3)
Determination of the (S)-Methacholine Conformation by NOESY and TRNOESY
274(4)
Methods for Determination of the Free (S)-Methacholine Conformation
275(2)
Methods for Determination of the Receptor-Bound (S)-Methacholine Conformation
277(1)
Assessment of (S)-Methacholine Conformation by Docking Studies
278(5)
Acknowledgment
280(1)
References
280(3)
Modeling of G Protein-Coupled Receptors for Drug Design
283(20)
Masaji Ishiguro
Introduction
283(2)
Structural Models of the Photointermediates, Batho, Lumi, and Meta I
285(4)
Structural Models of Photointermediates, Meta Ib and I380
289(1)
Structural Model of Fully Activated Photointermediate, Meta II
290(1)
Functional Structures of GPCRs
291(2)
Receptor-Ligand Complex Models for Muscarinic Acetylcholine Receptor
293(3)
The Ligand-Binding Modes of Adrenergic b2 Receptor
296(2)
Summary
298(5)
Abbreviations
299(1)
References
299(4)
Index 303


Tatsuya Haga, Shigeki Takeda