Signal Transduction: A Practical ApproachGraeme Milligan Since the publication of the first edition of Signal Transduction: A Practical Approach in 1992 there has been a great deal of new information about the processes of signal transduction and consequently many new methods have been developed. This new edition has therefore been updated and extended to include the major new methods now available. The first part of the book is mainly concerned with G protein-coupled receptors and covers structural studies of conformational changes and binding sites, phosphorylation and desensitisation, identification, receptor fusion proteins, and reporter gene systems. The second part includes methods for studying components of the other major families of signal transduction: adenylyl cylase and cAMP, phosphorylated inositol lipids, phosphinositide 3-kinases, phosphlipase D and phosphatidylcholine, sphingosine kinase, and inositol 1,4,5-triphosphate. Also included are chapters on baculoviral expression systems and the quantitative assay of mitogen activated protein kinases in intact cells and tissues. As with the previous edition Signal Transduction 2e covers a wide range of techniques and will be useful to both experienced researchers and newcomers. |
Contents
Direct assessment of conformational changes | 1 |
Phospholipase D and phosphatidylcholine | 12 |
Probing the structure of receptorbinding | 19 |
Posttranslational acylation of signal | 35 |
G proteincoupled receptor phosphorylation | 59 |
MULLANEY | 73 |
Department of Pharmacology University of Otago PO Box 913 Dunedin | 87 |
Construction and analysis of receptorG | 103 |
References | 249 |
Analysis of the polyphosphorylated | 255 |
Extraction of inositol lipids from yeast | 263 |
resulting watersoluble GroPInsP₁s | 271 |
References | 280 |
P T HAWKINS | 283 |
Measurement of the activation of PI3K | 287 |
Summary | 298 |
Expression of receptorG protein fusions in cultured cells | 110 |
Assays used for functional characterization of receptorG | 113 |
Summary and future perspectives | 134 |
The baculoviral life cycle and constructing recombinant | 144 |
Recombinant protein expression and purification | 156 |
References | 168 |
GIBBS | 171 |
Reporter gene systems for the study of GPCR signal | 178 |
Factors influencing the design of a mammalian cell | 189 |
Preparation of cells for reportergene assays | 197 |
Reporter protein assays | 214 |
Adenylyl cyclases and cyclic AMP | 223 |
metabolism | 301 |
PLD assays with brokencell preparations | 309 |
Acknowledgements | 317 |
How to study involvement of sphingosine kinase in | 325 |
Detection isolation and quantitative assay | 337 |
Measuring inositol 145trisphosphate | 361 |
37 | 363 |
U + + + + | 370 |
51 | 384 |
Appendix | 385 |
| 390 | |
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Common terms and phrases
acid activity adenylyl cyclase aequorin agonist amino antibody assay baculovirus binding Biochem Biol buffer Ca2+ cell lines centrifugation Chem column concentration containing COS-7 cells culture cysteine deacylated described in Protocol detection diluted EDTA Equipment and reagents expression extraction Figure firefly luciferase flask fluorescence fractions fusion protein G protein G₁₁a GPCR growth medium GTPase H₂O HPLC immunoprecipitation incubate inositol intracellular labelling ligand lipids lysis mammalian cells MAP kinase measured Method microcentrifuge ml¹ mmol molecular monolayers MTS reagents mutant NaCl palmitoylation pellet peptide pertussis pertussis toxin phase phosphoinositides phosphorylation PI3K plasma membrane plasmid plate promoter purified radioactivity reaction receptor receptor-G protein recombinant Renilla luciferase reporter enzyme reporter gene residues resuspend room temperature sample scintillation SDS-PAGE sequence Sf9 cells signal solubilized solution specific substrate supernatant toxin transcription factor transfection tube vector viral wash yeast µg ml-¹
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