Signal Transduction

1. Direct assessment of conformational changes in G protein-coupled receptors, using fluorescene spectroscopy. ; 1.1 Introduction ; 1.2 Expression and purification of the *b2 adrenergic receptor ; 1.3 Fluorescence labeling and spectroscopic analysis of the *b2 adrenergic receptor ; 1.4 Site-selective fluorescent labelling of the *b2 adrenergic receptor ; 1.5 Concluding remarks ; 2. Probing the structure of receptor-binding sites by the substituted-cysteine accessibility method ; 2.1 Introduction ; 2.2 Applications of the substituted-cysteine accessibility method (SCAM) ; 2.3 Critical parameters ; 2.4 Chemistry of SCAM ; 2.5 Interpretation of results ; 3. Post-translational acylation of signal transducing polypeptides: palmitoylation of G protein-coupled receptors ; 3.1 Introduction ; 3.2 Detection of receptor palmitoylation ; 3.3 Kinetics of palmitoylation ; 3.4 Assessing the palmitoylation state of the receptor at the cell surface ; 3.5 In vitro palmitoylation of synthetic peptides ; 4. G protein-coupled receptor phosphorylation and desensitization ; 4.1 Introduction ; 4.2 Techniques in the investigation of GPCR phosphorylation ; 4.3 Identification of desensitization of phospholiphase C-coupled receptors ; 5. G proteins and their identification ; 5.1 Introduction ; 5.2 Production of crude plasma membrane fractions for analysis of G proteins ; 5.3 Gel electrophoresis of G proteins ; 5.4 Immunological methods ; 5.5 Quantification of G protein *a subunits ; 5.6 Functional aspects of G protein signalling ; 6. Construction and analysis of receptor-G protein fusion proteins ; 6.1 Introduction ; 6.2 Methods to study G protein function ; 6.3 Receptor-G protein fusions ; 6.4 Expression of receptor-G protein fusions in cultured cells ; 6.5 Assays used for functional characterization of receptor-G protein fusions ; 6.6 Receptor-G protein fusions as research tools ; 6.7 Summary and future perspectives ; 7. Application of the baculoviral expression system to signal transduction ; 7.1 Introduction ; 7.3 The baculoviral life cycle, and constructing recombinant ; 7.4 Recombinant protein expression and purification ; 8. Reporter gene systems for the study of G protein-coupled receptor signal transduction in mammalian cells ; 8.1 Introduction ; 8.2 What is a reporter gene? ; 8.3 Reported gene systems for the study of GPCR signal transduciton ; 8.4 Factors influencing the design of a mammalian cell reporter-gene assay ; 8.5 Preparation of cells for reporter-gene assays ; 8.6 Reporter enzyme assays ; 8.7 Reporter protein assays ; 8.8 Summary and future perspectives ; 9. Adenylyl cyclases and cyclic AMP ; 9.1 Introduction ; 9.2 Mammalian expression of recombinant Acs ; 9.3 Measurement of intracellular cAMP level ; 9.4 CAMP-responsive bioluminescence assays using firefly luciferase