Transmembrane Signalling Protocols

About this book

This fully updated and expanded second edition of Transmembrane Signaling Protocols demonstrates the use of such techniques as single cell imaging, bioluminecence resonance energy transfer, and global proteomics in the study of transmembrane signaling events. Highlights include the functional expression and genetic selection of mutant mammalian transmembrane receptors in yeast, real-time analysis of transmembrane receptor signaling in live cells, and bioluminescence energy transfer to monitor protein-protein interaction. Additional chapters use state-of-the-art methods to analyze gene expression and proteomic profiles related to transmembrane signaling events, as well as to genetically reconstitute bone marrow for the study of signal transduction ex vivo. The protocols follow the successful Methods in Molecular BiologyT series format, each offering step-by-step laboratory instructions, an introduction outlining the principles behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.

Contents

Part I. Overviews. Peptide Recognition Mechanisms of Eukaryotic Signaling Modules C.-H. Lee D. Cowburn and J. Kuriyan. Protein-Protein Interactions in Signaling Cascades B. J. Mayer. Transmembrane Signaling by Receptor Oligomerization M. A. Lemmon and J. Schlessinger. Part II. Specific Topics. A. Use of Peptide Libraries to Study Transmembrane Signaling. Use of Peptide Libraries to Determine Optimal Substrates of Tyrosine Kinases P. M. Chan and W. T. Miller. Mapping the Specificity of SH3 Domains with Phage-Displayed Random-Peptide Libraries A. B. Sparks J. E. Rider and B. K. Kay. B. Use of Antisense Technology to Study Transmembrane Signaling. Selective Antagonism of Receptor Signaling Using Antisense RNA to Deplete G-Protein Subunits P. R. Albert and S. J. Morris. Microinjection of Antisense Oligonucleotides and Electrophysiological Recording of Whole-Cell Currents as Tools to Identify Specific G-Protein Subtypes Coupling Hormone Receptors to Voltage-Gated Calcium Channels V. E. Degtiar B. Wittig G. Schultz and F. Kalkbrenner. C. Use of Single-Cell Assays to Analyze Signal Transduction Pathways. Oocyte Microinjection Assay to Study the MAP-Kinase Cascade J. C. Lacal. Mammalian Cell Microinjection Assay to Study the Function of Rac and Rho A. J. Ridley. D. Reconstitution of Signaling Complexes. Identification and Functional Reconstitution of Effector Proteins for the GTPases Rac and CDC42Hs A. Abo. Cell-Free Assay System for Ras- and Rap1-Dependent Activation of MAP-Kinase Cascade K. Shimizu T. Ohtsuka and Y. Takai. Reconstitution System Based on Cytosol-Depleted Cells to Study the Regulation of Phospholipases C and D S. Cockcroft. E. Methods for Analyzing Protein-Protein Interactions. Two-Hybrid Analysis of Ras-Raf Interactions L. Van Aelst. Cloning and Mutational Analysis of the Sho-Phosphotyrosine Interaction/Phosphotyrosine- Binding Domain V. Yajnik P. Blaikie and B. Margolis. Use of Fluorescence Spectroscopy to Study the Regulation of Small G Proteins T. Nomanbhoy and R. A. Cerione. F. Posttranslational Processing of Signaling Proteins. Prenylation Assays for Small GTPases M. C. Seabra and G. L. James. Analysis of Myristoylated and Palmitoylated Src Family Proteins A. Wolven W. van't Hof and M. D. Resh. Ultracentrifugation Technique for Measuring the Binding of Peptides and Proteins to Sucrose-Loaded Phospholipid Vesicles C. A. Buser and S. McLaughlin. Biochemical and Biological Analyses of Farnesyl-Protein Transferase Inhibitors N. E. Kohl K. S. Koblan C. A. Omer A. Oliff and J. B. Gibbs. G. Kinases and Phosphatases in Signal Transduction. Identification and Characterization of Small GTPase-Associated Kinases E. Manser T. Leung and L. Lim. Functional Studies of Dual- Specificity Phosphatases H. Sun. Index.

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