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The previous edition of Transmembrane Signaling Protocols was published in 1998. Since then the human genome has been completely sequenced and new methods have been developed for the use of microarrays and proteomics to analyze global changes in gene expression and protein profiles. These advances have increased our ability to understand transmembrane signaling processes in much greater detail. They have also simultaneously enhanced our ability to determine the role of a large number of newly identified molecules in signaling events. In addition, novel video microscopy methods have been developed to image transmembrane signaling events in live cells in real time. In view of these major advances, it is time to update the previous edition. Because of the success of that volume, we have chosen to keep the essential character of the book intact. Introductory chapters from experts have been included to provide overall perspective and an overview of recent advances in signal transduction pathways. The individual chapters now include comp- hensive detailed methods, studies in genetically tractable systems, fluorescence microscopy in live single cells, ex vivo analysis of primary cells from tra- genic mice, as well as genomic and proteomic approaches to the analysis of transmembrane signaling events. We would like to express our deep gratitude to the coauthors of this publi- tion. We hope that Transmembrane Signaling Protocols, Second Edition will serve as a valuable resource for future progress in the study of signal transd- tion pathways.

The previous edition of Transmembrane Signaling Protocols was published in 1998. Since then the human genome has been completely sequenced and new methods have been developed for the use of microarrays and proteomics to analyze global changes in gene expression and protein profiles. These advances have increased our ability to understand transmembrane signaling processes in much greater detail. They have also simultaneously enhanced our ability to determine the role of a large number of newly identified molecules in signaling events. In addition, novel video microscopy methods have been developed to image transmembrane signaling events in live cells in real time. In view of these major advances, it is time to update the previous edition. Because of the success of that volume, we have chosen to keep the essential character of the book intact. Introductory chapters from experts have been included to provide overall perspective and an overview of recent advances in signal transduction pathways. The individual chapters now include comp- hensive detailed methods, studies in genetically tractable systems, fluorescence microscopy in live single cells, ex vivo analysis of primary cells from tra- genic mice, as well as genomic and proteomic approaches to the analysis of transmembrane signaling events. We would like to express our deep gratitude to the coauthors of this publi- tion. We hope that Transmembrane Signaling Protocols, Second Edition will serve as a valuable resource for future progress in the study of signal transd- tion pathways.

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The complete sequencing of the human genome and many new microarray and proteomic methods for analyzing global changes in both gene expression and protein profiles have greatly increased our ability to understand transmembrane signaling processes in close detail. In this fully updated and expanded second edition of Transmembrane Signaling Protocols, leading experts demonstrate the use of such techniques as single-cell imaging, bioluminescence 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. Introductory chapters provide a wide-ranging perspective, including an overview of the many recent advances in our understanding of signal transduction pathways. The protocols follow the successful Methods in Molecular Biology™ 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.
Cutting-edge and highly practical, Transmembrane Signaling Protocols, Second Edition, makes available to novice and experienced researchers alike the readily reproducible techniques needed to advance the study of signal transduction pathways today.

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Part I. Overviews
Transmembrane Signaling by G Protein-Coupled Receptors
Louis M. Luttrell
Crosstalk Coregulation Mechanisms of G Protein-Coupled Receptors and Receptor Tyrosine Kinases
Kanchana Natarajan and Bradford C. Berk
Protein-Protein Interactions in Signaling Cascades
Bruce J. Mayer
Part II. Specific Topics
A. Membrane Receptors and Signaling
Biological Role of the CXCR4-SDF-1 Axis in Normal Human Hematopoietic Cells
Marcin Majka and Mariusz Z. Ratajczak Functional Expression of CXCR4 in Saccharomyces cerevisiae in the Development of Powerful Tools for the Pharmacological Characterization of CXCR4
Zi-xuan Wang, James R. Broach, and Stephen C. Peiper
Characterization of Constitutively Active Mutants of G Protein-Coupled Receptors
Jean-Marc Navenot, Zi-xuan Wang, and Stephen C. Peiper
G Protein-Coupled Receptor Dimerization and Signaling
Mario Mellado, Antonio Serrano, Carlos Martinez-A, and Jose Miguel Rodriguez-Frade
Real-Time Analysis of G Protein-Coupled Receptor Signaling in Live Cell
Venkatakrishna R. Jala and Bodduluri Haribabu
B. Lipid Raft and Caveolae in Transmembrane Signaling
Isolation of Membrane Rafts and Signaling Complexes
Kathleen Boesze-Battaglia
Methods for the Study of Signaling Molecules in Membrane Lipid Rafts and Caveolae
Rennolds S. Ostrom and Paul A. Insel
C. Protein-Protein Interaction in Transmembrane Signaling
Bioluminescence Resonance Energy Transfer to Monitor Protein-Protein Interactions
Tarik Issad and Ralf Jockers
Identification of Interacting Proteins Using the Yeast Two-Hybrid Screen
Kelly L. Jordan-Sciutto and Marshall B. Montgomery
Analysis of PDZ Domain Interactions Using Yeast Two-Hybrid and Coimmunoprecipitation Assays
Hyun Woo Lee, Jaewon Ko, and EunjoonKim
Clustering Assay for Studying the Interaction of Membrane Proteins With PDZ Domain Proteins
Jaewon Ko and Eunjoon Kim
D. Rho Guanosine Triphosphatases and Reconstitution of Signaling Complexes
Mammalian Cell Microinjection Assay to Study the Function of Rho Family Guanosine Triphosphatases
Ritu Garg and Anne J. Ridley
Affinity-Based Assay of Rho Guanosine Triphosphatase Activation
Mary Stofega, Celine DerMardirossian, and Gary M. Bokoch
Assay of Phospholipase D Activity in Cell-Free Systems
Shankar S. Iyer and David J. Kusner
Reconstitution System Based on Cytosol-Depleted Cells to Study the Regulation of Phospholipase D
Amanda Fensome-Green and Shamshad Cockcroft
E. Genomics and Proteomics
Analysis of Global Gene Expression Profiles Activated by Chemoattractant Receptors
Fernando O. Martinez and Massimo Locati
Genetic Reconstitution of Bone Marrow for the Study of Signal Transduction Ex Vivo
Martha S. Jordan
Proteomic Analysis of Human Neutrophils
George Lominadze, Richard A. Ward, Jon B. Klein, and Kenneth R. McLeish
Index

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