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.

Overviews.- Transmembrane Signaling by G Protein-Coupled Receptors.- Crosstalk Coregulation Mechanisms of G Protein- Coupled Receptors and Receptor Tyrosine Kinases.- Protein-Protein Interactions in Signaling Cascades.- Specific Topics.- Biological Role of the CXCR4-SDF-1 Axis in Normal Human Hematopoietic Cells.- Functional Expression of CXCR4 in Saccharomyces cerevisiae in the Development of Powerful Tools for the Pharmacological Characterization of CXCR4.- Characterization of Constitutively Active Mutants of G Protein-Coupled Receptors.- G Protein-Coupled Receptor Dimerization and Signaling.- Real-Time Analysis of G Protein-Coupled Receptor Signaling in Live Cells.- Isolation of Membrane Rafts and Signaling Complexes.- Methods for the Study of Signaling Molecules in Membrane Lipid Rafts and Caveolae.- Bioluminescence Resonance Energy Transfer to Monitor Protein-Protein Interactions.- Identification of Interacting Proteins Using the Yeast Two-Hybrid Screen.- Analysis of PDZ Domain Interactions Using Yeast Two-Hybrid and Coimmunoprecipitation Assays.- Clustering Assay for Studying the Interaction of Membrane Proteins with PDZ Domain Proteins.- Mammalian Cell Microinjection Assay to Study the Function of Rho Family Guanosine Triphosphatases.- Affinity-Based Assay of Rho Guanosine Triphosphatase Activation.- Assay of Phospholipase D Activity in Cell-Free Systems.- Reconstitution System Based on Cytosol-Depleted Cells to Study the Regulation of Phospholipase D.- Analysis of Global Gene Expression Profiles Activated by Chemoattractant Receptors.- Genetic Reconstitution of Bone Marrow for the Study of Signal Transduction Ex Vivo.- Proteomic Analysis of Human Neutrophils.