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.

Part I. OverviewsTransmembrane Signaling by G Protein-Coupled ReceptorsLouis M. LuttrellCrosstalk Coregulation Mechanisms of G Protein-Coupled Receptors and Receptor Tyrosine KinasesKanchana Natarajan and Bradford C. BerkProtein-Protein Interactions in Signaling CascadesBruce J. MayerPart II. Specific TopicsA. Membrane Receptors and SignalingBiological Role of the CXCR4-SDF-1 Axis in Normal Human Hematopoietic CellsMarcin Majka and Mariusz Z. RatajczakFunctional Expression of CXCR4 in Saccharomyces cerevisiae in the Development of Powerful Tools for the Pharmacological Characterization of CXCR4Zi-xuan Wang, James R. Broach, and Stephen C. PeiperCharacterization of Constitutively Active Mutants of G Protein-Coupled ReceptorsJean-Marc Navenot, Zi-xuan Wang, and Stephen C. PeiperG Protein-Coupled Receptor Dimerization and SignalingMario Mellado, Antonio Serrano, Carlos Martínez-A, and José Miguel Rodríguez-FradeReal-Time Analysis of G Protein-Coupled Receptor Signaling in Live CellVenkatakrishna R. Jala and Bodduluri HaribabuB. Lipid Raft and Caveolae in Transmembrane SignalingIsolation of Membrane Rafts and Signaling ComplexesKathleen Boesze-BattagliaMethods for the Study of Signaling Molecules in Membrane Lipid Rafts and CaveolaeRennolds S. Ostrom and Paul A. InselC. Protein-Protein Interaction in Transmembrane SignalingBioluminescence Resonance Energy Transfer to Monitor Protein-Protein InteractionsTarik Issad and Ralf JockersIdentification of Interacting Proteins Using the Yeast Two-Hybrid ScreenKelly L. Jordan-Sciutto and Marshall B. MontgomeryAnalysis of PDZ Domain Interactions Using Yeast Two-Hybrid and Coimmunoprecipitation AssaysHyun Woo Lee, Jaewon Ko, and EunjoonKimClustering Assay for Studying the Interaction of Membrane Proteins With PDZ Domain ProteinsJaewon Ko and Eunjoon KimD. Rho Guanosine Triphosphatases and Reconstitution of Signaling ComplexesMammalian Cell Microinjection Assay to Study the Function of Rho Family Guanosine TriphosphatasesRitu Garg and Anne J. RidleyAffinity-Based Assay of Rho Guanosine Triphosphatase ActivationMary Stofega, Celine DerMardirossian, and Gary M. BokochAssay of Phospholipase D Activity in Cell-Free SystemsShankar S. Iyer and David J. KusnerReconstitution System Based on Cytosol-Depleted Cells to Study the Regulation of Phospholipase DAmanda Fensome-Green and Shamshad CockcroftE. Genomics and ProteomicsAnalysis of Global Gene Expression Profiles Activated by Chemoattractant ReceptorsFernando O. Martinez and Massimo LocatiGenetic Reconstitution of Bone Marrow for the Study of Signal Transduction Ex VivoMartha S. JordanProteomic Analysis of Human NeutrophilsGeorge Lominadze, Richard A. Ward, Jon B. Klein, and Kenneth R. McLeishIndex