Front Cover; Series Editors; Methods in Cell Biology G Protein-Coupled Receptors: Signaling, Trafficking and Regulation; Copyright; Contents; Contributors; Preface; 01 -- Trafficking, Localization and Imaging of GPCRs; 1 -- Localization and signaling of GPCRs in lipid rafts; INTRODUCTION; 1. LOCALIZATION OF GPCRS IN LIPID RAFTS; 1.1 Isolation of Lipid Rafts; 1.1.1 Detergent-free method; 1.1.2 Detergent-based method; 1.1.3 Immunoblotting and data interpretation; 1.2 Localization of GPCRs in Lipid Rafts; 1.2.1 Cells in suspension; 1.2.2 Adherent cells; 2. GPCR SIGNALING IN LIPID RAFTS

2.1 Perturbation of Raft Stability2.2 Changing the Cholesterol Content; 2.3 Fluorescence Imaging; REFERENCES; 2 -- Imaging GPCRs trafficking and signaling with total internal reflection fluorescence microscopy in cultured neurons; 1. IMAGE ACQUISITION; 1.1 Important Considerations before Imaging; 1.2 Materials; 1.2.1 Cell culture; 1.2.2 TIRF microscopy equipment and settings; 1.3 Imaging; 1.3.1 Cell culture; 1.3.2 Live cell imaging; 1.4 Notes; 2. IMAGE ANALYSIS; 3. FINAL COMMENTS; Acknowledgments; REFERENCES; 3 -- Trafficking of ciliary G protein-coupled receptors; INTRODUCTION

1. RHODOPSIN AS A CILIARY GPCR2. CILIARY GPCRS IN OTHER NEURONS; 3. OLFACTORY SENSORY NEURON CILIARY GPCRS; 4. DYNAMIC TRAFFICKING OF CILIARY GPCRS; CONCLUSIONS AND FUTURE DIRECTIONS; REFERENCES; 4 -- Single-molecule resolution of G protein-coupled receptor (GPCR) complexes; INTRODUCTION; 1. IMAGING METHODOLOGIES TO STUDY SINGLE GPCR MOLECULES; 1.1 Current Approaches to Visualize Individual GPCRs as Monomers, Dimers, and Oligomers; 1.2 Super-Resolution Imaging of Single Molecules: Resolving Lots and Lots of Spots ... ; 2. METHODS FOR PHOTOACTIVATED DYE-LOCALIZATION MICROSCOPY

2.1 Seeing Single Molecules2.1.1 Choice of probe and labeling; 2.1.2 Optimization steps-labeling cells, blocking buffers, and fixatives; 2.1.3 Imaging considerations and conditions; 2.2 Counting Single Molecules; 2.2.1 Resolving single-molecule data; 2.2.2 Custom quantification of GPCR monomers, dimers, and oligomers; CONCLUSIONS AND PERSPECTIVES; Acknowledgments; REFERENCES; 5 -- Quantification of the mRNA expression of G protein-coupled receptors in human adipose tissue; INTRODUCTION; 1. MATERIALS; 1.1 Handling of Adipose Tissue Biopsies; 1.2 Adipose Tissue Homogenization

1.3 Adipose Tissue RNA Purification and cDNA Generation1.4 qPCR Quantification of GPCR Expression Relative the Adipose Tissue Housekeeping Gene PPIA; 2. METHODS; 2.1 Handling of Adipose Tissue Biopsies; 2.2 Adipose Tissue Homogenization; 2.3 Adipose Tissue RNA Purification and cDNA Generation; 2.4 qPCR Quantification of GPCR Expression Relative to the Housekeeping Gene PPIA; 2.4.1 Screening of adipose tissue GPCR mRNA expression using pooled cDNA; 2.4.2 Quantification of GPCR expression relative to the expression of the adipose tissue housekeeping gene PPIA