Receptor Biochemistry and Methodology - Böcker

The past decade has seen tremendous advances in the study of G protein-coupled receptors (GPCRs), including the molecular cloning and identification of more than 100 hundred GPCR genes. But while GPCRs serve as targets for more than 300 medicines in the modern pharmacopoeia, the shrinking pool of known ligands and the continuing discovery of orphan GPCR genes have underscored the need for new approaches to ligand identification. Identification and Expression of G Protein-Coupled Receptors addresses this new direction in GPCR biochemistry-offering a definitive laboratory bench manual that emphasizes expression over primary cloning strategies. In a series of expert contributions by well-known researchers, this book provides detailed protocols for various expression systems-from bacteria to mammalian cells-as well as straightforward opinions on the advantages and shortcomings of each approach. Topics covered include:* Homology screening and the polymerase chain reaction in the cloning of GPCR genes* Cloning of GPCRs using mammalian cell expression* GPCR informatics and the orphan problem* The use of Xenopus laevis oocytes for the study of GPCRs* Stable expression of GPCRs in mammalian cells* Heterologous expression in primary cell cultures* Expression of GPCR in Escherichia coli* Large scale expression and purification of GPCRs in mammalian cells* High-level expression of GPCRs in the Baculovirus/Sf9 cell expression system* Expression of GPCRs in Drosophila Schneider 2 cells* Methods for genetic analysis and ligand identification using heterologous GPCRs expressed in Saccharomyces cerevisiae Supplemented with numerous photographs and illustrations, Identification and Expression of G Protein-Coupled Receptors is important reading for biochemists, pharmacologists, neuroscientists, structural biologists, and anyone involved in GPCR-based research. It delivers a wealth of useful advice, practical tips, and invaluable insight into trends at the cutting-edge of current research.

The detection of neurotransmitter receptor locations and distribution densities within the central nervous system and peripheral tissues is receiving intense attention within the neuroscience research community. Neurotransmitter receptors, which receive the chemical signals sent from one neuron to another, are critical links in a highly complex information-processing chain. Pinpointing receptor sites and systems is crucial for understanding neurological function as well as dysfunction. It is also essential for understanding how receptors process information when impacted by such substances as heroin or nicotine, or when affected by neurodegenerative disease. Receptor Localization: Laboratory Methods and Procedures is the first user-friendly guide to the latest techniques and approaches being employed to examine the localization of neurotransmitter receptors in the central nervous system and peripheral tissues. It covers detection methods that are applicable to a wide variety of receptor systems, ranging from genes and ligands to in vito receptors in individuals; and to numerous receptor subtypes, such as nicotine, muscarine, tackykinins, dopamine, adenosine, and GABA. The standard laboratory "recipes" or "tricks" employed in these detection methods are fully discussed, as are the advantages and limitations of each procedure. With contributions from leading experts and extensively illustrated, this book:* Discusses receptor ligand binding methods using irreversible and reversible compounds* Presents antireceptor antisera technology using synthetic peptides and fusion proteins at both the cellular and subcellular resolution levels* Examines molecular assessments of receptors* Describes in situ hybridization, reverse transcriptase-PCR, and fluorescent in situ transcription* Covers new visualization paradigms* Includes physiological analysis of receptor function, cellular detection in the brain slice, and cultured neurons* Discusses the use of PET and SPECT to assess in vivo receptor distributions in animals and humans Receptor Localization: Laboratory Methods and Procedures is an invaluable guide for researchers in the related fields of neurology, biochemistry, and pharmacology. Its lucid descriptions of new detection methods, inclusion of experimental examples, and emphasis on how these experimental approaches are applicable to particular research areas will appeal to both the experienced researcher and novice investigator.

This exciting volume elucidates how GPCRs organize signal transduction and control intracellular activities. Chapters written by the recognized expert in each respective topic offer state-of-the-art updates on the principles and technology, as well as stepwise protocols for methods currently applied to the analysis of protein-receptor interactions.

G protein-coupled receptors (GPCRs) are the largest single class of receptors in biology, often playing key roles in a remarkably large number of physiological and pathophysiological conditions. GPCRs or GPCR-dependent signalling pathways are the targets of a very large number of therapeutically useful drugs. Detailed knowledge about the molecular structure of GPCRs should therefore pave the way for the design of novel drugs with increased efficacy and specificity. This volume provides a concise, up-to-date presentation of methods (including molecular genetic, biochemical, and biophysical) which have been used successfully in studying the structure and function of GPCRs. With contributions from international leaders in the field, the editor provides overviews of various techniques, followed by in-depth descriptions of basic procedures and discussions of critical experimental parameters. Divided into specific, accessible sections, Structure-Function Analysis of G Protein-Coupled Receptors includes:* An overview of mutagenesis techniques* Examples of molecular modeling techniques* Using peptides as tools for the study of GPCR interactions* Site-Directed Spin Labeling (SDSL) studies of the GPCR rhodopsin* A complete description of the electron-crystalographic analysis of two-dimensional rhodopsin crystals* The use of nuclear magnetic resonance techniques to study GPCR structure