Methods in Signal Transduction, CRC Press LLC, 2000 Corporate Blvd., NW Boca Raton FL 33431 USA, 1999, 301 pp
The first five chapters of this book deal with the expression and functional analysis of mammalian G protein subunits in cells of bacterial, insect, mammalian, and yeast origin. Escherichia coli and Sf9 insect cells are key sources of subunits for work in the areas of reconstitution, structural and biochemical analyses, and microinjection. Bacterial expression systems are amenable to large-scale production of recombinant subunits whose purification is relatively straightforward. The next four chapters cover the techniques of evaluating G protein regulation. A critical endeavor in this field is the deployment of antibodies to assess subunit expression and covalent modification within cells. The production of antibodies and their use for Western blots and immunoprecipitation therefore assumes a prominent place in any set of techniques. Procedures of measuring the basic parameters of GTP binding and hydrolysis are also quite important, as they provide an understanding of the activation and deactivation of G proteins at a biochemical level. Superimposed on this is the need to evaluate the regulation imposed by the growing array of RGS proteins. Fatty-acid acylation has also emerged as a critical determinant in G protein function. The remaining five chapters focus on techniques for mapping pathways established between receptors, G proteins, and effectors. These chapters address a critical need for defining how the cell sets up specific linkages among the three proteins and determining how these linkages change from cell to cell and in response to regulatory pressures. Utilization of mutants of G sub(i) and G sub(o) resistant to pertussis toxin holds considerable potential in discriminating subtypes of this widely used family of G proteins. Microinjection of antibodies that disrupt interactions between receptors and G proteins represents a means of blocking pathways of transduction that can be analyzed in single cells. Expression of antisense molecules represents another technique for ablation, and can be extended from the intact cell to a transgenic animal. Each chapter is designed to provide a step-by-step description of how a given technique is performed. Each presents an overview of the technique, an account of the technique, and a section on commonly encountered problems. The overview is intended to provide useful background on the subject, together with references and any necessary theory. The Problems section covers troubleshooting and alternative methodologies. Each chapter concludes with examples of data obtained, ranges of values, and controls.