Reconstitution of G protein-coupled receptors with recombinant G protein α and βγ subunits

Publisher Summary This chapter describes experiments that can probe the first steps in receptor–G protein interaction using defined, recombinant receptors and G proteins. A basic issue in cell signaling is the identification of those receptors that interact with specific isoforms of the multiple G protein α and βγ subunits. At the molecular level, it is important to understand which domains of the G protein α and βγ subunits interact with the intracellular loops and transmembrane helices of receptors. Thus, it is useful to have methods to study the receptor–G protein interaction with recombinant proteins. To date, many techniques have been developed to examine this question, which use intact cells, isolated plasma membranes, and/or purified receptors, and G proteins. The protocols have the advantages that the receptors are inserted properly in a cell membrane and that the investigator has complete control of the proteins reconstituted with the receptor. Specific mutations in the receptors or G proteins are studied easily and the protocols allow precise examination of the stoichiometry of the receptor–α–βγ interaction.