Identification of receptor-activated G proteins: Selective immunoprecipitation of photolabeled G-protein α subunits

Reconstitution and cotransfection experiments are powerful tools to study the interaction of receptors, G proteins, and effectors. To examine receptor–G-protein coupling at natural concentrations of the individual components, methods for in situ determination of receptor–G-protein interaction have been developed. These approaches are based on the action of specific antisense oligonucleotides or on the functional effects of subtype-specific antibodies. In some cases, G proteins coupled to receptors can be identified with specific antibodies in isolated receptor–G-protein complexes or by receptor-stimulated adenosine diphosphate (ADP)-ribosylation of subunits by cholera toxin. This chapter describes the application of [α-³²P]guanosine-5'-triphosphate (GTP) azidoanilide as a tool to label α subunits of receptor-activated G proteins. Because the number of known G proteins, which are defined by their α-subunits, has constantly increased, it was necessary to improve the selective identification of G-protein α subunits photolabeled in response to receptor activation. This was achieved by combining photolabeling of receptor-activated G proteins by [α-³²P]GTP azidoanilide in membranes with immunoprecipitation of the photolabeled G-protein α subunits by subtype-specific antisera. Whereas this experimental approach is not suitable for identification of βγ complexes involved in receptor-G-protein coupling, it allows the exact identification of receptor-activated G proteins provided that specific precipitating antisera are available.