; In most cases, it is not known which receptors activate which G proteins to modulate which effectors in vivo. Reconstitution approaches have begun to provide invaluable information on the potential of specific G protein alpha or beta-gamma subunits to interact with particular receptors or effectors in vitro. Nevertheless, it is clear that there is a far wider range of potential interactions that occur in reconstituted systems than actually do occur in native membrane or cellular systems. This is exemplified by the finding that beta-adrenergic receptor activates both Gs and Gi equally in a reconstituted system, whereas this receptor activates Gs exclusively in an intact cardiac cell system. From these and other studies, it has become increasingly clear that although a receptor can activate several types of G proteins in vitro, the signaling pathways that the receptor regulates in vivo is much more restricted. This suggests that homology among signaling components and/or the artificial assay systems that have been employed in vitro do not provide the conditions necessary to distinguish what may be subtle, but important, mechanistic differences among signaling components that operate in vivo. There is increasing evidence that these mechanistic differences reflect not only biochemical, but also spatial differences, among signaling components. Hence, ascribing functional roles for G protein alpha and beta-gamma subunits in vivo remains a critical and formidable task. The focus of this application is on the development and application of new approaches to allow the role of the specific beta-gamma dimer in a particular receptor-effector pathway to be determined in vivo. Multiple approaches, including reverse genetics, biochemical, and immunological strategies, will be taken. Validation of results with all of these approaches will provide strong support for the role of a specific beta-gamma dimer in a particular receptor signaling pathway.
