31] Fluorescence assays for G-protein interactions

Abstract

Several fluorescence spectroscopic approaches have been investigated over the past several years for monitoring the guanosine-5'-triphosphate (GTP)-binding/GTPase cycle of G proteins and for monitoring the interactions of G-protein α and βγ subunits with one another and with the receptor and effector proteins. The vertebrate phototransduction system is used as a model for investigations because it is possible to isolate each of the components of this system in milligram quantities and in highly purified form. This system is comprised of the receptor protein rhodopsin, the G protein transducin, and the effector enzyme, the cyclic guanosine-monophosphate (GMP) phosphodiesterase; light activation of rhodopsin stimulates the exchange of guanosine-diphosphate (GDP) for GTP on transducin, and it leads to the stimulation of cyclic GMP hydrolysis by the phosphodiesterase. It is expected that the development of fluorescence approaches for studying the phototransduction system will be generally applicable to a number of G-protein signaling systems, especially as expression systems are developed for the large-scale generation of various signaling proteins. This chapter describes techniques that have been used to monitor the GTP-binding and the GTPase activities of G-protein a subunits through changes in intrinsic tryptophan fluorescence and the use of extrinsic reporter groups to read out G-protein interactions with other signal transduction components.