Receptor-mediated activation of heterotrimeric G-proteins in living cells

Receptor-mediated activation of heterotrimeric CTP-binding proteins (C-prot eins) was visualized in living Dictyostelium discoideum cells by monitoring fluorescence resonance energy transfer (FRET) between alpha- and beta- sub units fused to cyan and yellow fluorescent proteins. The G-protein heterotr imer rapidly dissociated and reassociated upon addition and removal of chem oattractant, During continuous stimulation, C-protein activation reached a dose-dependent steady-state Level. Even though physiological responses subs ided, the activation did not decline. Thus, adaptation occurs at another po int in the signaling pathway, and occupied receptors, whether or not they a re phosphorylated, catalyze the C-protein cycle. Construction of similar en ergy-transfer pairs of mammalian G-proteins should enable direct in situ me chanistic studies and applications such as drug screening and identifying L igands of newly found C-protein-coupled receptors.