EXPERIMENTAL FIGURE 15-15 Activation of a G protein occurs within seconds of ligand binding to its cell-surface G protein–coupled receptor. In the amoeba Dictyostelium discoideum, cAMP acts as an extracellular signaling molecule and binds to and signals via a G protein–coupled receptor; it is not a second messenger. Amoeba cells were transfected with genes encoding two fusion proteins: a G_α fused to cyan fluorescent protein (CFP), and a G_β fused to yellow fluorescent protein (YFP). CFP normally fluoresces 490-nm light; YFP, 527-nm light. (a) When CFP and YFP are close to each other, as in the resting G_α·G_βγ complex, fluorescence energy transfer can occur between them (left). As a result, irradiation of resting cells with 440-nm light (which directly excites CFP but not YFP) causes emission of 527-nm (yellow) light, characteristic of YFP, because of fluorescence energy transfer from CFP to YFP. However, if ligand binding leads to dissociation of the G_α and G_βγ subunits, then fluorescence energy transfer cannot occur. In this case, irradiation of cells at 440 nm causes emission of 490-nm (cyan) light, characteristic of CFP (right). (b) Plot of the emission of yellow light (527 nm) from a single transfected amoeba cell before and after addition of extracellular cAMP (arrow). The drop in yellow fluorescence, which results from the dissociation of the G_α-CFP fusion protein from the G_β-YFP fusion protein, occurs within seconds of cAMP addition.

[Data from C. Janetopoulos et al., 2001, Science 291:2408.]