Perturbing the Linker Regions of the α-Subunit of Transducin

Abstract

The GDP-GTP exchange activity of the retinal G protein, transducin, is markedly accelerated by the photoreceptor rhodopsin in the first step of visual transduction. The x-ray structures for the α subunits of transducin (α_T) and other G proteins suggest that the nucleotide-binding (Ras-like) domain and a large helical domain form a “clam shell” that buries the GDP molecule. Thus, receptor-promoted G protein activation may involve “opening the clam shell” to facilitate GDP dissociation. In this study, we have examined whether perturbing the linker regions connecting the Ras-like and helical domains of Gα subunits gives rise to a more readily exchangeable state. The sole glycine residues in linkers 1 and 2 were individually changed to proline residues within an α_T/α_i1 chimera (designated ). Both linker mutants showed significant increases in their basal rates of GDP-GTP exchange when compared either to retinal α_T or recombinant . The linker mutants were responsive to aluminum fluoride, which binds to α-GDP complexes and induces changes in Switch 2. Although both linker mutants were further activated by light-activated rhodopsin together with the βγ complex, their activation was not influenced by βγ alone, arguing against the idea that the βγ complex helps to pry apart the helical and Ras-like domains of Gα subunits. Once activated, the linker mutants were able to stimulate the cyclic GMP phosphodiesterase. Overall, these findings highlight a new class of activated Gα mutants that constitutively exchange GDP for GTP and should prove valuable in studying different G protein-signaling systems.