Communication between Switch II and Switch III of the transducin α subunit is essential for target activation

Abstract

Comparisons of the tertiary structures of the GDP-bound and guanosine 5'-O-(thiotriphosphate) (GTPγS)-bound forms of the α subunit of transducin (α(T)) indicate that there are three regions that undergo changes in conformation upon α(T) activation. Two of these regions, Switch I and Switch II, were originally identified in Ras, while Switch III appears to be unique to trimeric GTP-binding proteins (G proteins). We find that replacement of the Switch III region (aspartic acid 227 through asparagine 237) with a single alanine residue yields an α(T) subunit that fully binds and hydrolyzes GTP but no longer stimulates the activity of the cyclic GMP phosphodiesterase (PDE), the physiological target for transducin. We also show that changing glutamic acid 232 of α(T) to a leucine (E232L) had no effect on rhodopsin-stimulated GTP-GDP exchange nor on the GTP hydrolytic activity of α(T). However, the GTPγS-bound form of the α(T)E232L mutant was unable to stimulate the activity of the cyclic GMP PDE. The lack of stimulation was not due to an inability of the α(T)E232L mutant to bind to the target. Taken together, these results indicate that glutamic acid 232 mediates a conformational coupling between Switch II and Switch III, which is essential for converting GTP-dependent G protein-target interactions into a stimulation of target/effector activity.