Mutating Arg-238 to Glu (R238E) in the switch 3 region of a transducin alpha(*T alpha) in which 27 aa of the GTPase domain have been replaced with those of the a-subunit of the inhibitory G protein 1 (Gil alpha), was reported to create an alpha-subunit that is resistant to activation by GTP gamma S, is devoid of resident nucleotide, and has dominant negative (DN) properties. In an attempt to create a DN stimultory G protein a (Gs alpha) with a single mutation we created Gs alpha-R265E, equivalent to *T alpha-R238E. Gs alpha-R265E has facilitated activation by GTP gamma S, a slightly facilitated activation by GTP but much reduced receptor plus GTP stimulated activation, and an apparently unaltered ability to interact with receptor as seen in ligand binding studies. Further, the activity profile of Gs alpha-R265E is that of an alpha-subunit with unaltered or increased GTPase activity. The only change in Gs alpha that is similar to that in *T alpha is that the apparent affinity for guanine nucleotides is decreased in both proteins. The molecular basis of the changed properties are discussed based on the known crystal structure of Gs alpha and the changes introduced by the same mutation in a *T alpha (Gt alpha*) with only 23 aa from Gil alpha. Gt alpha*-R238E, with four fewer mutations in switch 3, was reported to show no evidence of DN properties, is activated by GTP gamma S, and has reduced GTPase activity. The data highlight a critical role for the switch 3 region in setting overall properties of signal-transducing GTPases.
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