A mutation in the heterotrimeric stimulatory guanine nucleotide binding protein α-subunit with impaired receptor-mediated activation because of elevated GTPase activity

It has been reported that substitution of Arg(258), a residue within the GTPase domain of the heterotrimeric guanine nucleotide binding protein (G protein) α-subunit (α(s)), to alanine (α(s)-R258A) results in decreased activation by receptor or aluminum fluoride (AlF(4)(−)) and increased basal GDP release. Arg(258) interacts with Gln(170) in the helical domain, and, presumably, loss of this interaction between the GTPase and helical domain leads to more rapid GDP release, resulting in decreased activation by AlF(4)(−) and increased thermolability. In this study, we mutate Gln(170) to alanine (α(s)-Q170A) and demonstrate that this mutant, like α(s)-R258A, has decreased activation by AlF(4)(−), increased thermolability (both reversed in the presence of excess guanine nucleotide), and an increased rate of GDP release. However, unlike α(s)-R258A, α(s)-Q170A does not have impaired receptor-mediated activation. Therefore, this interdomain interaction is critical to maintain normal guanine nucleotide binding (and hence normal activation by AlF(4)(−)) but is not important for receptor-mediated activation. In single turnover GTPase assays, the catalytic rate for GTP hydrolysis of α(s)-R258A was 14-fold higher than normal whereas that of α(s)-Q170A was unaffected. Examination of the α(s) crystal structure suggests that Arg(258), through interactions with Glu(50), might constrain the position of Arg(201), a residue critical for catalyzing the GTPase reaction. This is an example of a mutation in a heterotrimeric G protein that results in an increased intrinsic GTPase activity and provides another mechanism by which G protein mutations can impair signal transduction.