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

It has been reported that substitution of Arg(258), a residue within the GT Pase domain of the heterotrimeric guanine nucleotide binding protein (G pro tein) alpha-subunit (alpha(s)), to alanine (alpha(s)-R258A) results in decr eased activation by receptor or aluminum fluoride (AlF4-) and increased bas al GDP release. Arg(258) interacts with Gln(170) in the helical domain, and , presumably, Loss of this interaction between the GTPase and helical domai n leads to more rapid GDP release, resulting in decreased activation by AlF 4- and increased thermolability. In this study, me mutate Gln(170) to alani ne (alpha(s)-Q170A) and demonstrate that this mutant, like alpha(s)-R258A, has decreased activation by AlF4-, increased thermolability (both reversed in the presence of excess guanine nucleotide), and an increased rate of GDP release. However, unlike alpha(s)-R258A, alpha(s)-Q170A does not have impa ired receptor-mediated activation. Therefore, this interdomain interaction is critical to maintain normal guanine nucleotide binding (and hence normal activation by AlF4-) but is not important for receptor-mediated activation . In single turnover GTPase assays, the catalytic rate for GTP hydrolysis o f alpha(s)-R258A was 14-fold higher than normal whereas that of alpha(s)-Q1 70A was unaffected. Examination of the cr, crystal structure suggests that Arg(258), through interactions with Glu(50), might constrain the position o f 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 i ncreased intrinsic GTPase activity and provides another mechanism by which G protein mutations can impair signal transduction.