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.sup.258], a residue within the GTPase domain of the heterotrimeric guanine nucleotide binding protein (G protein) [Alpha]-sub-unit ([[Alpha].sub.s]), to alanine ([[Alpha].sub.s]-R258A) results in decreased activation by receptor or aluminum fluoride (Al[[F.sub.4].sup.-]) and increased basal GDP release. [Arg.sup.258] interacts with [Gln.sup.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 Al[[F.sub.4].sup.-] and increased thermolability. In this study, we mutate [Gin.sup.170] to alanine ([[Alpha].sub.s]-Q170A) and demonstrate that this mutant, like [[Alpha].sub.s]-R258A, has decreased activation by Al[[F.sub.4].sup.-], increased thermolability (both reversed in the presence of excess guanine nucleotide), and an increased rate of GDP release. However, unlike [[Alpha].sub.s]R258A, [[Alpha].sub.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 Al[[F.sub.4].sup.-]) but is not important for receptor-mediated activation. In single turnover GTPase assays, the catalytic rate for GTP hydrolysis of [[Alpha].sub.s]-R258A was 14-fold higher than normal whereas that of [[Alpha].sub.s]-Q170A was unaffected. Examination of the [[Alpha].sub.s] crystal structure suggests that [Arg.sup.258], through interactions with [Glu.sup.50], might constrain the position of [Arg.sup.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.