Abstract
The complex between the photoreceptor-specific regulator of G protein signaling (RGS) protein, RGS9-1, and type 5 G protein beta-subunit, Gbeta5L, regulates the duration of the cellular response to light by stimulating the GTPase activity of G protein, transducin. An important property of RGS9-1.Gbeta5L is that it interacts specifically with transducin bound to its effector, cGMP phosphodiesterase, rather than with transducin alone. The minimal structure within the RGS9-1.Gbeta5L complex capable of activating transducin GTPase is the catalytic domain of RGS9. This domain itself is also able to discriminate between free and effector-bound transducin but to a lesser degree than RGS9-1.Gbeta5L. The goal of this study was to determine whether other, noncatalytic domains of RGS9-1.Gbeta5L enhance the intrinsic specificity of the catalytic domain or whether they set the specificity of RGS9-1.Gbeta5L regardless of the specificity of its catalytic domain. We found that a double L353E/R360P amino acid substitution reversed the specificity of the recombinant catalytic domain but did not reverse the specificity of RGS9-1.Gbeta5L. However, the degree of discrimination between free and effector-bound transducin was reduced. Therefore, noncatalytic domains of RGS9-1.Gbeta5L play a decisive role in establishing its substrate specificity, yet the high degree of this specificity observed under physiological conditions requires an additional contribution from the catalytic domain.
Highlights
RGS1 proteins regulate the duration of signaling in many G protein pathways [1,2,3]
The goal of this study was to determine whether other, noncatalytic domains of RGS9-11⁄7G5L enhance the intrinsic specificity of the catalytic domain or whether they set the specificity of RGS9-11⁄7G5L regardless of the specificity of its catalytic domain
They have shown that normally RGS7d activates the GTPase of free G␣t better than that of the G␣t1⁄7PDE␥ complex, but substituting only two residues within RGS7d for corresponding residues from RGS9-11⁄7G5L but also for its catalytic domain (RGS9d) reverses the effect of PDE␥, from inhibition of the RGS7d activity to its stimulation
Summary
RGS1 proteins regulate the duration of signaling in many G protein pathways [1,2,3]. They act by stimulating the GTP hydrolysis on G protein ␣-subunits, which results in termination of signaling events conferred by activated G proteins. There are indications that these noncatalytic domains may participate in modulation of the RGS catalytic activity or may allow RGS proteins to interact with components of other intracellular pathways (reviewed in Ref. 4). Another putative role for the noncatalytic domains of RGS proteins is to contribute to the recognition of their specific G protein ␣-subunit targets. The degree of discrimination between G␣t and G␣t1⁄7PDE␥ observed with RGS9d is much lower than that with the fulllength RGS9-11⁄7G5L (ϳ2-fold versus ϳ20-fold; see Ref. 7). This suggests that both catalytic and noncatalytic domains contribute to this important property of RGS9-11⁄7G5L. Mutant RGS9-11⁄7G5L discriminated between G␣t and G␣t1⁄7PDE␥ to a lower degree than did the wild type
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