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KA Martemyanov, VY Arshavsky; Noncatalytic Domains of RGS9-Gß5 Are Critical for its Selective Interaction With the Transducin-phosphodiesterase Complex in Photoreceptors . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1410.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: RGS9-Gß5 regulates the lifetime of activated transducin by stimulating its GTPase activity. A unique property of RGS9-Gß5 is to bind more efficiently to the complex of transducin with its effector, the γ subunit of cGMP phosphodiesterase (PDEγ), than to free activated transducin. This allows RGS9-Gß5 to inactivate transducin only after its function of effector activation is fulfilled. Other RGS proteins are inhibited by PDEγ. Our goal was to elucidate which part of the RGS9-Gß5 complex - its catalytic domain (RGS9d) or noncatalytic domains - play a decisive role in establishing positive cooperativity with PDEγ. Methods: Our approach was to mutate RGS9d in such a way that the effect of PDEγ would be reversed from potentiation to inhibition of the RGS9d ability. We then tested the direction of PDEγ effect with the full-length RGS9-Gß5 bearing the same mutations. The study by Sowa et al. (Nature Struct. Biol., 2001, v.8, p.234) showed that a substitution of two amino acids in the catalytic domain of RGS7 for corresponding residues of RGS9 switches the effect of PDEγ on the RGS7 from inhibition to activation. Following this finding, we introduced two amino acid substitutions (L353E and R360P) into recombinant RGS9d and full-length RGS9-Gß5. We then studied catalytic properties of both mutant and wild type proteins. Results: We found that the mutant RGS9d is indeed inhibited by PDEγ whereas the wild-type RGS9d is stimulated. We further demonstrated that this reversal results from the loss of the mutant's affinity for the transducin-PDEγ complex without changes in its affinity for free activated transducin. As a result, the affinity of mutant RGS9d for free transducin is higher than its affinity for the transducin-PDEγ complex, whereas the affinity of the wild type RGS9d for transducin-PDEγ is higher than that for free transducin. Mutations have also increased the maximal catalytic activity of RGS9d. To the contrary, both wild type and mutant RGS9-Gß5 complexes have higher affinities for transducin bound to PDEγ than to free transducin. Their catalytic parameters observed with PDEγ are nearly identical, although the degree of mutant's activity potentiation by PDEγ is lower than that of the wild type. This difference is due to the mutant's higher catalytic activity observed in the absence of PDEγ. Conclusion: We conclude that the direction of the PDEγ effect on the RGS9-Gß5 activity is determined by the noncatalytic domains of this protein complex. However, the degree of the PDEγ effect, determined by the RGS9 catalytic efficiency without PDEγ, is determined by properties of the catalytic domain.
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