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Regulation of Transducin GTPase Activity by Human Retinal RGS
Journal article   Open access   Peer reviewed

Regulation of Transducin GTPase Activity by Human Retinal RGS

Michael Natochin, Alexey E Granovsky and Nikolai O Artemyev
The Journal of biological chemistry, Vol.272(28), pp.17444-17449
07/11/1997
DOI: 10.1074/jbc.272.28.17444
PMID: 9211888
url
https://doi.org/10.1074/jbc.272.28.17444View
Published (Version of record) Open Access

Abstract

The intrinsic GTPase activity of transducin controls inactivation of the effector enzyme, cGMP phosphodiesterase (PDE), during turnoff of the visual signal. The inhibitory γ-subunit of PDE (Pγ), an unidentified membrane factor and a retinal specific member of the RGS family of proteins have been shown to accelerate GTP hydrolysis by transducin. We have expressed a human homologue of murine retinal specific RGS (hRGSr) in Escherichia coli and investigated its role in the regulation of transducin GTPase activity. As other RGS proteins, hRGSr interacted preferentially with a transitional conformation of the transducin α-subunit, GtαGDPAlF4−, while its binding to GtαGTPγS or GtαGDP was weak. hRGSr and Pγ did not compete for the interaction with GtαGDPAlF4−. Affinity of the Pγ-GtαGDPAlF4−interaction was modestly enhanced by addition of hRGSr, as measured by a fluorescence assay of GtαGDPAlF4−binding to Pγ labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (PγBC). Binding of hRGSr to GtαGDPAlF4−complexed with PγBC resulted in a maximal ∼40% reduction of BC fluorescence allowing estimation of the hRGSr affinity for GtαGDPAlF4−(Kd 35 nm). In a single turnover assay, hRGSr accelerated GTPase activity of transducin reconstituted with the urea-stripped rod outer segment (ROS) membranes by more than 10-fold to a rate of 0.23 s−1. Addition of Pγ to the reconstituted system reduced the GTPase level accelerated by hRGSr (kcat 0.085 s−1). The GTPase activity of transducin and the PDE inactivation rates in native ROS membranes in the presence of hRGSr were elevated 3-fold or more regardless of the membrane concentrations. In ROS suspensions containing 30 μm rhodopsin these rates exceeded 0.7 s−1. Our data suggest that effects of hRGSr on transducin's GTPase activity are attenuated by Pγ but independent of a putative membrane GTPase activating protein factor. The rate of transducin GTPase activity in the presence of hRGSr is sufficient to correlate it with in vivo turnoff kinetics of the visual cascade.

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