The intrinsic GTPase activity of transducin controls inactivation of the effector enzyme, cGMP phosphodiesterase (PDE), during turnoff of the visual signal, The inhibitory gamma-subunit of PDE (P gamma), an unidentified membrane factor and a retinal specific member of ta-Le RGS family of proteins have been shown to accelerate GTP hydrolysis by transducin, ae 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 cu-subunit, G(t alpha)GDPAlF(4)(-), while its binding to G(t alpha)GTP gamma S or G(t alpha)GDP was weak, hRGSr and P gamma did not compete for the interaction with G(t alpha)GPAlF(4)(-) Affinity of the P gamma-G(t alpha)GDPAlF(4)(-) interaction was modestly enhanced by addition of hRGSr, as measured by a fluorescence assay of G(t alpha)DPAlF(4)(-) binding to P gamma labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (P gamma BC), Binding of hRGSr to G(t alpha)GDPAlF(4)(-) complexed with P gamma BC resulted in a maximal similar to 40% reduction of BC fluorescence allowing estimation of the hRGSr affinity for G(t alpha)GDPAlF(4)(-) (K-d 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 P gamma to the reconstituted system reduced the GTPase level accelerated by hRGSr (K-cat 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 mu M rhodopsin these rates exceeded 0.7 s(-1), Our data suggest that effects of hRGSr on transducin's GTPase activity are attenuated by P gamma 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.
