REGULATION OF TRANSDUCIN GTPASE ACTIVITY BY HUMAN RETINAL RGS

The intrinsic GTPase activity of transducin controls inactivation of t he effector enzyme, cGMP phosphodiesterase (PDE), during turnoff of th e visual signal, The inhibitory gamma-subunit of PDE (P gamma), an uni dentified membrane factor and a retinal specific member of ta-Le RGS f amily of proteins have been shown to accelerate GTP hydrolysis by tran sducin, ae have expressed a human homologue of murine retinal specific RGS (hRGSr) in Escherichia coli and investigated its role in the regu lation of transducin GTPase activity, As other RGS proteins, hRGSr int eracted preferentially with a transitional conformation of the transdu cin 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 comp ete for the interaction with G(t alpha)GPAlF(4)(-) Affinity of the P g amma-G(t alpha)GDPAlF(4)(-) interaction was modestly enhanced by addit ion of hRGSr, as measured by a fluorescence assay of G(t alpha)DPAlF(4 )(-) binding to P gamma labeled with 3-(bromoacetyl)-7-diethylaminocou marin (P gamma BC), Binding of hRGSr to G(t alpha)GDPAlF(4)(-) complex ed with P gamma BC resulted in a maximal similar to 40% reduction of B C fluorescence allowing estimation of the hRGSr affinity for G(t alpha )GDPAlF(4)(-) (K-d 35 nM). In a single turnover assay, hRGSr accelerat ed 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 acti vity of transducin and the PDE inactivation rates in native ROS membra nes in the presence of hRGSr were elevated 3-fold or more regardless o f the membrane concentrations, In ROS suspensions containing 30 mu M r hodopsin these rates exceeded 0.7 s(-1), Our data suggest that effects of hRGSr on transducin's GTPase activity are attenuated by P gamma bu t. independent of a putative membrane GTPase activating protein factor , The rate of transducin GTPase activity in the presence of hRGSr is s ufficient to correlate it with in vivo turnoff kinetics of the visual cascade.