Comparison of the effect of mRGS-r protein and RGS-4 protein on GTP hydrolysis by TD. The hydrolysis of GTP (10 μM) by 200 nM TD plus 10 μg of urea-treated ROS membranes was determined at the indicated concentrations of mRGS-r protein (•) or RGS-4 protein (□) after 10 min of incubation at 30°C. The data shown are mean ± SD of triplicate determinations.

Northern blot analysis of 1 μg of poly(A)⁺ RNA from various rat tissues (CLONTECH) hybridized with rRGS-r probe (Upper). Rat glyceraldehyde-3-phosphate dehydrogenase (G3PDH) probe (CLONTECH) was used as a control (Lower).

Sequence alignment of mouse RGS-r (mRGS-r), rat RGS-r (rRGS-r), rat RGS-4 (rRGS-4), and GAIP.

Complex formation of mRGS-r protein with TDα_GDP·AMF analyzed by gel filtration. Purified TDα_GDP (6 μM) was incubated with 18 μM mRGS-r for 10 min at 30°C in the absence (○ and Middle) and presence of AMF (• and Bottom). Fractions in the protein profile (Top) were analyzed by SDS/PAGE, followed by Coomassie brilliant blue staining. Complex formation of mRGS-r protein with TDα was observed in the presence of AMF. The calculated molecular sizes were 40 kDa, 35 kDa, and 72.5 kDa for TD, mRGS-r protein, and the complex, respectively.

Complex formation of mRGS-r protein with TDα_GDP·AMF analyzed by binding to Ni²⁺-NTA. Experiments were performed as described. Only the TDα_GDP·AMF was found to bind to the matrix by its interaction with mRGS-r protein.

Enhanced GTP hydrolysis of TDα by mRGS-r protein without affecting the loading of GTP. (A) mRGS-r protein increases the P_i release from TDα. The hydrolysis of GTP (10 μM) by 0.5 μM TD, 10 μg of urea-treated ROS membranes (R*, bleached rhodopsin in rod outer segment membranes), 0.7 μM mRGS-r, or combinations of these components was measured for 3 min at 30°C. Hydrolysis of GTP was observed in the presence of R* and TD. mRGS-r protein increased the P_i release from TD ≈7-fold. (B) Dose–response curve of mRGS-r protein. Half-maximal and maximal stimulation by mRGS-r protein was observed at 50 and 200 nM, respectively. (C and D) The binding of nonhydrolyzable GTP analogs to TD was not affected by mRGS-r protein. GTP[γS] (100 nM) or Gpp[NH]p (1 μM) was used as described. (E) TD activated by nonhydrolyzable GTP analogs does not bind to mRGS-r protein. TD loaded with labeled nonhydrolyzable GTP analogs was prepared by centrifugation of the reaction mixtures from C and D. The supernatants, containing the labeled TD and mRGS-r protein, were applied to Ni²⁺-NTA columns. The amount of protein-bound radioactivity in the flow-through and the 400 mM imidazole eluate was determined. In all parts of this figure, means ± SD of triplicate determinations are shown.