Modulation of opsin apoprotein activity

Purpose: To characterize the modulation of the opsin apoprotein activation of transducin by 11-cis-retinal or phosphorylation, and determine the role of the cytoplasmic loops in opsin activity. Methods: Bovine opsin and transducin were as described previously (Surya et al. J. Biol. Chem. 270, 5024). Phosphorylated opsin was prepared from ROS that were incubated in the light with ATP for 2 h. Regeneration with molar excess 11-cis-retinal was performed either at 0°C (forming R⁰) or at 22°C (forming R²²). Synthetic peptides corresponding to bovine rhodopsin cytoplasmic loop 2, loop 3 and loop 4 were added to nucleotide exchange assays. Results: Incubation of opsin with 11-cis-retinal had dramatic effects on its ability to activate transducin, depending upon the temperature of incubation. Regenerations performed at 22°C caused >80% inhibition of apoprotein activity The inhibition paralleled the formation of the 500 nm-absorbing species. In contrast, regenerations at 0°C had little effect on opsin activity, even though formation of 50C nm species occurred to a similar extent as at 22°C. Thus, formation of a protonated Schiff base does not by itself inactivate opsin. The R⁰ conformation did not depend upon an intact ROS membrane, since solubilization with dodecyl maltoside had no effect on the R⁰ activity. R⁰ could be converted to R²² by incubation at 22°C (t_1/2=45 min). Experiments are underway including L2, L3 and L4 at 100 μM in the assay. Conclusion: These results support the idea that part of the transducin binding site of metarhodopsinII is preserved in opsin, leading to apoprotein activity. Regeneration consists of at least two separable steps: 1) binding of 11-cis-retinal and formation of the Schiff base and 2 conformational changes in the cytoplasmic loops to reduce photoreceptor noise.