Abstract
Purpose: :
To define the role of RGR in the visual cycle.
Methods: :
We studied mice of four genotypes: (i) 129/SvEv wild–type mice homozygous for the rpe65 L450 allele (WT); (ii) rgr –/– knockout mice, with the L450 allele of rpe65 (rgr –/–); (iii) rpe65 –/– knockout mice; and (iv) rgr –/–, rpe65 –/– double knockout mice. Mice were age–matched and exposed to similar light conditions for each experiment. Enzyme assays (LRAT and isomerase) were performed using homogenates or microsomes from RPE–containing eyecups. Retinoids were extracted from mouse homogenates, microsomes, cultured cells, or assay mixtures and analyzed by HPLC.
Results: :
Eyecups from dark adapted rgr –/– mice contained two–fold higher all–trans–retinyl esters (atRE's) and three–fold higher 13–cis–retinyl esters (13cRE's) compared to WT mice. Although the levels of 11–cis–retinaldehyde (11cRAL) were similar in eyes from dark–adapted WT and rgr –/– mice, the rate of 11cRAL regeneration following a photobleach was several–fold slower in rgr –/– mice. Previously, we observed dramatic light–dependent mobilization of atRE's in eyecups from rpe65 –/– mice, which are blind due to lack visual chromophore. Interestingly, this light–dependent mobilization was abolished in rgr –/–, rpe65 –/– double knockout mice. Levels of atRE's were similar in eyecups from dark–adapted rgr –/– and rgr –/–, rpe65 –/– mice. We also observed light–dependent mobilization of atRE's from hRPE cells previously incubated with atROL. This mobilization was reduced significantly when hRPE cells were exposed to RGR siRNA's, but not control siRNA's. Kinetic analysis showed similar KM and Vmax values for LRAT in eyecup microsomes from WT and rgr –/– mice. We also observed similar LRAT activity in light–exposed 293T cells that express LRAT plus RGR versus LRAT alone. Finally, retinoid isomerase activity was unchanged in homogenates from dark or light–adapted 293T cells expressing LRAT plus Rpe65 compared to cells expressing Rpe65 alone.
Conclusions: :
(1) RPE cells are inherently light sensitive. (2) Light exposure results in mobilization of atRE's from normal RPE cells. (3) This light–dependent mobilization is lost when RGR is missing, suggesting that RGR may mediate the light sensitivity of RPE. (4) The effect of RGR on atRE levels is unlikely to involve modulation of LRAT activity. (5) RGR does not affect the isomerase activity of Rpe65 when both proteins are co–expressed in 293T cells. (6) A possible function for RGR that explains the phenotype in rgr –/– mice will be presented.
Keywords: retinal pigment epithelium • retinoids/retinoid binding proteins • opsins