
Transgenic photoreceptors expressing bovine P23H rhodopsin, demonstrating differential reactivity of N- and C-terminal antibody epitopes. While the C-terminus (1D4) is abundant in the outer segments, the N-terminal epitope (2B2) is largely absent from the outer segments, and relatively speaking, is much more abundant in inner segments. This is due to the fact that the N-terminus of P23H rhodopsin is cleaved on exit from the ER (confirmed in western blot to the right). In this image, the outer segments are counterstained with wheat germ agglutinin (green).

Complete rescue of retinal degeneration in animals expressing bovine P23H rhodopsin by dark rearing. The photoreceptors in the top panel appear quite normal, while most of the rods are missing in the lower panel. Retinal degeneration in animals expressing bovine rhodopsin is strongly influenced by light exposure, and chromophore binding is involved in the rescuing effects of dark rearing (see panel to the right). We are currently investigating whether this effect is also linked to the phenomenon of N-terminal cleavage.

We obtained similar differential labeling with N- and C-terminal directed antibodies with human P23H rhodopsin expressed in X. laevis photoreceptors. The effect is not as dramatic, but clearly present.

We obtained a similar rescuing effect of dark rearing with human P23H rhodopsin. Again, the effect is not as dramatic, but clearly present. Dark rearing did not completely protect these animals from retinal degeneration.

Greater quantities of cleaved P23H rhodopsin are transported to the rod outer segment under dark rearing conditions. Labeling with the C-terminal antibody 1D4 (left panels) shows more consistent and higher levels of transport of P23H rhodopsin to rod outer segments in the dark (bottom) than in cyclic light (top). Banding in the top panel also suggests a relationship between this transport and the light/dark cycle. However, virtually all of the rhodopsin transported to the outer segments is cleaved at the N-terminus, and cannot be labeled with the N-terminal antibody 2B2 (right panels). In total, our results suggest a synergy between chromophore binding and proteolytic cleavage that allows transport of P23H rhodopsin to the outer segment in the absence of light exposure.
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