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Visual arrestin (48 kDa) plays a role in the deactivation of rhodopsin by binding to the light-activated, phosphorylated form of the receptor. In bovine rod outer segments that were prepared in the presence of protease inhibitors, two faster migrating forms of arrestin, with apparent molecular masses of 46 and 44 kDa, were observed by Western blot analysis. The 46-kDa form was more evident in rod outer segments of eyes kept in the light than those placed in darkness and was found to be identical to that generated by in vitro proteolysis of arrestin by pure retinal calpain II. In vitro analysis showed that arrestin was proteolyzed only when bound to rhodopsin; soluble arrestin was not significantly cleaved by calpain. Proteolysis involves sequential cleavage at two, possibly three sites, resulting in the removal of 27 amino acids from the COOH terminus. The remaining 46-kDa protein was resistant to further proteolysis by calpain. Unlike intact arrestin, the 46-kDa truncated arrestin was not readily released from the receptor after the receptor had lost its chromophore, nor was it released upon the addition of 11-cis-retinal to regenerate the receptor. Truncated arrestin was found to inhibit receptor dephosphorylation to the same extent as intact arrestin. In conclusion, these results provide evidence that a 46-kDa form of arrestin in rod outer segments is a product of selective proteolysis by calpain. Furthermore, they suggest that this proteolysis may provide a mechanism for prolonging the phosphorylated state of the visual receptor.

Calpains are calcium-activated proteinases which have been implicated in tissue differentiation and degeneration. The aims of the present study were: (1) to determine the relationship between postnatal age and calpain activity in the rat retina; (2) to test if calpain activity was aberrant in the RCS retina at different postnatal ages. Calpain activity was measured by a standard in vitro assay in fractions of retinas of rats, ranging in postnatal age of 2 to 42 days. Most retinal calpain activity was in the cytosolic fraction. Specific calpain activity declined with age. In the Long Evans rat, it was 8-fold higher on postnatal day 2 than on postnatal day 42. Comparison between RCS rats and their congenic controls showed that calpain activity was lower in the retinas of neonatal RCS rats. Specific calpain activity in RCS rat retinas was 46% lower on postnatal day 2 and 22% lower on postnatal day 3. It is concluded that during postnatal development of the retina, marked changes occurred in calpain activity. In addition, calpain activity is abnormal in the retina of the neonatal RCS rat--well before the onset of any morphological deterioration and preceding any other previously detected abnormality. Aberrant calpain activity appears to be a manifestation of very early events in processes that lead to retinal degeneration in the RCS rat.

Calpain II was purified to apparent homogeneity from bovine neural retinas. It was found to be biochemically similar to brain calpain II, purified by the same procedure, with respect to: subunit mobility in SDS-polyacrylamide gel electrophoresis; Ca2+ sensitivity; inhibition by calpeptin and other cysteine protease inhibitors; and optimal pH. Semithin cryosections were immuno-labeled with antibodies specific for the catalytic subunit of calpain II. Calpain II was detected in most layers of the retina, with the most pronounced label present in the plexiform layers (synaptic regions) and the photoreceptor outer segments. In dark-adapted retinas, the label was distributed throughout the outer segments. In light-adapted retinas, outer segment labeling was concentrated in the connecting cilium, and the inner segments were labeled. A partially pure preparation of calpain II from isolated rod outer segments was found to have the same biochemical characteristics as calpain II prepared in the same way from the whole retina. The enzyme was distributed fairly evenly between the cytosolic and cytoskeletal fractions of isolated rod outer segments. Immunoblots of the rod outer segment cytoskeleton were used to determine the susceptibility of known components of the actin-based cytoskeleton to proteolysis by calpain II in vitro. Actin was not proteolyzed at all, alpha-actinin was only slowly degraded, but myosin II heavy chain was rapidly proteolyzed. Actin filaments have been shown previously to be associated with myosin II and alpha-actinin in a small domain within the connecting cilium, where they play an essential role in the morphogenesis of new disk membranes. The localization of calpain II in the connecting cilium after light exposure, combined with the in vitro proteolysis of myosin II, suggests that calpain II could be involved in light-dependent regulation of disk membrane morphogenesis by proteolysis of myosin II.