Selective proteolysis of arrestin by calpain. Molecular characteristics and its effect on rhodopsin dephosphorylation.
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.
Azarian
S M
SM
School of Optometry, Indiana University, Bloomington 47405, USA.
King
A J
AJ
Hallett
M A
MA
Williams
D S
DS
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000941:Antigens / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D019393:Arrestin; D015153:Blotting, Western; D002154:Calpain / Q000378:metabolism*; D002417:Cattle; D003624:Darkness; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008027:Light; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D051381:Rats / Q000276:immunology; D012160:Retina / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000941:Antigens; D019393:Arrestin; D005136:Eye Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D012243:Rhodopsin; D002154:Calpain