Selective Proteolysis of Arrestin by Calpain

doi:10.1074/jbc.270.41.24375

Selective Proteolysis of Arrestin by Calpain

MOLECULAR CHARACTERISTICS AND ITS EFFECT ON RHODOPSIN DEPHOSPHORYLATION (*)

From the (1)School of Optometry, Indiana University, Bloomington, Indiana 47405 and the
(2) Departments of Pharmacology and Neurosciences, University of California San Diego School of Medicine, La Jolla, California 92093

^¶ To whom correspondence should be addressed:
Dept. of Pharmacology, UCSD School of Medicine, 9500 Gilman Dr., La Jolla, CA 92093-0983.
Tel.: 619-546-9439; Fax: 619-546-9389; dswilliams{at}ucsd.edu

↵^§ Present address: Dept. of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX 75235.

Abstract

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.

Footnotes

↵* This work was supported by National Institutes of Health Grant EY07042 (to D. S. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

mAb

monoclonal antibody

DTT

dithiothreitol

PAGE

polyacrylamide gel electrophoresis

ROS

rod outer segment

Rh

rhodopsin, unexposed to light

Rh*

light-activated rhodopsin

Rh-P

phosphorylated rhodopsin

Rh*-P

phosphorylated and light-activated rhodopsin

Op

opsin (rhodopsin without chromophore)

Op-P

phosphorylated opsin.
↵²Y.-K. Ho, personal communication.
↵³N. Mangini, personal communication.
↵⁴In the present study, 2 mM Ca was used for the experiments shown in Fig. 2(and then throughout in the other in vitro studies for consistency).
- Received June 30, 1995.