Characterization of a truncated form of arrestin isolated from bovine rod outer segments

Abstract

The inactivation of photolyzed rhodopsin requires phosphorylation of the receptor and binding of a 48-kDa regulatory protein, arrestin. By binding to phosphorylated photolyzed rhodopsin, arrestin inhibits G protein (G,) activation and blocks premature dephosphorylation, thereby preventing the reentry of photolyzed rhodopsin into the phototransduction pathway. In this study, we isolated a 44-kDa form of arrestin, called p4, from fresh bovine rod outer segments and characterized its structure and function. A partial primary structure of p44 was established by a combination of mass spectrometry and automated Edman degradation of proteolytic peptides. The amino acid sequence was found to be identical with arrestin, except that the C-terminal35 residues (positions 370-404) are replaced by a single alanine. p44 appeared to be generated by alternative mRNA splicing, because intron 15 interrupts within the nucleotide codon for 369Ser in the arrestin gene. Functionally, p44 binds avidly to photolyzed or phosphorylated and photolyzed rhodopsin. As a consequence of its relatively high affinity for bleached rhodopsin, pa blocks G, activation. The binding characteristics of p4 set it apart from tryptic forms of arrestin (truncated at the N-and C-termini), which require phosphorylation of rhodopsin for tight binding. We propose that p44 is a novel splice variant of arrestin that could be involved in the regulation of G, activation.

Phototransduction in mouse rods and cones

“…At least two splice variants exist for rod arrestin: a fulllength (p48) and a C-terminal truncated form (p44) [134]. P44 has a faster on-rate than p48 for binding R* and R*-P [135,136]. In addition, p44 is more efficient than p48 in turning off R* in vitro [137,138].…”

Responses of the phototransduction cascade to dim light.

“…HoloTransducin is extracted from bovine ROS and purified on a Superose-12 gel filtration column (Pharmacia Biotech) and concentrated on Centricon-30. Arrestin and its splice variant, p44, are purified from bovine ROS (7,13). Rhodopsin kinase is expressed in a baculovirussf9 system and purified on a recoverin affinity column (14).…”

Site-specific Cleavage of G Protein-coupled Receptor-engaged β-Arrestin

“…Azarian et al (9) found that a short form (p46) of visual arrestin generated by light exposure of bovine rod outer segments is identical to that generated by in vitro proteolysis of visual arrestin by calpain. The short form of visual arrestin (p44) resulting from alternative mRNA splicing (7,8) differs in biochemical characteristics compared with the full-length visual arrestin. For instance, the full-length visual arrestin binds only to phosphorylated rhodopsin, but p44 binds to both nonphosphorylated and phosphorylated rhodopsin with similar affinity (7).…”

“…In the visual system, splice variants of the visual arrestin that lack the C-terminal tail have been reported (7,8). A splice variant of visual arrestin, termed p44, is identical to full-length arrestin except that the last exon encoding the C-terminal 35 amino acids is replaced by a single alanine residue and binds to both nonphosphorylated and phosphorylated rhodopsin, whereas the full-length visual arrestin binds to only phosphorylated rhodopsin (7).…”

“…A splice variant of visual arrestin, termed p44, is identical to full-length arrestin except that the last exon encoding the C-terminal 35 amino acids is replaced by a single alanine residue and binds to both nonphosphorylated and phosphorylated rhodopsin, whereas the full-length visual arrestin binds to only phosphorylated rhodopsin (7). In purified bovine rod outer segments, visual arrestin was shown to be proteolyzed in light conditions, and the proteolysis occurred only when the visual arrestin was bound to rhodopsin (9).…”