Functional differences in the interaction of arrestin and its splice variant, p44, with rhodopsin

Arrestin quenches signal transduction in rod photoreceptors by blocking the catalytic activity of photoactivated phosphorylated rhodopsin toward the G protein, transducin (G t). Rod cells also express a splice variant of arrestin, termed p 44 , in which the last 35 amino acids are replaced by a single Ala. In contrast to arrestin, this protein has been reported to bind to both the phosphorylated and nonphosphorylated forms of the activated receptor. In this study, we analyzed formation of the rhodopsin-p 44 complex in Vitro. Like arrestin, p 44 stabilized the meta II (MII) photoproduct relative to forms MI and MIII and did not interact measurably with the apoprotein opsin. However, several differences between p 44 and its parent protein were found: (i) p 44 binds to nonphosphorylated MII with a much lower affinity (KD ) 0.24 IM) than to phosphorylated MII (P-MII) (KD ) 12 nM); arrestin binds only to P-MII (KD ) 20 nM); (ii) p 44 interacted also with truncated MII ( 329 G-Rho MII), which lacked the sites of phosphorylation; (iii) with both MII and P-MII, the activation energy of complex formation with p 44 was lower than that found for arrestin (70 kJ/mol instead of 140 kJ/mol); and (iv) InsP 6 inhibited poorly the interaction between p 44 and P-MII, but it strongly inhibited the interaction between arrestin and P-MII. Extrapolation of the measured on-rates to physiological conditions yielded reaction times for the binding of p 44 to activated rhodopsin. The data suggest that the splice variant, p 44 , and its parent protein, arrestin, play different roles in phototransduction. The physiological significance of these differences remains to be determined.