Sulfhydryl reactivity demonstrates different conformational states for arrestin, arrestin activated by a synthetic phosphopeptide, and constitutivelyactive arrestin

The sulfhydryl groups of the three cysteines in bovine arrestin react with DTNB very slowly (over a period of several hours). In the presence of the s ynthetic phosphopeptide comprising the fully phosphorylated carboxyl-termin al 19 amino acids of bovine rhodopsin, the reactivity of one of the sulfhyd ryls was enhanced while that of another was greatly reduced. Since this syn thetic peptide was shown to activate arrestin with respect to its binding t o unphosphorylated, light-activated rhodopsin, the reactivity of the sulfhy dryl groups of a constitutively active R175Q arrestin mutant was examined. All three of the sulfhydryl groups of the mutant arrestin R175Q reacted rap idly with DTNB, but not as rapidly as with SDS-denatured arrestin, The arre stin mutant R175Q bound to light-activated, unphosphorylated rhodopsin in R OS disk membranes. The arrestin mutant R175Q also inhibited the light-activ ated PDE activity with an IC50 of 1.3 mu M under the experimental condition s that were used. These data indicate that each of these forms of arrestin is a different conformation. The activated conformation of arrestin that bi nds to phosphorylated rhodopsin in vivo may be yet another conformation. We conclude that arrestin is a flexible molecule that is able to attain sever al different conformations, all of which are able to attain the activated f unctional state of arrestin.