Interactions of metarhodopsin II - Arrestin peptides compete with arrestinand transducin

Arrestin blocks the interaction of rhodopsin with the G protein transducin (G(t)). To characterize the sites of arrestin that interact with rhodopsin, we have utilized a spectrophotometric peptide competition assay. It is bas ed on the stabilization of the active intermediates metarhodopsin II (MII) and phosphorylated MII by G(t) and arrestin, respectively (extra MII monito r). The protocol involves native disc membranes and three sets of peptides 10-30 amino acids in length spanning the arrestin sequence, In the absence of arrestin, not one of the peptides by itself had an effect on the amount of MII formed. However, inhibition of arrestin-dependent extra MII was foun d for the peptides at residues 11-30 and 51-70 (IC50 < 100 <mu>M) and resid ues 231-260 (IC50 < 200 <mu>M) A similar pattern of inhibition by arrestin peptides was seen when arrestin was replaced by G(t) or the farnesylated G( t)gamma C-terminal peptide. Only arrestin-(11-30) inhibited MII.G(t) less ( IC50 = 300 muM) than phosphorylated MII.arrestin. We interpreted the data b y competition of the arrestin peptides for interaction sites at rhodopsin, exposed in the MPI conformation and specific for both arrestin and G(t). Th e arrestin sites are located in both the C- and N-terminal domains of the a rrestin structure.