Identification of regions of arrestin that bind to rhodopsin

Arrestin facilitates phototransduction inactivation through binding to phot oactivated and phosphorylated rhodopsin (R*P). However, the specific portio ns of arrestin that bind to R*P are not known. In this study, two different approaches were used to determine the regions of arrestin that bind to rho dopsin: panning of phage-displayed arrestin fragments against R*P and cGMP phosphodiesterase (PDE) activity inhibition using synthetic arrestin peptid es spanning the entire arrestin protein. Phage display indicated the predom inant region of binding was contained within amino acids 90-140. A portion of this region (residues 95-140) expressed as a fusion protein with glutath ione S-transferase is capable of binding to rhodopsin regardless of the act ivation or phosphorylation state of the receptor. Within this region, the s ynthetic peptide of residues 109-130 was shown to completely inhibit the bi nding of arrestin to rhodopsin with an IC50 of 1.1 mM. The relatively high IC50 of this competition suggests that this portion of the molecule may be only one of several regions of binding between arrestin and R*P. A survey o f synthetic arrestin peptides in the PDE assay indicated that the two most effective inhibitors of PDE activity were peptides of residues 111-130 and 101-120. These results indicate that at least one of the principal regions of binding between arrestin and R*P is contained within the region of resid ues 109-130.