This application proposes to identify regions on the surfaces of rhodopsin and arrestin that interact when arrestin binds photoactivated phosphorylated rhodopsin. There are seven specific aims: (1) peptides corresponding to phosphorylated forms of the rhodopsin C-terminus will be synthesized and their structures and activities will be examined; (2) another peptide corresponding to the rhodopsin third cytoplasmic loop will be produced in a cyclic form and its structure and activity will be analyzed; (3) phage display technology will be used to identify sequences in arrestin that bind to rhodopsin; (4) phage display will also be used to identify sequences in rhodopsin that bind to arrestin; (5) antibody-variable regions will be expressed in bacteria and used to map the surface of arrestin in the presence and absence of phosphopeptides; (6) site(s) of interaction between rhodopsin and arrestin will be mapped by cross-linking; and (7) arrestins from several species will be isolated or expressed and used to grow crystals.
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