Upon photoexcitation, rhodopsin's surface changes conformation to a """"""""signalling state"""""""" that binds transducin, rhodopsin kinase and arrestin. We propose to determine these regions of rhodopsin's surface, their important amino acids and their arrangement in space that constitute this """"""""signalling state"""""""". To do this we will map the surfaces of rhodopsin, arrestin and transducin that interact with one another. (1) We will determine the surface regions of photoexcited phosphorylated rhodopsin that interact with arrestin by direct chemical modification and by peptide competition using rhodopsin peptides and their analogs. (2) In complementary experiments we will determine the surface regions of arrestin that interact with photoexcited phosphorylated rhodopsin by direct chemical modification, by peptide competition using arrestin peptides and their analogs, and by the use of anti-arrestin antibodies of defined site specificity. In order to do this we will raise anti-arrestin monoclonal antibodies, determine their specificity, and use them for assays and other experiments. (3) We will determine the strength of interactions between arrestin peptides and phosphorylated rhodopsin, and between rhodopsin peptides and arrestin. (4) We will extend our knowledge of the interaction between the transducin Alpha-subunit and photoexcited rhodopsin to determine all sites that interact and the important amino acids in each region, and their strengths of interaction. Complementary studies will be completed for the interactive sites of rhodopsin with Alpha-transducin. (5) We will use the above information to synthetically simplify the interactive peptide regions from the rhodopsin surface and to assemble them to simulate the measured activities of the surface of photoactivated rhodopsin.
