After absorbing a photon, rhodopsin is multiply phosphorylated by an intrinsic kinase specific for rhodopsin. The in vivo rate of dephosphorylation approximately corresponds to the time course of dark adaptation. Rhodopsin phosphorylation apparently, at least partially, regulates the c-GMP phosphodiesterase (PDE) activity in the rod outer segment. The long-term objective of this application is to determine the role rhodopsin phosphorylation plays in the biochemistry and physiology of the rod. Rhodopsin with 0, 2, 4, 6, 8, and 9 phosphates per rhodopsin have been isolated. However, under many conditions (e.g., low pH and low divalent metal ion concentration), phosphate is lost from the rhodopsin during the processing. In order to test theories involving rhodopsin phosphorylation, we must be able to purify and reconstitute the separate components of the system. We plan to investigate the divalent metal ion requirement of phosphorylated rhodopsin. We will establish conditions necessary for maintaining the phosphorylation level of each of the rhodopsin species and will isolate and reconstitute each within membrane vesicles. The hypothesis that phosphorylation of opsin is involved in the turn-off mechanism in the light activation of the PDE will be tested using the purified extractable components proposed in the activation-deactivation cycle along with vesicles containing the phosphorylated species of rhodopsin. The possible further involvement of phosphorylation sites in a more """"""""fine control"""""""" manner will be examined by using these vesicles and preactivated GTPase with extracted PDE to determine if various levels of PDE activity can be obtained using the different phosphorylated rhodopsins. If the GTP binding protein can be activated by the phosphorylated rhodopsin, we will determine if variable activity can be imparted by the various forms of rhodopsin. The relative binding affinities of the peripheral membrane proteins in the ROS for the phosphorylated forms of rhodopsin will be investigated. We will investigate the time course of the appearance of specific phosphorylated species and determine the species that are generated under various Ca++ concentrations. We will further characterize the phosphorylation of unbleached rhodopsin after a series of 0.1 to 0.4% bleaches, determining if solution conditions can be found in which rhodopsin phosphorylation is enhanced relative to opsin phosphorylation or vice versa.
