In rod visual cells, light absorption by the receptor protein, rhodopsin, initiates an enzyme cascade that leads to a decrease in intracellular levels of cGMP. This transient decrease in [cGMP]i causes closure of cation channels on the plasma membrane of the rod outer segment and a hyperpolarization of the visual cell (i.e., a photoresponse). Sensitivity and adaptation of the photoresponse under varying conditions of illumination depend critically on the deactivation of this enzyme cascade, and the recovery of cGMP level. An impairment in the ability to deactivate processes underlying the light-induced signal will result in abnormal levels of cGMP, and can lead to poor vision or even photoreceptor cell death. The emphasis of the present application is on arrestin, one of the proteins involved in deactivating the enzyme cascade of phototransduction. The goal of proposed studies is to determine the mechanism of arrestin turnover, and to examine how this mechanism relates to driving forces behind light-dependent movements of arrestin between rod inner- (IS) and outer segments (OS). The rational is preliminary data suggesting enhanced degradation of arrestin upon light exposure. If such degradation of arrestin occurs, then arrestin's half-life in rods may be shorter than that of opsin, and a mechanism for degradation -- in addition to disk shedding - - might be involved. Targeting of arrestin by cytosolic proteases (e.g., calpain) is hypothesized as one possible mechanism. A novel analysis of the arrestin sequence identifying PEST regions (i.e., conditional sites of proteolysis signalling by cytosolic proteases) is consistent with this hypothesis. On this basis, studies examining (i) arrestin biosynthesis and (ii) limited proteolysis as a mechanism for arrestin """"""""deactivation"""""""" are proposed. Experiments employing biochemical, immunocytochemical and morphological methods are planned.
Specific aims of the proposed studies include:
(AIM 1 a) determining the turnover of arrestin in rods as a function of light exposure, and (AIM 1b) examining the possible involvement of microfilaments in the transport of arrestin between IS and OS;
(AIM 2) examining whether arrestin is degraded by an ROS (cytosolic) protease (e.g., calpain, and/or ubiquitin), and (AIM 3) examining the functional consequences of limited proteolysis of arrestin. Experiments to determine whether proteolysis occurs at a PEST region of the arrestin sequence are also planned. Continued renewal -- not only of arrestin -- but all proteins within rods, is critical for normal visual cell function. A long-term goal of proposed studies is to determine whether defects in this renewal process may underlie retinal degenerative diseases such as Retinitis Pigmentosa.
