33K/BETA-Gamma Transducin Complex of Mammalian Rod Photoreceptors

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This research project aims to characterize further the mechanisms which regulate the phosphorylation in darkness and the light-activated dephosphorylation of the visual cell specific, 33K/beta-gamma transducin (33K/TBY) complex. The 33K (33 Kilodalton) protein which is phosphorylated by cyclic nucleotide- dependent protein kinase, in vitro, has been purified to near homogeneity and shown to form a complex with the beta-gamma subunits of transducin. In situ, the level of phosphorylated 33K in dark-adapted ROS is enhanced further by incubating retinas with drugs which increase the concentration of cyclic GMP. Immunocytochemistry using monospecific antibodies against bovine 33K or the beta/gamma subunits of transducin shows that both 33K and T-beta/gamma are localized in rod outer segments as well as the inner segments of bovine and mouse photoreceptors. Biochemical fractionation shows further that the 33K protein is bound firmly to ROS membranes and that the 33K is probably phosphorylated whereas the soluble 33K/TBY complex is unphosphorylated. These findings suggest that the binding of 33K and/or the 33K/TBY complex to ROS membranes is controlled by the phosphorylation of 33K. The P.I.s will evaluate this hypothesis and find the role of 33K/TBY complex in a series of related projects that (1) characterize the membrane-bound species of 33K (m33K); (2) evaluate the partitioning of 33K between the soluble and membrane fractions of ROS; (3) investigate the light- initiated dephosphorylation of m33K; (4) identify and characterize the protein kinase which phosphorylates m33K; and (5) determine whether 33K and/or the 33K/TBY complex interacts with transducin and/or modulates the participation of transducin in the PDE-activation cascade. This systematic study of the 33K/TBY complex will provide a fuller understanding of light- activated events in rod visual cells and project a role for the 33K/TBY complex in ROS function.