Molecular components regulating mammalian phototransduction

Monoclonal antibody 7G6 specifically labels primate and bovine cone photoreceptors, but not cones of rodents. Immunoblot analysis indicated that 7G6 recognized a single protein band around 45 kD. The antigen was purified from human retina by immunoprecipitation. Partial peptide sequencing identified the antigen as cone arrestin. The identity was confirmed by Western blotting and competition experiments. The epitope of 7G6 was mapped within a divergent loop exposed at the surface of the cone arrestin N-domain. Similar to rod arrestin, cone arrestin exhibited light-dependent relocalization in light-adapted and dark-adapted bovine eyes (ex-vivo).; To explore the mechanisms of light-dependent redistribution of phototransduction components, we examined the redistribution of visual arrestins and transducin within retina from various knockout mice defective in phototransduction. Our results indicated that Arr and cArr redistributed in the light to the outer segment in transducin α subunit knockout (Gnat1−/− ) and rhodopsin knockout (Grk1−/−) mice as well as in Grk1−/−/Gnat1−/− double knockout retinas. Immunoblotting revealed that approximately 25–50% of Arr associated with the membrane in light-adapted wild-type, Gnat1−/− and Gnat1−/−/Grk1 −/− mouse retinas. In contrast, cArr did not associate stably with light-adapted membranes in wild-type and Grk1−/− retinas, but redistributed to the outer segments in a light-dependent manner. The redistribution of transducin subunits to the inner segments in light occurred in both wild-type and Grk1−/−/Arr −/− double knockout photoreceptors. However, Tβγ subunits did not redistribute in the absence of Tα, suggesting that transducin only translocates as an intact heterotrimer.; We also studied the function of PDEδ in photoreceptors. Immunocytochemistry with a PDEδ specific antibody indicated that PDEδ was present in rods, cones and other cell types of the bovine retina. We found by yeast-two hybrid that PDEδ could interact with farnesylated rhodopsin kinase (GRK1), and that prenylation is essential for this interaction. GST pulldowns indicated that both recombinant farnesylated GRK1 and geranylgeranylated GRK7 coprecipitated with a GST-PDEδ fusion protein. Using fluorescence resonance energy transfer (FRET) techniques, we showed that PDEδ specifically binds geranylgeranyl and farnesyl moieties with a Kd of 19.06 μM and 0.70 μM, respectively. Our experiments establish that PDEδ functions as a prenyl binding protein.