Characterization of 3',5' cyclic nucleotide phosphodiesterase activity in Y79 retinoblastoma cells: absence of functional PDE6

The phototransduction cascade within photoreceptors ismediated primarily through the action of three proteins: thereceptor, rhodopsin, the G-protein, transducin, and 3',5' cyclicnucleotide phosphodiesterase type 6 (PDE6). PDE6 isoformsare the only PDE family enzymes [1,2] in the rod and conephotoreceptor outer segments. Rod photoreceptor PDE6 iscomposed of two catalytic subunits, α and β, and two identi-cal partially inhibitory subunits, γ with calculated molecularmasses of 99 kDa, 98 kDa, and 11 kDa, respectively [3]. Conephotoreceptor PDE6 is composed of two α’ catalytic subunits,and a γ’ inhibitory subunit with calculated molecular massesof 93 kDa and 13 kDa, respectively [4,5]. A 14 kDa protein(δ) originally called a fourth subunit of PDE6 was identified[6] that can solubilize PDE6 activity from the rod outer seg-ment membrane and alter cGMP binding to noncatalytic bind-ing sites [7,8] and reduce light stimulated hydrolysis of cGMP[9]. Other retinal and non-retinal proteins in other tissues havebeen shown to interact with the δ-subunit [10-12]. PDE6 isreadily purified from isolated outer segments and can be ex-pressed in a weakly active or inactive state; however, expres-sion of a fully active stable enzyme that can be purified andremains active has not been demonstrated [13-18].The retinoblastoma derived cell lines Y79 and WERI[19,20] have been shown to exhibit biochemical characteris-tics of the retinal photoreceptors as well as other retinal celltypes [21,22]. Rod PDE6 α, β, and cone PDE6 α’ transcriptswere reported in Y79 cells at levels that could be detected byRNA blot analysis [23], however no protein analysis was per-formed. Early studies demonstrated that a Ca