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

[摘要] Purpose: Previous studies identified rod photoreceptor cyclic GMPphosphodiesterase (PDE6) transcripts in the human Y79 retinoblastomacell line. To assess the potential to utilize this cell line forstructure/function studies of PDE6, we analyzed 3',5' cyclic nucleotidephosphodiesterase activity focusing on expression of PDE6.Methods: DEAE-chromatography was used to fractionate PDE activityfrom Y79 cell homogenates. PCR was performed on cDNA generated from Y79cells and retina with PDE isoform specific primers. Western blots wereperformed with antibodies to PDE1, PDE4, or rod PDE6. DNA sequencing andprotein truncation tests were performed with plasmids containing theentire coding region of Y79 rod PDE6 transcripts. Proteasome mediateddegradation of PDE6 subunits was analyzed with a pathway specificinhibitor. Polysome isolation was performed by fractionation on sucrosegradients followed by RT-PCR for the PDE6 transcripts.Results: Of three peaks of PDE activity, peaks 1 and 2 wereactivated by Ca2+/calmodulin, inhibited by dipyridamole andzaprinast, and were reactive with a PDE1 antibody. Peak 3 hydrolyzedonly cAMP and was rolipram sensitive, indicative of PDE4. Transcriptsfor rod and cone PDE6 isoforms were detected in Y79 total RNA, howeverPDE6 antibodies recognized only a single 99 kDa polypeptide fromimmunoprecipitated 35S labeled Y79 extracts. DNA sequencing ofPDE6 α, β, γ, and PDE6 associated δ-subunit cDNArevealed some polymorphism, but no apparent mutations. Each of the PDE6transcripts could be translated into protein of the correct length. Theconcentration of cGMP in the cells was greatly reduced in comparison tothat reported in the photoreceptor cell. Addition of cyclic nucleotideanalogues, zinc, or butyrate did not enhance the expression of PDE6.Transduction into Y79 cells of adenovirus expressing PDE6 subunitsfailed to produce functional enzymeConclusions: PDE1 and PDE4 enzyme activities predominate in Y79cells. Despite the presence of PDE6 transcripts and the ability totranslate each into protein in vitro, a functional PDE6 enzyme could notbe detected. Attempts to enhance expression with cell culture or withintroduction of virus expressing PDE6 were not successful. The resultsindicate that expression of a fully active stable PDE6 enzyme requiresother post-transcriptional events that do not occur or are inhibited inY79 cells.

[学科分类] 生物化学/生物物理