Molecular and biochemical characterization of photoreceptor phosphodiesterase in retina and retinoblastoma cells.

摘要

The 3′, 5′-cyclic nucleotide phosphodiesterases (PDE) are composed of a family of 11 distinct enzymes (PDE1-PDE11) and several isozymes. These enzymes function in signal transduction cascades involving the second messengers 3′, 5′-cyclic adenosine monophosphate (cAMP) and 3′, 5′ -cyclic guanosine monophosphate (cGMP), through hydrolysis of a phosphodiester bond generating the corresponding 5′ nucleotide monophosphate. The PDE6 family consists of isozymes located exclusively in the rod and cone photoreceptor cells (rPDE6 and cPDE6, respectively) of the retina and plays a central role in the visual transduction cascade. Activated rPDE6 hydrolyzes cGMP, yielding 5′-GMP, which, in turns, leads to the closure of a photoreceptor-specific cGMP-gated cation channel. When the channel closes, hyperpolarization of the photoreceptor results, leading to a signal being generated and transmitted to the visual centers of the brain. Recovery to the dark-adapted state results from activation of a photoreceptor-specific guanylate cyclase replenishing cGMP and from inhibition of rhodopsin through phosphorylation and subsequent binding of arrestin.; Defects in rPDE6 have been shown to cause human hereditary retinal diseases that lead to photoreceptor degeneration that can lead to blindness. Animal models for retinal diseases have also been shown to contain mutations in the α- and β-subunits of rPDE6, further indicating that this enzyme is crucial for proper photoreceptor function.; The goal of this research was to gain an understanding of the molecular and biochemical mechanisms involved in the regulation of rPDE6 gene expression and functional enzyme activity. Promoter analysis is beneficial for an understanding of expression patterns and levels of expression and has already been proven to be a useful benefit for targeted gene delivery in the treatment of human disease. Structure and function studies of this enzyme have also been hampered because of the lack of a suitable system to produce recombinant rPDE6.; The retina-derived cell line Y79 was used because it has been shown to contain rPDE6 transcripts. The total complement of phosphodiesterase activities was characterized to determine the relative contribution of rPDE6. Finally, this cell line was used to gain an understanding of the regulatory components of the rPDE6A gene necessary for cell-specific expression through in vitro analysis. This project establishes the usefulness of the Y79 cell line for expression of rPDE6 and furthers our understanding of the mechanisms that regulate rPDE6 gene expression through the characterization of the rPDE6A promoter in this cell line.