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L. L. Wong, S. Sezate, J. F. McGinnis; Analysis of the Molecular Basis for Translocation/Compartmentalization of Rod Transducin-Alpha. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4650.
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Rod Transducin-alpha (rTa) mediates the downstream signaling from activated rhodopsin molecules for dim light vision. However, under normal lighting, rTa is not available for signal transduction because it is almost completely absent from the rod outer segment (ROS) where photo-transduction occurs. Because rTa is present in the ROS in the dark and absent in the light, we think the translocation process by which these changes occur must be important for rod photoreceptor cell function and/or health. However, the molecular mechanism by which rTa translocation/compartmentalization are achieved is not well understood. Therefore we have initiated a structure/function analysis to investigate the portion of rTa that is sufficient to mediate these processes in mammalian rod photoreceptor cells.
We used PCR cloning to fuse the entire mouse gnat1 or one-third partial segments (N1: N-ter; N2: middle; and N3: C-ter) to the C-terminal of EGFP in a mammalian expression vector under control of the CMV promoter (pEGFP-rTa, -rTN1-3). The constructs were transfected into HEK 293 cells to test the expression of the fusion proteins. The DNA was then transfected into P0-4 rat pup retinas via subretinal injection and electroporation. Pups were either dark- or light-adapted before their retinas were harvested at P21 or older. Sections from GFP-positive retinas were immunolabeled with antibodies against Rhodopsin, rTa, rTbeta, Recoverin, and cone Opsins to characterize the expression pattern of EGFP-fused constructs. Confocal microscopy was used to analyze the labeled sections.
The EGFP alone construct expressed optimally in HEK 293 cells and in the retina where GFP was found throughout the soma and processes of Müller glia, bipolar and rod cells. The expression of fusion proteins were confirmed by Western blots. All four EGFP constructs were expressed in HEK 293 cells and in vivo in the retina, although expression patterns and efficiencies were very different. Some of the EGFP-fused proteins were only expressed in rods, despite the CMV promoter. The different subcellular localization patterns in light-and dark-adapted retinas will be presented in detail.
We have fused EGFP to rTa and truncated rTa and have shown them to express in HEK 293 cells and in rat retinas via transient transfection. Their differential patterns of expression should reveal the portions of rTa that enable its subcellular compartmentalization. Our data will contribute to our understanding of the maintenance of photoreceptor cell health.
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