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X Song, F Meng, H Qian; White Perch GABA rho Subunits Interact With a Homologue of Endocytosis Protein RME-8 . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3784.
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Purpose: The GABAC receptors are composed of GABA ρ subunits and are expressed predominately in retinal neurons. The amount of GABAC receptors on cell membrane is thought to be dynamically regulated through endocytosis pathway. In this study, we used yeast two-hybrid systems to identify the intracellular proteins that interact with the GABA ρ subunits. Methods: The large intracellular domain of white perch GABA ρ2B subunit was cloned in frame into a bait vector pAS2-1. The construct was co-transfected into yeast (AH109) with a Zebrafish cDNA library constructed in the Gal4 activation domain carrying vector pGAD-10. Potential positives were selected on triple dropout medium (-Leu-Trp-His), and evaluated by liquid ß-galactosidase assay. Northern blots were performed with 5 µg of total RNA isolated from white perch retina and probed with 32P-labelled 2B-A3 fragment. Results: Sixty-five clones were selected with SD-Leu-Trp-His from 1x106 recombinants of the cDNA library. Fourteen of them exhibit positive reaction in liquid assay for ß-galactosidase activities. One clone (2B-A3) shows high homology (57% at amino acid level) to RME-8, a protein in C. elegans that participates in the trafficking of endocytotic vesicles. The expression of 2B-A3 in white perch retina was detected by Northern blot analysis. When tested in yeasts, 2B-A3 selectively interacts with other perch ρ subunits: it binds to ρ3 subunit, but not to ρ1A or ρ1B subunit. Conclusion: We identified a new protein that interacts with GABA ρ subunits, and such interactions might play important roles in mediating the endocytosis of the GABAC receptors on retinal neurons.
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