Abstract
by 1-by-1 Y2H interaction assay on solid growth under restrictive growth medium conditions. The interaction between hSVCT1 and hGRHPR was further confirmed by in vitro GST-pulldown assay and in vivo co-immunoprecipitation and mammalian two-hybrid firefly luciferase assays. In addition, hSVCT1-YFP and DsRed-hGRHPR co-expressing human liver HepG2 cells demonstrated co-localization of these two proteins in intracellular trafficking vesicles. Co-expression of hGRHPR with hSVCT1 led to a marked increase in 14C-AA uptake. On the other hand, knock down of endogenous hGRHPR with gene-specific siRNA led to a marked inhibition in 14C-AA uptake. This effect was dependent upon a marked reduction in GRHPR mRNA, while, hSVCT1 and hSVCT2 mRNA levels were unaffected. Conclusion: This study demonstrates hGRHPR is an hSVCT1 interacting partner in human liver cells and this interaction is important for the function and cell biology of hSVCT1. (Supported by grants from the DVA and NIH DK 71538, DK 84094, DK 56061).
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