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A.L. Rao, C.–K. Chen; Instability of RGS7 in COS7 cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1275.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To investigate the instability of GGL (G–protein Gamma–Like) containing RGS (Regulator of G–protein signaling) proteins in the absence of Gß5. We test the hypothesis that a protein degradation signal conferring instability resides in the primary sequence of these RGS proteins. Methods: Full–length RGS7 cDNA was engineered to contain an in–frame EGFP and was expressed in COS7 cells in the presence and absence of Gß5. To identify the region of RGS7 that confers instability, we tested the stability of a library of EGFP–tagged, truncated, and over–lapping RGS7 fragments of approximately 180 amino acids. Western blot analysis and fluorescent microscopy were used to determine the stability of the fusion proteins. MG132 (proteasome inhibitor) was used to test the involvement of the proteosome degradation pathway. In vitro transcription/translation was used to examine all of our plasmid constructs. Results: Full length RGS7–EGFP fusion protein was not stable in COS7 cells. However, it could be stabilized by Gß5 co–expression or by MG132 treatment. Truncated RGS7–EGFP fusion proteins containing the GGL domains were not stable, but could be stabilized by MG132 and not by Gß5 co–transfection. MG132 treatment could enhance the steady state levels of RGS7–EGFP fusion proteins containing the N–terminal DEP domain. Conclusion: We conclude that the GGL domain of RGS7 contains a protein degradation signal that confers instability in the absence of Gß5. Our data also suggest that N–terminal DEP domain of RGS7 may also involve in the degradation of RGS7.
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