Quantitative interactome analysis of RGS9‐2 degradation mechanisms in mammalian brain.

Abstract

RGS9‐2 is a striatal‐specific protein involved into regulation of movement and reward through negative regulation of muopioid and D2 dopamine receptor signaling. Changing RGS9‐2 expression is an important way to achieve the plasticity of signaling regulation and behavioral outcomes. R7 binding protein (R7BP) is an interaction partner of RGS9‐2 that sets the expression of RGS9‐2 by controlling proteolytic stability of the complex. Uncoupling of RGS9‐2 from R7BP is thought to be a mechanism of RGS9‐2 levels regulation.The aim of the study was to elucidate the molecular mechanism of RGS9‐2 degradation upon loss of R7BP.We developed a proteomic approach for quantitative analysis of interactome changes in mouse knockout models and used it to identify interactions of RGS9‐2 upregulated in R7BP knockout mice. The screen resulted in the identification of 21 proteins among which chaperone Hsc70 was found to exhibit one of the biggest changes in binding to RGS9‐2. The interaction was validated both in vivo and in transfected 293 cells and was found to be mediated by the intrinsically disordered C‐terminal domain of RGS9‐2. Down‐regulation of Hsc70 expression using siRNA and a dominant negative approach increased levels of RGS9‐2.We conclude that Hsc70 is a negative regulator of RGS9‐2 levels in the striatum.NIH NIDA DA021743