Identification of a Deubiquitinating Enzyme as a Novel AGS3-Interacting Protein

Abstract

Activator of G protein Signaling 3 (AGS3) is a receptor-independent G protein activator that has been implicated in multiple biological events such as brain development, neuroplasticity and addiction, cardiac function, Golgi structure/function, macroautophagy and metabolism. However, how AGS3 is regulated is little known. We demonstrate here that AGS3 interacts with a ubiquitin specific protease USP9x, and this interaction is at least partially mediated through the C-terminal G protein regulatory domain of AGS3. Knockdown of USP9x causes a moderate reduction in the level of AGS3. In contrast, overexpression of either USP9x or its deubiquitinating domain UCH increases the amount of AGS3, whereas expression of the mutant UCH domain that lacks deubiquitinating activity does not have the same effect. As previously observed in AGS3 knockdown cells, the localization of several marker proteins of the late Golgi compartments is disturbed in cells depleted of USP9x. Taken together, our study suggests that USP9x can modulate the level of a subpopulation of AGS3, and this modulation plays a role in regulating the structure of the late Golgi compartments. Finally, we have found that levels of AGS3 and USP9x are co-regulated in the prefrontal cortex of rats withdrawn from repeated cocaine treatment. In conjunction with the above data, this observation indicates a potential role of USP9X in the regulation of the AGS3 level during cocaine-induced neuroplasticity.

Rapamycin attenuates the expression of cocaine‐induced place preference and behavioral sensitization

“…The total protein concentrations and were equalized across samples for each brain region and denatured in SDS-PAGE sample loading buffer. Immunoblotting was performed as described previously (Xu et al, 2010). Equal amounts of total protein (30–35ug) were loaded into each well of 10% acrylamide gels, separated by SDS-PAGE, and transferred to Immobilon 0.45μM PVDF membranes (Millipore, Billerica, MA) using a Semi Dry Electroblotting System (Thermo Scientific Owl Separation Systems, Rochester, NY).…”

WD Repeat-containing Protein 5 (WDR5) Localizes to the Midbody and Regulates Abscission

“…Production of Lentiviral Particles-Production of lentiviral particles and infection of HeLa and RPE1 cell lines were performed as described previously (40). HEK293T cells were grown to 70% confluence in 100-mm plates, and the spent medium was replaced with fresh medium 2 h before transfection.…”

Neuronal Functions of Activators of G Protein Signaling

“…AGS3 is found to be coregulated with USP9x (ubiquitin specific protease 9x) in the rat PFC after cocaine withdrawal [42]. USP9x interacts with the GPR motifs of AGS3 and stabilizes AGS3 by de-ubiquitination.…”