In-frame fusion of SUMO1 enhances ßarrestin2's association with activated GPCRs as well as with nuclear pore complexes.

ABSTRACT

Small ubiquitin like modifier (SUMO) conjugation or SUMOylation of ßarrestin2 promotes its association with the clathrin adaptor protein AP2 and facilitates rapid ß2 adrenergic receptor (ß2AR) internalization. However, disruption of the consensus SUMOylation site in ßarrestin2, did not prevent ßarrestin2's association with activated ß2ARs, dopamine D2 receptors (D2Rs), angiotensin type 1a receptors (AT1aRs) and V2 vasopressin receptors (V2Rs). To address the role of SUMOylation in the trafficking of ßarrestin and GPCR complexes, we generated and characterized a yellow fluorescent protein (YFP) tagged ßarrestin2-SUMO1 chimeric protein, which is resistant to de-SUMOylation. In HEK-293 cells, YFP-SUMO1 predominantly localized in the nucleus, whereas YFP-ßarrestin2 is cytoplasmic. YFP-ßarrestin2-SUMO1 in addition to being cytoplasmic, is localized at the nuclear membrane. Nonetheless, ßarrestin2-SUMO1 associated robustly with agonist-activated ß2ARs as evaluated by co-immunoprecipitation, confocal microscopy and bioluminescence resonance energy transfer (BRET). ßarrestin2-SUMO1 associated strongly with the D2R, which forms transient complexes with ßarrestin2. But, ßarrestin2-SUMO1 and ßarrestin2 showed equivalent binding with the V2R, which forms stable complexes with ßarrestin2. ßarrestin2 expression level directly correlated with the steady state levels of the unmodified form of RanGAP1, which upon SUMOylation associates with nuclear membrane. On the other hand, ßarrestin2-SUMO1 not only localized at the nuclear membrane, but also formed a macromolecular complex with RanGAP1. Taken together, our data suggest that SUMOylation of ßarrestin2 promotes its protein interactions at both cell and nuclear membranes. Furthermore, ßarrestin2-SUMO1 presents as a useful tool to characterize ßarrestin2 recruitment to GPCRs, which form transient and unstable complex with ßarrestin2.