In-frame fusion of SUMO1 enhances βarrestin2's association with activated GPCRs as well as with nuclear pore complexes.
Small ubiquitin like modifier (SUMO) conjugation or SUMOylation of βarrestin2 promotes its association with the clathrin adaptor protein AP2 and facilitates rapid β2 adrenergic receptor (β2AR) internalization. However, disruption of the consensus SUMOylation site in βarrestin2, did not prevent βarrestin2's association with activated β2ARs, dopamine D2 receptors (D2Rs), angiotensin type 1a receptors (AT1aRs) and V2 vasopressin receptors (V2Rs). To address the role of SUMOylation in the trafficking of βarrestin and GPCR complexes, we generated and characterized a yellow fluorescent protein (YFP) tagged βarrestin2-SUMO1 chimeric protein, which is resistant to de-SUMOylation. In HEK-293 cells, YFP-SUMO1 predominantly localized in the nucleus, whereas YFP-βarrestin2 is cytoplasmic. YFP-βarrestin2-SUMO1 in addition to being cytoplasmic, is localized at the nuclear membrane. Nonetheless, βarrestin2-SUMO1 associated robustly with agonist-activated β2ARs as evaluated by co-immunoprecipitation, confocal microscopy and bioluminescence resonance energy transfer (BRET). βarrestin2-SUMO1 associated strongly with the D2R, which forms transient complexes with βarrestin2. But, βarrestin2-SUMO1 and βarrestin2 showed equivalent binding with the V2R, which forms stable complexes with βarrestin2. βarrestin2 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, βarrestin2-SUMO1 not only localized at the nuclear membrane, but also formed a macromolecular complex with RanGAP1. Taken together, our data suggest that SUMOylation of βarrestin2 promotes its protein interactions at both cell and nuclear membranes. Furthermore, βarrestin2-SUMO1 presents as a useful tool to characterize βarrestin2 recruitment to GPCRs, which form transient and unstable complex with βarrestin2.
Nagi, K; Kaur, S; Bai, Y; Shenoy, SK
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