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 (AT_1aRs) 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.

βarrestin2 的小泛素样修饰剂 (SUMO) 缀合或 SUMOylation 促进其与网格蛋白接头蛋白 AP2 的结合，并促进 β ₂肾上腺素能受体 (β ₂ AR) 的快速内化。然而，βarrestin2 共有 SUMOylation 位点的破坏并没有阻止 βarrestin2 与活化的 β ₂ AR、多巴胺 D ₂受体（D ₂ Rs）、血管紧张素 1a 型受体（AT _1a Rs）和 V ₂加压素受体（V ₂卢比）。为了解决 SUMOylation 在 βarrestin 和 GPCR 复合物运输中的作用，我们生成并表征了一种黄色荧光蛋白 (YFP) 标记的 βarrestin2-SUMO1 嵌合蛋白，该蛋白对去 SUMOylation 具有抗性。在 HEK-293 细胞中，YFP-SUMO1 主要位于细胞核中，而 YFP-βarrestin2 位于细胞质中。YFP-βarrestin2-SUMO1 除了位于细胞质外，还定位于核膜。尽管如此，通过免疫共沉淀、共聚焦显微镜和生物发光共振能量转移 (BRET) 评估，βarrestin2-SUMO1 与激动剂激活的 β ₂ AR密切相关。βarrestin2-SUMO1 与 D ₂密切相关R，与 βarrestin2 形成瞬时复合物。但是，βarrestin2-SUMO1 和βarrestin2 显示出与V ₂ R 的等效结合，后者与βarrestin2 形成稳定的复合物。βarrestin2 表达水平与未修饰形式 RanGAP1 的稳态水平直接相关，后者在 SUMO 化后与核膜相关。另一方面，βarrestin2-SUMO1不仅定位于核膜，还与RanGAP1形成大分子复合物。总之，我们的数据表明 βarrestin2 的 SUMO 化促进了其在细胞膜和核膜上的蛋白质相互作用。此外，βarrestin2-SUMO1 是表征 βarrestin2 募集到 GPCR 的有用工具，GPCR 与 βarrestin2 形成短暂且不稳定的复合物。