The glucagon receptor (GCGR) activated by the peptide hormone glucagon is a seven-transmembrane G protein–coupled receptor (GPCR) that regulates blood glucose levels. Ubiquitination influences trafficking and signaling of many GPCRs, but its characterization for the GCGR is lacking. Using endocytic colocalization and ubiquitination assays, we have identified a correlation between the ubiquitination profile and recycling of the GCGR. Our experiments revealed that GCGRs are constitutively ubiquitinated at the cell surface. Glucagon stimulation not only promoted GCGR endocytic trafficking through Rab5a early endosomes and Rab4a recycling endosomes, but also induced rapid deubiquitination of GCGRs. Inhibiting GCGR internalization or disrupting endocytic trafficking prevented agonist-induced deubiquitination of the GCGR. Furthermore, a Rab4a dominant negative (DN) that blocks trafficking at recycling endosomes enabled GCGR deubiquitination, whereas a Rab5a DN that blocks trafficking at early endosomes eliminated agonist-induced GCGR deubiquitination. By down-regulating candidate deubiquitinases that are either linked with GPCR trafficking or localized on endosomes, we identified signal-transducing adaptor molecule–binding protein (STAMBP) and ubiquitin-specific protease 33 (USP33) as cognate deubiquitinases for the GCGR. Our data suggest that USP33 constitutively deubiquitinates the GCGR, whereas both STAMBP and USP33 deubiquitinate agonist-activated GCGRs at early endosomes. A mutant GCGR with all five intracellular lysines altered to arginines remains deubiquitinated and shows augmented trafficking to Rab4a recycling endosomes compared with the WT, thus affirming the role of deubiquitination in GCGR recycling. We conclude that the GCGRs are rapidly deubiquitinated after agonist-activation to facilitate Rab4a-dependent recycling and that USP33 and STAMBP activities are critical for the endocytic recycling of the GCGR.

中文翻译：

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激动剂激活的胰高血糖素受体在早期内涵体中被两种不同的去泛素化酶去泛素化，以促进 Rab4a 依赖性回收。

由肽激素胰高血糖素激活的胰高血糖素受体 (GCGR) 是一种七跨膜 G 蛋白偶联受体 (GPCR)，可调节血糖水平。泛素化影响许多 GPCR 的运输和信号传导，但缺乏 GCGR 的表征。使用内吞共定位和泛素化测定，我们确定了泛素化谱与 GCGR 回收之间的相关性。我们的实验表明 GCGR 在细胞表面组成性泛素化。胰高血糖素刺激不仅促进 GCGR 通过 Rab5a 早期内体和 Rab4a 回收内体进行内吞运输，而且还诱导 GCGR 快速去泛素化。抑制 GCGR 内化或破坏内吞运输可防止激动剂诱导的 GCGR 去泛素化。此外，阻止循环内体转运的 Rab4a 显性失活 (DN) 能够实现 GCGR 去泛素化，而阻止早期内体转运的 Rab5a DN 消除了激动剂诱导的 GCGR 去泛素化。通过下调与 GPCR 运输相关或位于核内体上的候选去泛素酶，我们确定了信号转导衔接分子结合蛋白 (STAMBP) 和泛素特异性蛋白酶 33 (USP33) 作为 GCGR 的同源去泛素酶。我们的数据表明，USP33 组成性地使 GCGR 去泛素化，而 STAMBP 和 USP33 都使早期核内体中激动剂激活的 GCGR 去泛素化。与 WT 相比，所有 5 个细胞内赖氨酸均变为精氨酸的突变 GCGR 仍处于去泛素化状态，并且显示出向 Rab4a 回收内体的运输增加，从而肯定了去泛素化在 GCGR 回收中的作用。我们得出的结论是，GCGR 在激动剂激活后迅速去泛素化，以促进 Rab4a 依赖性回收，并且 USP33 和 STAMBP 活性对于 GCGR 的内吞回收至关重要。