Agonist-activated glucagon receptors are deubiquitinated at early endosomes by two distinct deubiquitinases to facilitate Rab4a-dependent recycling.
Agonist-activated glucagon receptors are deubiquitinated at early endosomes by two distinct deubiquitinases to facilitate Rab4a-dependent recycling.
复制标题
激动剂活化的胰高血糖素受体在早期内体被两种不同的去泛素酶去泛素化，以促进RAB4A依赖性回收。
DOI：
10.1074/jbc.ra120.014532
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发表时间：
2020-12-04
期刊：
影响因子：
--
通讯作者：
Shenoy SK
中科院分区：
文献类型：
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作者：
Kaur S;Chen Y;Shenoy SK
胰高血糖素受体(GCGR)是一种七跨膜G蛋白偶联受体，调节血糖水平。泛素化影响许多GPCR的贩运和信号传递，但缺乏对GCGR的表征。利用细胞内共定位和泛素化分析，我们已经确定了泛素化特征和GCGR循环之间的相关性。我们的实验表明，GCGRs在细胞表面有结构性的泛素化。胰高血糖素刺激不仅促进GCGR通过Rab5a早期内体和Rab4a循环内体转运，而且诱导GCGR快速去泛素化。抑制GCGR内化或干扰细胞内转运可阻止激动剂诱导的GCGR去泛素化。此外，Rab4a显性阴性(DN)可以阻止GCGR去泛素化，而Rab5a dN可以阻止激动剂诱导的GCGR去泛素化。通过下调与GPCR转运相关或定位在内体上的候选脱泛素酶，我们确定信号转导适配器分子结合蛋白(STAMBP)和泛素特异性蛋白酶33(USP33)是GCGR的同源脱泛素酶。我们的数据表明，USP33结构性地去泛素化GCGR，而STAMBP和USP33去泛素化激动剂激活的GCGR在早期内体。突变的GCGR将细胞内的五种赖氨酸全部改变为精氨酸，仍然是去泛素化的，与WT相比，GCGR向Rab4a循环内体的转运增加，从而肯定了去泛素化在GCGR循环中的作用。我们的结论是，GCGR在激动剂激活后迅速去泛素化，以促进Rab4a依赖的循环，并且USP33和STAMBP活性对GCGR的内吞循环至关重要。
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.