Yang Jingwen, Shen Zhangfei, Jiang Xue, Yang Huipeng, Huang Haishan, Jin Lili, Chen Yajie, Shi Liangen, Zhou Naiming
National Engineering Research Center of Marine Facilities Aquaculture, Zhejiang Ocean University , Zhoushan, Zhejiang 316022, China.
Department of Economic Zoology, College of Animal Sciences, Zhejiang University , Hangzhou, Zhejiang 310058, China.
Biochemistry. 2016 Jul 19;55(28):3874-87. doi: 10.1021/acs.biochem.6b00250. Epub 2016 Jul 8.
Agonist-induced internalization plays a key role in the tight regulation of the extent and duration of G protein-coupled receptor signaling. Previously, we have shown that the Bombyx corazonin receptor (BmCrzR) activates both Gαq- and Gαs-dependent signaling cascades. However, the molecular mechanisms involved in the regulation of the internalization and desensitization of BmCrzR remain to be elucidated. Here, vectors for expressing BmCrzR fused with enhanced green fluorescent protein (EGFP) at the C-terminal end were used to further characterize BmCrzR internalization. We found that the BmCrzR heterologously expressed in HEK-293 and BmN cells was rapidly internalized from the plasma membrane into the cytoplasm in a concentration- and time-dependent manner via a β-arrestin (Kurtz)-dependent and clathrin-independent pathway in response to agonist challenge. While most of the internalized receptors were recycled to the cell surface via early endosomes, some others were transported to lysosomes for degradation. Assays using RNA interference revealed that both GRK2 and GRK5 were essentially involved in the regulation of BmCrzR phosphorylation and internalization. Further investigations indicated that the identified cluster of Ser/Thr residues ((411)TSS(413)) was responsible for GRK-mediated phosphorylation and internalization. This is the first detailed investigation of the internalization and trafficking of Bombyx corazonin receptors.
激动剂诱导的内化作用在G蛋白偶联受体信号传导的程度和持续时间的严格调控中起着关键作用。此前，我们已经表明，家蚕促心肽受体（BmCrzR）可激活Gαq和Gαs依赖性信号级联反应。然而，BmCrzR内化和脱敏调控所涉及的分子机制仍有待阐明。在此，利用在C末端融合增强型绿色荧光蛋白（EGFP）的BmCrzR表达载体，进一步表征BmCrzR的内化过程。我们发现，在HEK-293和BmN细胞中异源表达的BmCrzR，在激动剂刺激下，通过β-抑制蛋白（库尔茨）依赖性且网格蛋白非依赖性途径，以浓度和时间依赖性方式从质膜迅速内化至细胞质中。虽然大多数内化的受体通过早期内体循环至细胞表面，但其他一些则被转运至溶酶体进行降解。使用RNA干扰的实验表明，GRK2和GRK5均基本参与BmCrzR磷酸化和内化的调控。进一步研究表明，所鉴定的Ser/Thr残基簇（(411)TSS(413)）负责GRK介导的磷酸化和内化。这是对家蚕促心肽受体内化和转运的首次详细研究。
