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Internalization of AMPA-type Glutamate Receptor in the MIN6 Pancreatic β-cell Line.
Cell Structure and Function ( IF 2.0 ) Pub Date : 2020-08-21 , DOI: 10.1247/csf.20020
The Mon La 1 , Hiroshi Yamada 1 , Sayaka Seiriki 1 , Shun-Ai Li 2 , Kenshiro Fujise 1 , Natsuho Katsumi 1 , Tadashi Abe 1 , Masami Watanabe 2 , Kohji Takei 1
Affiliation  

The activity of AMPA-type glutamate receptor is involved in insulin release from pancreatic β-cells. However, the mechanism and dynamics that underlie AMPA receptor-mediated insulin release in β-cells is largely unknown. Here, we show that AMPA induces internalization of glutamate receptor 2/3 (GluR2/3), AMPA receptor subtype, in the mouse β-cell line MIN6. Immunofluorescence experiments showed that GluR2/3 appeared as fine dots that were distributed throughout MIN6 cells. Intracellular GluR2/3 co-localized with AP2 and clathrin, markers for clathrin-coated pits and vesicles. Immunoelectron microscopy revealed that GluR2/3 was also localized at plasma membrane. Surface biotinylation and immunofluorescence measurements showed that addition of AMPA caused an approximate 1.8-fold increase in GluR2/3 internalization under low-glucose conditions. Furthermore, internalized GluR2 largely co-localized with EEA1, an early endosome marker. In addition, GluR2/3 co-immunoprecipitated with cortactin, a F-actin binding protein. Depletion of cortactin by RNAi in MIN6 cells altered the intracellular distribution of GluR2/3, suggesting that cortactin is involved in internalization of GluR2/3 in MIN6 cells. Taken together, our results suggest that pancreatic β-cells adjust the amount of AMPA-type GluR2/3 on the cell surface to regulate the receptive capability of the cell for glutamate.



中文翻译:



AMPA 型谷氨酸受体在 MIN6 胰腺 β 细胞系中的内化。



AMPA 型谷氨酸受体的活性参与胰腺 β 细胞释放胰岛素。然而,β 细胞中 AMPA 受体介导的胰岛素释放的机制和动力学在很大程度上尚不清楚。在这里,我们证明 AMPA 在小鼠 β 细胞系 MIN6 中诱导谷氨酸受体 2/3 (GluR2/3)(AMPA 受体亚型)的内化。免疫荧光实验表明,GluR2/3 呈细点状分布在整个 MIN6 细胞中。细胞内 GluR2/3 与 AP2 和网格蛋白共定位,网格蛋白包被的小凹和囊泡的标记物。免疫电镜显示 GluR2/3 也位于质膜上。表面生物素化和免疫荧光测量表明,添加 AMPA 导致低血糖条件下 GluR2/3 内化增加约 1.8 倍。此外,内化的 GluR2 很大程度上与早期内体标记物 EEA1 共定位。此外,GluR2/3 与 Cortactin(一种 F-肌动蛋白结合蛋白)进行免疫共沉淀。 MIN6 细胞中 RNAi 消耗的 cortactin 改变了 GluR2/3 的细胞内分布,表明 cortactin 参与了 MIN6 细胞中 GluR2/3 的内化。综上所述,我们的结果表明胰腺 β 细胞调节细胞表面 AMPA 型 GluR2/3 的量,以调节细胞对谷氨酸的接受能力。

更新日期:2020-09-12
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