Mundell Stuart J, Pula Giordano, Carswell Katy, Roberts Peter J, Kelly Eamonn
Department of Pharmacology, School of Medical Sciences, University of Bristol, UK.
J Neurochem. 2003 Jan;84(2):294-304. doi: 10.1046/j.1471-4159.2003.01515.x.
To investigate the role of the intracellular C-terminal tail of the rat metabotropic glutamate receptor 1a (mGlu1a) in receptor regulation, we constructed three C-terminal tail deletion mutants (Arg847stop, DM-I; Arg868stop, DM-II; Val893stop, DM-III). Quantification of glutamate-induced internalization provided by ELISA indicated that DM-III, like the wild-type mGlu1a, underwent rapid internalization whilst internalization of DM-I and DM-II was impaired. The selective inhibitor of protein kinase C (PKC), GF109203X, which significantly reduced glutamate-induced mGlu1a internalization, had no effect on the internalization of DM-I, DM-II, or DM-III. In addition activation by carbachol of endogenously expressed M1 muscarinic acetylcholine receptors, which induces PKC- and Ca2+-calmodulin-dependent protein kinase II-dependent internalization of mGlu1a, produced negligible internalization of the deletion mutants. Co-expression of a dominant negative mutant form of G protein-coupled receptor kinase 2 (DNM-GRK2; Lys220Arg) significantly attenuated glutamate-induced internalization of mGlu1a and DM-III, whilst internalization of DM-I and DM-II was not significantly affected. The glutamate-induced internalization of mGlu1a and DM-III, but not of DM-I or DM-II, was inhibited by expression of DNM-arrestin [arrestin-2(319-418)]. In addition glutamate-induced rapid translocation of arrestin-2-Green Fluorescent Protein (arr-2-GFP) from cytosol to membrane was only observed in cells expressing mGlu1a or DM-III. Functionally, in cells expressing mGlu1a, glutamate-stimulated inositol phosphate accumulation was increased in the presence of PKC inhibition, but so too was that in cells expressing DM-II and DM-III. Together these results indicate that different PKC mechanisms regulate the desensitization and internalization of mGlu1a. Furthermore, PKC regulation of mGlu1a internalization requires the distal C terminus of the receptor (Ser894-Leu1199), whilst in contrast glutamate-stimulated GRK- and arrestin-dependent regulation of this receptor depends on a region of 25 amino acids (Ser869-Val893) in the proximal C-terminal tail.
为了研究大鼠代谢型谷氨酸受体1a（mGlu1a）细胞内C末端尾巴在受体调节中的作用，我们构建了三个C末端尾巴缺失突变体（Arg847stop，DM-I；Arg868stop，DM-II；Val893stop，DM-III）。通过酶联免疫吸附测定（ELISA）对谷氨酸诱导的内化进行定量分析，结果表明，与野生型mGlu1a一样，DM-III能快速内化，而DM-I和DM-II的内化则受到损害。蛋白激酶C（PKC）的选择性抑制剂GF109203X可显著减少谷氨酸诱导的mGlu1a内化，但对DM-I、DM-II或DM-III的内化没有影响。此外，卡巴胆碱对内源表达的M1毒蕈碱型乙酰胆碱受体的激活可诱导mGlu1a的PKC和Ca2+ - 钙调蛋白依赖性蛋白激酶II依赖性内化，但对缺失突变体的内化作用可忽略不计。G蛋白偶联受体激酶2（DNM-GRK2；Lys220Arg）的显性负突变体形式的共表达可显著减弱谷氨酸诱导的mGlu1a和DM-III的内化，而DM-I和DM-II的内化则未受到显著影响。DNM-抑制蛋白[抑制蛋白-2(319 - 418)]的表达可抑制谷氨酸诱导的mGlu1a和DM-III的内化，但不能抑制DM-I或DM-II的内化。此外，仅在表达mGlu1a或DM-III的细胞中观察到谷氨酸诱导的抑制蛋白-2-绿色荧光蛋白（arr-2-GFP）从胞质溶胶快速转运至细胞膜。在功能上，在表达mGlu1a的细胞中，PKC抑制时谷氨酸刺激的肌醇磷酸积累增加，在表达DM-II和DM-III的细胞中也是如此。这些结果共同表明，不同的PKC机制调节mGlu1a的脱敏和内化。此外，PKC对mGlu1a内化的调节需要受体的远端C末端（Ser894-Leu1199），而相比之下，谷氨酸刺激的该受体的GRK和抑制蛋白依赖性调节则取决于近端C末端尾巴中的25个氨基酸区域（Ser869-Val893）。
