González-Maeso Javier, Wise Alan, Green Andrew, Koenig Jennifer A
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, UK.
Eur J Pharmacol. 2003 Nov 14;481(1):15-23. doi: 10.1016/j.ejphar.2003.09.002.
gamma-Aminobutyric acid B (GABA(B)) receptor is the first discovered G protein-coupled receptor that requires two subunits, GB1 and GB2, to form a functional receptor. Whereas the molecular and functional characteristics of GABA(B) receptors have been recently extensively studied, the mechanisms underlying receptor desensitization and endocytosis are still poorly understood. We have investigated the effect of continuous agonist exposure on the human GABA(B) receptor functional response and redistribution when expressed in Chinese hamster ovary (CHO-K1) cells. The wild-type GABA(B) receptor-mediated inhibition of the adenylate cyclase activity appeared desensitized after 2 h in the presence of GABA (100 microM). Fusion proteins were generated by attachment of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to GB1 and GB2, respectively, and confocal microscopy experiments in intact living cells semi-stably expressing the constructs were performed. Incubation of co-expressing CFP-GB1 and YFP-GB2 cells in the presence of GABA (100 microM) for 2 h induced a profound receptor internalization, and CFP-GB1 and YFP-GB2 appeared co-localized in the endosome (labelled with Cy3-transferrin). The internalization was blocked by a selective GABA(B) receptor antagonist. These results represent the first clear visualization of agonist-induced internalization of the unique heterodimeric GABA(B) receptor.
γ-氨基丁酸B（GABA(B)）受体是首个被发现的G蛋白偶联受体，它需要GB1和GB2两个亚基才能形成功能性受体。尽管最近对GABA(B)受体的分子和功能特性进行了广泛研究，但受体脱敏和内吞作用的潜在机制仍知之甚少。我们研究了在中华仓鼠卵巢（CHO-K1）细胞中表达时，持续暴露于激动剂对人GABA(B)受体功能反应和再分布的影响。在存在GABA（100微摩尔）的情况下，野生型GABA(B)受体介导的腺苷酸环化酶活性抑制在2小时后出现脱敏。分别通过将青色荧光蛋白（CFP）和黄色荧光蛋白（YFP）连接到GB1和GB2上生成融合蛋白，并在半稳定表达构建体的完整活细胞中进行共聚焦显微镜实验。在存在GABA（100微摩尔）的情况下，将共表达CFP-GB1和YFP-GB2的细胞孵育2小时会诱导受体大量内化，并且CFP-GB1和YFP-GB2似乎在内体中共同定位（用Cy3-转铁蛋白标记）。这种内化被选择性GABA(B)受体拮抗剂阻断。这些结果首次清晰地显示了激动剂诱导的独特异二聚体GABA(B)受体的内化。
