School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.
PLoS One. 2012;7(10):e47016. doi: 10.1371/journal.pone.0047016. Epub 2012 Oct 8.
Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2) receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions. To examine the contributions of host cell and epitope tag on receptor expression and activity, epitope-tagged AT2 receptor variants were transiently or stably expressed in HEK293, CHO-K1 and PC12 cells. The epitope-tagged AT2 receptor variants were detected both on the cell membrane and in the perinuclear region. In transiently transfected HEK293 cells, Myc-AT2 existed predominantly as monomer. Additionally, a ladder of ubiquitinated AT2 receptor proteins was detected. By contrast, stably expressed epitope-tagged AT2 receptor variants existed as both monomer and high molecular weight complexes, and the latter was enriched in cell surface. Glycosylation promoted cell surface expression of Myc-AT2 but had no effect on AT2-GFP in HEK293 cells. In cells that stably expressed Myc-AT2, serum starvation induced apoptosis in CHO-K1 cells but not in HEK293 or PC12 cells. Instead, HEK293 and PC12 cells stably expressing Myc-AT2 exhibited partial cell cycle arrest with cells accumulating at G1 and S phases, respectively. Taken together, these results suggest that expression levels, subcellular distributions and ligand-independent constitutive activities of AT2 receptor were cell type-dependent while posttranslational processing of nascent AT2 receptor protein was modulated by epitope tag and mode of expression.
尽管异源表达表位标记的 GPCR 被广泛用于功能表征，但对于表达宿主和表位标记对 GPCR 表达的影响缺乏系统分析。血管紧张素 II（AT2）受体表现出激动剂依赖性和非依赖性活性，与一系列信号分子偶联。然而，对于 AT2 受体的亚细胞分布、信号级联和受体介导的作用，尚未达成共识。为了研究宿主细胞和表位标记对受体表达和活性的贡献，瞬时或稳定表达了表位标记的 AT2 受体变体在 HEK293、CHO-K1 和 PC12 细胞中。在瞬时转染的 HEK293 细胞中，Myc-AT2 主要以单体形式存在。此外，还检测到了泛素化的 AT2 受体蛋白的阶梯。相比之下，稳定表达的表位标记的 AT2 受体变体既存在单体又存在高分子量复合物，后者在细胞表面富集。糖基化促进了 Myc-AT2 的细胞表面表达，但对 HEK293 细胞中的 AT2-GFP 没有影响。在稳定表达 Myc-AT2 的细胞中，血清饥饿诱导 CHO-K1 细胞凋亡，但不诱导 HEK293 或 PC12 细胞凋亡。相反，稳定表达 Myc-AT2 的 HEK293 和 PC12 细胞表现出部分细胞周期阻滞，分别有细胞在 G1 和 S 期积累。总之，这些结果表明，AT2 受体的表达水平、亚细胞分布和配体非依赖性组成性活性依赖于细胞类型，而新生 AT2 受体蛋白的翻译后加工受表位标记和表达模式的调节。
