Program in Molecular and Integrative Physiological Sciences, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115, USA.
FASEB J. 2012 May;26(5):1995-2007. doi: 10.1096/fj.11-193870. Epub 2012 Jan 25.
The β2-adrenergic receptor (β2AR) plays important physiological roles in the heart and lung and is the primary target of β-agonists, the mainstay asthma drugs. Activation of β2AR by β-agonists is attenuated by receptor down-regulation, which ensures transient stimulation of the receptor but reduces the efficacy of β-agonists. Here we report the identification, through a functional genome-wide RNA interference (RNAi) screen, of new genes critically involved in β2AR down-regulation. We developed a lentivirus-based RNAi library consisting of 26-nt short-hairpin RNAs (shRNAs). The library was generated enzymatically from a large collection of expressed sequence tag (EST) DNAs corresponding to ∼20,000 human genes and contains on average ∼6 highly potent shRNAs (>75% knockdown efficiency) for each gene. Using this novel shRNA library, together with a robust cell model for β2AR expression, we performed fluorescence-activated cell sorting and isolated cells that, as a consequence of shRNA-mediated gene inactivation, exhibited defective agonist-induced down-regulation. The screen discovered several previously unrecognized β2AR regulators, including farnesyl diphosphate synthase (FDPS). We showed that inactivation of FDPS by shRNA, small interfering RNA, or the highly specific pharmaceutical inhibitor alendronate inhibited β2AR down-regulation. Notably, in human airway smooth muscle cells, the physiological target of β-agonists, alendronate treatment functionally reversed agonist-induced endogenous β2AR loss as indicated by an increase in cAMP production. FDPS inactivation interfered with β2AR internalization into endosomes through disrupting the membrane localization of the Rab5 small GTPase. Furthermore, Rab5 overexpression reversed the deficient receptor down-regulation induced by alendronate, suggesting that FDPS regulates receptor down-regulation in a Rab5-dependent manner. Together, our findings reveal a FDPS-dependent mechanism in the internalization and down-regulation of β2AR, identify FDPS as a potential target for improving the therapeutic efficacy of β-agonists, and demonstrate the utility of the unique EST-derived shRNA library for functional genetics studies.
β2-肾上腺素能受体（β2AR）在心脏和肺部中发挥重要的生理作用，是β-激动剂的主要靶标，β-激动剂是治疗哮喘的主要药物。β-激动剂激活β2AR 会导致受体下调，从而确保受体的短暂刺激，但降低β-激动剂的疗效。在这里，我们通过功能全基因组 RNA 干扰（RNAi）筛选鉴定了新的关键基因，这些基因参与β2AR 下调。我们开发了一种基于慢病毒的 RNAi 文库，该文库由 26 个核苷酸的短发夹 RNA（shRNA）组成。该文库通过酶促反应从大量表达序列标签（EST）DNA 中生成，这些 EST DNA 对应于约 20000 个人类基因，平均每个基因包含约 6 个高效的 shRNA（>75%的敲低效率）。我们使用这种新型 shRNA 文库，以及一个稳健的β2AR 表达细胞模型，进行了荧光激活细胞分选，并分离出由于 shRNA 介导的基因失活而导致激动剂诱导下调缺陷的细胞。该筛选发现了几个以前未被识别的β2AR 调节剂，包括法呢基二磷酸合酶（FDPS）。我们表明，shRNA、小干扰 RNA 或高度特异性药物抑制剂阿仑膦酸钠（alendronate）对 FDPS 的失活抑制了β2AR 的下调。值得注意的是，在人类气道平滑肌细胞中，β-激动剂的生理靶标，阿仑膦酸钠处理在功能上逆转了激动剂诱导的内源性β2AR 损失，这表现为 cAMP 产生的增加。FDPS 失活通过破坏 Rab5 小 GTPase 的膜定位干扰β2AR 内吞进入内体。此外，Rab5 过表达逆转了阿仑膦酸钠诱导的受体下调缺陷，表明 FDPS 以 Rab5 依赖的方式调节受体下调。总之，我们的发现揭示了 FDPS 依赖的β2AR 内化和下调机制，鉴定 FDPS 作为提高β-激动剂治疗效果的潜在靶标，并证明了独特的 EST 衍生 shRNA 文库在功能遗传学研究中的实用性。
