A novel EST-derived RNAi screen reveals a critical role for farnesyl diphosphate synthase in β2-adrenergic receptor internalization and down-regulation

The beta 2-adrenergic receptor (beta 2AR) plays important physiological roles in the heart and lung and is the primary target of beta-agonists, the mainstay asthma drugs. Activation of beta 2AR by beta-agonists is attenuated by receptor down-regulation, which ensures transient stimulation of the receptor but reduces the efficacy of beta-agonists. Here we report the identification, through a functional genome-wide RNA interference (RNAi) screen, of new genes critically involved in beta 2AR 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 similar to 20,000 human genes and contains on average similar to 6 highly potent shRNAs (>75% knockdown efficiency) for each gene. Using this novel shRNA library, together with a robust cell model for beta 2AR 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 beta 2AR 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 beta 2AR down-regulation. Notably, in human airway smooth muscle cells, the physiological target of beta-agonists, alendronate treatment functionally reversed agonist-induced endogenous beta 2AR loss as indicated by an increase in cAMP production. FDPS inactivation interfered with beta 2AR 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 beta 2AR, identify FDPS as a potential target for improving the therapeutic efficacy of beta-agonists, and demonstrate the utility of the unique EST-derived shRNA library for functional genetics studies.-Jiang, X., Pan, H., Nabhan, J. F., Krishnan, R., Koziol-White, C., Panettieri, R. A., Lu, Q. A novel EST-derived RNAi screen reveals a critical role for farnesyl diphosphate synthase in beta 2-adrenergic receptor internalization and down-regulation. FASEB J. 26, 1995-2007 (2012). www.fasebj.org