Dataset Information

A novel EST-derived RNAi screen reveals a critical role for farnesyl diphosphate synthase in ?2-adrenergic receptor internalization and down-regulation.

ABSTRACT: The ?2-adrenergic receptor (?2AR) plays important physiological roles in the heart and lung and is the primary target of ?-agonists, the mainstay asthma drugs. Activation of ?2AR by ?-agonists is attenuated by receptor down-regulation, which ensures transient stimulation of the receptor but reduces the efficacy of ?-agonists. Here we report the identification, through a functional genome-wide RNA interference (RNAi) screen, of new genes critically involved in ?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 ?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 ?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 ?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 ?2AR down-regulation. Notably, in human airway smooth muscle cells, the physiological target of ?-agonists, alendronate treatment functionally reversed agonist-induced endogenous ?2AR loss as indicated by an increase in cAMP production. FDPS inactivation interfered with ?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 ?2AR, identify FDPS as a potential target for improving the therapeutic efficacy of ?-agonists, and demonstrate the utility of the unique EST-derived shRNA library for functional genetics studies.

SUBMITTER: Jiang X

PROVIDER: S-EPMC3336790 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Jiang Xiaofeng X Pan Hui H Nabhan Joseph F JF Krishnan Ramaswamy R Koziol-White Cynthia C Panettieri Reynold A RA Lu Quan Q

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20120125 5

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 ...[more]

PMID: 22278941

Similar Datasets

Project description:BackgroundIsoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites.MethodsThe isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis-Menten; also, inhibition assays were performed using risedronate.ResultsThe expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68?±?5 ?M, 7.8?±?1.3 ?M and 2.06?±?0.4 ?M. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate.ConclusionsThe rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria.

Dataset Information

A novel EST-derived RNAi screen reveals a critical role for farnesyl diphosphate synthase in ?2-adrenergic receptor internalization and down-regulation.

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets