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EST‐based shRNA library screen identifies a critical role for the cholesterol biosynthetic pathway in beta2‐adrenergic receptor degradation
Author(s) -
Pan Hui,
Nabhan Joseph F,
Lu Quan
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.lb324
Subject(s) - small hairpin rna , biology , cell sorting , receptor , cdna library , cell culture , microbiology and biotechnology , cell , chemistry , biochemistry , complementary dna , rna , gene , genetics
This study describes the development of a novel expressed sequence tag (EST)‐based short hairpin RNA (shRNA) library and its application in screening for genes regulating the agonist‐induced degradation of the beta2‐adrenergic receptor (beta2‐AR). We established an efficient procedure for making a large number of shRNA expressing clones from the cDNA fragments. Using this procedure, we made a lentiviral library which could express about 600,000 individual shRNA based on a collection of approximate 40,000 human ESTs. We transduced the shRNA library into a human cell line stably expressing an epitope‐tagged beta2‐AR. We used fluorescence‐activated cell sorting to repeatedly enrich cells that retain more beta2‐AR on the cell surface after treatment with the receptor agonist isoproterenol. We have isolated and sub‐cloned the shRNAs expressing constructs from the enriched cell population. Interestingly, several clones target common cellular pathways related to lipid metabolism. One of these clones targeted the farnesyl diphosphate synthase (FDPS), a key enzyme in cholesterol and sterol biosynthesis. We found that a chemical inhibitor of FDPS, alendronate, and the cholesterol depleting reagent, methyl‐beta‐cyclodextrin, both had inhibitory effects on beta2‐AR degradation. Our results thus reveal a novel role for the cholesterol biosynthetic pathway in regulating beta2‐AR degradation.