A high-throughput screening system for G-protein-coupled receptors using β-lactamase enzyme complementation technology

AIM: To establish a system for monitoring the activation of G-protein-coupled receptors (GPCRs) using β-lactamase enzyme fragment complementation (EFC) technology. METHODS: Two inactive β-lactamase deletion fragments, bla(a) and bla(b), were fused to β-arrestin and GPCR, respectively. A stable cell line named HEK/293-β2a2, which expressed two fusion proteins, GPCR/bla(b) and β-arrestin2/bla(a), was generated under antibiotic selection. A natural compound library of high performance liquid chromatography (HPLC)-fractionated samples from the ethanol extracts of Chinese medicinal herbs was used for high-throughput screening (HTS) of β2-adrenoceptor (β2AR) agonists against the cell line HEK/293-β2a2. The interested hits were validated by the measurement of second-messenger cyclic adenosine monophosphate (cAMP) production. RESULTS: The stable cell line HEK/293-β2a2 responded to β2AR agonist/antagonist in a dose-dependent manner. The EC(50) value obtained for isoproterenol was 15.5 nmol/L, and the IC(50) value obtained for propranolol was 51.9 nmol/L. Furthermore, HTS was performed to identify β2AR agonists from the natural compound library we established. The Z′ factor value was determined to be 0.68. Three hits were identified from primary screening and found to be as potent as isoproterenol in a cAMP assay. CONCLUSION: A cell-based high-throughput functional assay was established to directly monitor the activation of GPCRs based on the interaction between agonist-activated GPCR and β-arrestin using β-lactamase EFC technology, which can be used to search for leads in the natural compound library.