High throughput GPCR characterisation utilising Presto-Tango
- 1 December 2023 to 30 September 2024
- Awards: Pump-priming Awards
This project aims to establish the Presto-Tango system within the BHF CRE. Presto-Tango is designed for high-throughput screening of GPCR receptors through β-arrestin recruitment. This assay has been developed to identify inhibitors of GPCRs, and to identify agonists of orphan GPCRs. The Presto-Tango system, once established, will be used within our laboratory to identify anti-inflammatory molecules targeting chemokine receptor GPCRs. This project will support the further characterisation of prospective anti-inflammatory peptide therapeutics against previously untested chemokine receptors. The Presto-Tango kit contains 300+ GPCR constructs and is available as a resource for BHF CRE researchers interested in GPCRs. Details of the kit can be found here: Addgene: Roth Lab PRESTO-Tango GPCR Kit
Schematic depiction of the Presto-Tango β-arrestin recruitment assay. HTLA cells, expressing a β-arrestin2-TEV protease fusion protein and a luciferase reporter gene, are transiently transfected with a Tango GPCR construct. 1) The G-protein coupled receptor (GPCR) is activated through agonist ligand binding. 2) β-arrestin2- TEV (tobacco virus nuclear inclusion a endopeptidase) is recruited to the GPCR C-terminus. 3) The TEV protease bound to the β-arrestin2 cleaves the TEV cleavage site, releasing the tTA (tetracycline transactivator protein). 4) The tTA translocates to the nucleus, activating luciferase production.