Fluorescent assay technologies for G-protein interactions.

Abstract

Assay technologies for GPCRs and their associated G-proteins are in demand for drug screening and other biotechnology applications such as biosensors for diagnostic purposes or odorant/flavour assessment as well as for elucidating the remaining controversial mechanisms in G-protein mediated signalling. This study aims to make progress towards developing a TR-FRET assay for G-protein interactions that could be used as a generic assay platform for GPCR signalling that would be fluorescent, homogeneous and amenable to miniaturization. The first chapter of this study investigates the use of small molecule labels, CS124-DTPA-EMCH:Tb and Alexa546 in a TR-FRET assay. This TR-FRET pair had previously been applied to Gα, Gβγ and RGS4 proteins and during the characterization of this assay, the protein CrV2 was observed to interact with the G-protein. Using TR-FRET, it was demonstrated that a high affinity interaction appears to occur between Gαi1 and CrV2 (Apparent Kd 6.2 nM). CrV2 is encoded by a polydnavirus from endoparasitoid wasps, which is thought to mediate immune suppression, and the interaction with Gα could have important implications in the regulation of the immune system of invertebrates. Improvements to the labelling strategy used in this assay are then attempted through the creation of various G-protein subunit fusions with small, genetically encoded lanthanide binding tags (LBTs) or tetracysteine motifs (TCMs) for site-specific labelling with terbium or FlAsH, respectively. The consequence of the fusions on maintaining G-protein subunit integrity and on the affinity of the tags for their labels is characterized, and then the utility of these constructs as TR-FRET partners is demonstrated. TCM:FlAsH complexes could successfully be used as TR-FRET acceptors for CS124-DTPA-EMCH:Tb labelled binding partners. The interaction between Gβγ2-TCM:FlAsH and Gα:Tb could be measured using TR-FRET and generated an apparent Kd of 3.6 nM. Likewise, LBT:Tb complexes could be used as TR-FRET donors to Alexa546 labelled binding partners which was demonstrated using the chimeric, promiscuous Gα subunit, LBT2:Tb-GαS25 and Gβγ:Alexa. Furthermore, the two site-specific labelling strategies can be used together in TR-FRET studies and an interaction between LBT2:Tb-Gα[subscript]S25 and Gβγ₂-TCM:FlAsH was shown to have an apparent Kd of 2.3 nM. The TRFRET assays were further validated using protease treatments and the addition of unlabelled binding partners reduced the TR-FRET signal. Finally, the feasibility of fusing lanthanide binding tags to GPCRs for alternate assay platforms or other applications was investigated. The β₂- adrenergic and M₂-muscarinic receptors were fused to LBTs and the integrity of the receptors determined using ligand binding and [[superscript]35 S]GTPγS signalling assays. Terbium binding to the LBT was then demonstrated. The utility of these constructs in alternative TR-FRET platforms with Gproteins was then explored.