Neuropeptide Y Y1 receptor association with beta-arrestin measured by bimolecular fluorescence complementation

We are developing a new way to measure protein interactions involved in signalling from cell surface receptors, using powerful fluorescent microscopy techniques.

The receptor proteins are called G protein coupled receptors, present in every cell in the body, which recognise chemical neurotransmitters and hormones. We will focus on their association with important partners called arrestins. Arrestins regulate receptor activity, contributing to drug tolerance, and also organise signalling leading to cell survival or differentiation.

In our technique we add two halves of a fluorescent protein to the receptor and arrestin molecules, so that the light from it will only be seen when the segments are brought together by a receptor-arrestin complex. We will then identify these complexes by a range of imaging methods, some of which can examine behaviour at a single molecule level. This will tell us more about the molecular controls that govern the formation of different receptor-arrestin combinations, and how these controls generate alternative patterns of signalling.

We hope that this will help a wide scientific community, including those interested in the particular receptors under study (for the neurotransmitter Neuropeptide Y) as targets to treat obesity, and those who see potential for receptor drugs that selectively alter arrestin signalling.

Beta-arrestins bind G protein coupled receptors (GPCRs) to initiate their desensitization and internalization, and are also signalling adaptors for protein kinase cascades. Efficient interaction with GPCRs involves recognition of activated and phosphorylated GPCR domains by two modular sensors in the arrestin protein. However it is now apparent that the molecular nature of the GPCR-arrestin complex varies with ligand or arrestin isoform, and that this variation is functionally important. For example, there is evidence for some GPCRs that antagonists can stimulate certain signalling cascades via arrestins but independently of G protein activation. In this application the technique of bimolecular fluorescence complementation (BiFC) will be used to evaluate at the single living cell level the nature of the interaction between the neuropeptide Y Y1 (NPY1) receptor and beta-arrestins by different ligands. In this technique, the association of NPY1 receptors with beta-arrestins can be followed by the complementation of fragments of either yellow or cyan fluorescent proteins (YFP / CFP; which themselves are non fluorescent) attached to the protein partners to regenerate YFP or CFP fluorescence. This will then be detected and quantitified in living cells using both confocal microscopy and (for detailed pharmacological analysis) a confocal plate reader. Using these techniques we will determine whether agonists (endogenous peptides e.g. neuropeptide Y and synthetic ligands) can direct association with specific arrestin isoforms, and whether Y1 receptor antagonists (of G protein coupled responses) can stimulate arrestin binding (!?dual efficacy!?). Arrestin binding determinants used by these different complexes will be determined by mutagenesis of the Y1 receptor activation and phosphorylation sites. In addition we will investigate whether GPCRs and arrestins interact as dimers. Finally, we will use
BiFC fluorescence complementation in association with fluorescence correlation spectroscopy to monitor the diffusional characteristics of individual Y1 receptor-arrestin complexes in membrane microdomains (e.g. clathrin coated pits) of single living cells.
Findings from this research will provide new insight into the mechanism of GPCR-arrestin interaction, and into signalling from the neuropeptide Y1 receptor which has widespread physiological roles and therapeutic potential.