Investigation of the novel pharmacology of Neuropeptide Y (NPY) receptor dimers constrained by bimolecular fluorescence complementation (BiFC)

Abstract

Dimerisation of GPCRs potentially influences their pharmacology via binding site cooperativity or modified signalling. We have used bimolecular fluorescence complementation (BiFC; Kilpatrick et al. (2012) Methods Mol Biol 897) to constrain Neuropeptide Y (NPY) Y1 receptor (Y1R) homo and heterodimers of known composition. Using quantitative platereader imaging we measured the internalisation of BiFC receptor dimers simultaneously with endocytosis of SNAP tagged Y1R protomer to compare the pharmacology of Y1/Y1, Y1/Y4 or Y1/Y5 BiFC dimers.Y1/Y1 and Y1/Y4 BiFC dimers internalised in response to NPY or PP (Y1/Y4), with potencies comparable to individually expressed YR subtypes. Endocytosis of Y1/Y1 or Y1/Y4 dimers to NPY was also inhibited by the Y1 antagonist BIBO3304, with expected pKb values ((8.2–8.5) from NPY concentration‐response curve shifts). However, the Y5 selective agonist PP(1–17)(Ala31, Aib32) NPY(18– 36) showed reduced potency for constrained Y1/Y5 BiFC dimers, compared to Y5‐GFP internalisation. BIBO3304 demonstrated a lower apparent affinity (pKb 7.4 ± 0.1) for Y1/Y5 BiFC dimers compared to the SNAP‐Y1 sub‐population (pKb 8.2 ± 0.4). This suggests an interaction between Y1 and Y5 orthosteric binding sites of Y1/Y5 BiFC dimers, with implications for selective targeting of this dimer as a mediator of central NPY appetite responses. LK is supported by the British Pharmacological Society.