Neuropeptide Y (NPY) Y1/Y5 receptor heterodimers constrained by bimolecular fluorescence complementation (BiFC) exhibit altered Y5 antagonist pharmacology

LE Kilpatrick, ND Holliday. University of Nottingham, Nottingham, UK

Heterodimerisation between class A G protein coupled receptors (GPCRs) potentially offers novel pharmacology through co-operativity between orthosteric ligand binding sites, or by altered coupling to effector proteins1. We have used bimolecular fluorescence complementation (BiFC) to constrain Neuropeptide Y (NPY) Y1 and Y5 receptor subtypes, both implicated in central appetite responses2, as heterodimers of known composition. This allowed quantitative investigation of heterodimer-specific antagonist pharmacology by measuring effects on NPY-promoted internalisation of the BiFC dimer3.

A stably transfected HEK293 cell line (Y1/Y5) was established co-expressing SNAP-tagged Y1 receptor-Yn (Y1R) and Y5R-Yc3. Irreversible BiFC between the YFP fragments generated a constrained Y1/Y5 dimer identified by recomplemented YFP. Control cells stably expressed Y5R-GFP. For internalisation, cells were labelled with SNAPsurface AF647, followed by antagonist (30 min) pretreatment, and then NPY stimulation (30 min, 37°C) in HBS/0.1 % BSA. Following paraformaldehyde fixation, cellular YFP BiFC and SNAP647 fluorescence were imaged (MDC IX Ultra platereader). Internalisation of the BiFC Y1/Y5 dimer, and SNAP-Y1R populations, was quantified by granularity analysis on a per cell basis3. For comparison, [125I]PYY competition binding assays, and NPY inhibition of 30 µM forskolin stimulated [3H]cAMP accumulation were performed4. All data were expressed as mean ± s.e.m (n = 3-4). pEC50 values were calculated by curve fitting to pooled data (Graphpad Prism), and antagonist pKb estimates calculated by the Gaddum equation.

The Y5 subtype selective ligand CGP71683 (30nM) was a surmountable antagonist of NPY stimulated Y5-GFP internalisation (pKb 8.4 ± 0.1; control NPY pEC50 8.2 ± 0.1). In Y1/Y5 cells, NPY stimulated internalisation of both the SNAP-Y1R (pEC50 8.0 ± 0.1) and Y1/Y5 BiFC dimer populations (pEC50 7.6 ± 0.1). CGP71683 pretreatment (30-300nM) had no effect on NPY-induced internalisation of the SNAP-Y1R population. However CGP71683 became a non-surmountable antagonist of Y1/Y5 BiFC dimer internalisation, significantly reducing maximal 1 µM NPY responses (with 30nM CGP71683, 37.9 ± 8.0 % control; *** P < 0.001 Student’s t test), without significantly changing NPY potency. Higher CGP71683 concentration (300nM) did not further reduce maximal NPY responses. In Y1/Y5 membrane preparations the Y1 antagonist BIBO3304 but not CGP71683 displaced [125I]PYY binding (data not shown). The presence of 30nM CGP7683 had no significant effect on NPY potency or its maximum inhibition of forskolin induced cAMP accumulation in whole cells (control pEC50 9.3 ± 0.1).

We conclude that, in comparison to cell population assays, the measurement of Y1/Y5 BiFC dimer internalisation can isolate its specific pharmacology. Our data suggest that interaction between Y1 and Y5 orthosteric binding sites in a Y1/Y5 dimer modifies its interactions with Y5 antagonists, consistent with a previous report5, and with implications for targeting this receptor pair as central regulators of appetite.

LEK is a BPS AJ Clark student.

1. Smith & Milligan (2010) Pharmacol Rev 62, 701-25. 2. Nguyen et al. (2012) PLoS One 7, e40191.

3. Kilpatrick et al. (2012) Methods Mol Biol 897, 109-38. 4. Donaldson et al. (1988) Biochem Pharmacol 37, 715-23. 5. Gehlert et al. (2007) Biochem Pharmacol 74, 1652-64.