Neuropeptide Y (NPY) Y1 receptors, constrained as dimers by bimolecular fluorescence complementation, undergo rapid agonist-promoted internalisation

Laura Kilpatrick, Nick Holliday. School of Biomedical Sicences, University of Nottingham, NG7 2UH, United Kingdom.

The significance of G protein coupled receptor dimerisation remains controversial, although dimers are implicated in regulating both ligand binding and G protein/arrestin effector interactions (Milligan, 2008). In this study we use bimolecular fluorescence complementation (BiFC, Rose et al., 2010) to constrain dimers of the NPY Y1 receptor, which can then be specifically identified by imaging complemented YFP fluorescence. We investigate the function of Y1 receptor BiFC dimers through quantitative measurement of their internalisation, as an indirect readout of β-arrestin recruitment (Kilpatrick et al., 2010).

A stable clonal HEK293 cell line (ssY1-Yn) expressed the Y1 receptor cDNA fused to (i) an N terminal signal sequence and SNAP-tag (New England Biolabs) and (ii) a C terminal Venus vYFP N fragment (1-173). As appropriate this cell line was additionally co-transfected with cDNA encoding FLAG tagged Y1 receptors fused to the complementary vYFP C fragment (155-238), giving mixed co-transfected populations (e.g. ssY1-Yn/Y1-Yc). For platereader imaging (96 well), surface ssY1-Yn was labelled with membrane impermeant SNAPsurface BG-AF647 (NEB; 0.2 µM), before cells were treated with vehicle or agonist (NPY/PYY) and fixed. SNAP labelled receptors (Cy5 channel, 630nm excitation), and ssY1-Yn/Y1-Yc BiFC dimers (FITC channel, 488nm excitation) were imaged in the same field of cells (MDC IX Ultra), and for each channel internalisation was assessed by granularity analysis (Kilpatrick et al.,2010) Concentration response curves from pooled triplicate data were normalised to 100 nM NPY responses (Prism v5).

SNAP fluorophore labelled ssY1-Yn receptors in untreated HEK293 cells, expressed alone or in combination with Y1-Yc constructs, were localised to the plasma membrane with some basal receptor internalisation. A similar distribution was observed for BiFC receptor dimers generated by co-expression of either Y1-Yc, or Y1Y99A-Yc, a mutant which does not bind NPY (Kilpatrick et al.,2010). 100 nM NPY stimulated rapid internalisation of the SNAP-labelled ssY1-Yn populations, and also ssY1-Yn/Y1-Yc or ssY1-Yn/Y1Y99A-Yc BiFC dimers, with similar kinetics (Table 1). Moreover agonists NPY and PYY exhibited similar potencies for inducing ssY1-Yn/Y1-Yc and ssY1-Yn/Y1Y99A-Yc BiFC dimer internalisation (pEC50 range: 7.7 – 8.2; n = 2-3). These were also equivalent to potencies derived from measuring the SNAP-tagged Y1-Yn population alone in each cell line (pEC50 range: 7.8 – 8.2; n = 2-3). Thus constraining Y1 receptors as BiFC dimers does not prevent rapid agonist-induced internalisation, and one functioning agonist binding site within a BiFC dimer is sufficient for this response.

Table 1 Kinetics of 100 nM NPY-induced internalisation of SNAP-labelled Y1Yn receptors and Y1 BiFC dimers (half time (min), with 95 % confidence limits).

Kilpatrick, LE et al., (2010) Br J Pharmacol 160, 892-906.

Milligan, G (2008) Br J Pharmacol 153, S216-S229.`

Rose, RH et al., (2010) Br J Pharmacol 159, 738-750.

Laura Kilpatrick is an AJ Clark student (2010).

This work was also supported by the MRC.