Splice variants of the human free fatty acid G protein coupled receptor GPR120 exhibit distinct signalling, but similar intracellular trafficking profiles

Sarah-Jane Watson1, Alastair Brown2, Nick Holliday1. 1School of Biomedical Sciences, University of Nottingham, NG7 2UH, United Kingdom, 2AstraZeneca, Alderley Park, SK10 4TG, United Kingdom.

The free fatty acid receptor GPR120 is implicated in incretin release from colonic endocrine cells (Hirasawa et al., 2005), and in macrophage and adipocyte function (Oh Da et al., 2010). Long (L) and short (S) human GPR120 splice variants differ by insertion of 16 amino acids in intracellular loop three. Here we compare signalling and trafficking of GPR120S and GPR120L in HEK293 cells, using calcium mobilisation assays and quantitative measurements of receptor internalisation.

Human GPR120S and GPR120L cDNAs in pcDNA4TO (Invitrogen) were modified to include an N-terminal signal sequence and SNAP-tag (New England Biolabs). Stably transfected HEK293TR cells (Invitrogen) were seeded onto poly D-lysine coated 96 well plates, and GPR120 receptor expression was induced by 18 h pre-treatment with 1 µg/ml tetracycline. All agonist incubations were in HBS/0.02 % fatty acid free BSA. Calcium responses were quantified as peak change in fluorescence using Fluo4 measurements (10-20s following agonist addition, 37°C; May et al., 2010). For platereader imaging, surface SNAP-tagged GPR120 was labelled with SNAPsurface BG-AF488 (NEB; 0.1 µM), before cells were treated with vehicle or agonist (30min at 37°C) and fixed. SNAP labelled receptors were imaged (MDC IX Ultra), and receptor internalisation was assessed by granularity analysis (Kilpatrick et al., 2010) Concentration response curves from pooled triplicate data were normalised to 300 µM oleic acid (OA) responses (Prism v5.02).

Calcium responses were observed in GPR120S cells to OA (pEC50 4.0±0.3, n = 5) and the synthetic agonist GW9508 (Briscoe et al., 2006; pEC50 5.3±0.2, 10µM response, 158±17 % of 300 µM OA n = 5), but only after tetracycline induction. Pertussis toxin (PTx, 100 ng/ml for 24 h) increased agonist potency, without altering maximal response (e.g. GW9508 with PTx; pEC50 5.9 ± 0.1). Surprisingly no OA or GW9508 responses were observed in cells expressing GPR120L. Both GPR120S and GPR120L were present at the cell surface and underwent robust internalisation to OA and GW9508. For example respective half times for internalisation were 21±5 min (GPR120S) and 9±2 (GPR120L) for 100 µM GW9508, and 20±2 min (GPR120S) and 30±2 min (GPR120L) for 300 µM OA. Following 30 min 300 µM OA treatment and wash steps including 0.1 % BSA, internalised GPR120S and 120L failed to recycle (e.g. GPR120S 73 ± 8% still internalised after 60 min wash, n = 5). Additionally, confocal microscopy demonstrated internal GPR120S and GPR120L colocalisation with lysotracker red, a marker for the lysosomal pathway (n = 3).

Thus the third intracellular loop insertion in GPR120L prevents Gi/o-independent intracellular calcium mobilisation, but agonist-stimulated GPR120S and GP120L receptors both undergo internalisation to non-recycling compartments. This differential signalling of GPR120S and 120L isoforms has significance given the recent identification of selective, physiologically relevant GPR120 signalling through G protein independent pathways (Oh Da et al., 2010).

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Hirasawa, A et al. (2005) Nat Med 11, 90-94.

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

May, LT et al. (2010) Mol Pharmacol 77, 678-686.

Oh Da, Y et al. (2010) Cell 142, 687-698.

SJW is an EPSRC PhD student and we thank AstraZeneca (Alderley Park) for financial support.