Investigating the pharmacology of the fatty acid receptors GPR440, GPR41 and GPR43 using fusion proteins

Leigh Stoddart1, Andrew Brown2 and Graeme Milligan1. 1Glasgow University, Glasgow G12 8QQ, UK, 2GlaxoSmithKline, Harlow, Essex, CM19 5AW.

GPR40, GPR41 and GPR43 have recently been identified as receptors for fatty acids. GPR40 has been shown to be activated by medium to long chain fatty acids of between 6 and 20 carbons in length (Briscoe et al., 2003; Itoh et al., 2003) whereas GPR41 and GPR43 are both activated by short chain fatty acids of between 1 and 6 carbons in length (Brown et al., 2003; Le Poul et al., 2003; Nilsson et al., 2003). An important tool in the study of GPCRs is the ability to fuse them, either at the N or C terminus, to epitope tags, fluorescent proteins or G protein alpha subunits. Without the availability of a radiolabelled ligand or a specific antibody for these receptors the ability to tag them to investigate their function and pharmacology is especially important.

Using a PCR based method various tags were fused to the three receptors. To determine the effect of tagging the receptors [35S]GTPS binding in response to increasing concentrations of fatty acid were measured in HEK293 membranes expressing the required receptor or fatty acid stimulated Ca2+ release was measured using the Ca2+ sensitive dye Fura-2.

GPR41 and GPR43 were tagged at the N terminus with either the Flag or c-myc epitope tags. N-terminally tagging GPR41 and GPR43 with c-myc abolished expression of the receptors. Tagging GPR43 with Flag did not affect its expression and only slightly impaired its ability to cause a rise in intracellular Ca2+ levels in response to stimulation by the short chain fatty acid, acetate. GPR41, when co-transfected with the G protein alpha subunit, Gi3 causes only a 60 ± 9% rise in [35S]GTPS binding in the presence of 1mM propionate whereas when GPR41 is fused to G i3 a 500 ± 53% increase in binding was observed. The pEC50 for propionate was not changed (GPR41: 3.4, GPR41-G i3 : 3.7). GPR40 and GPR43 were both fused to CFP at their C-terminus and this allowed cells expressing the receptor to be selected for measuring intracellular [Ca2+]. Using these fluorescent fusions the effect of fatty acid free BSA on the ability of the receptors to respond to various fatty acids was determined. In the presence of 0.1% BSA 100 µM of lauric acid, palmitic acid or elaidic acid still caused a rise in intracellular [Ca2+]. As with GPR40, GPR43 could still respond to 10 mM acetate or propionate when 0.1% BSA was added.

Brown, A.J. et al., (2003) J.Biol.Chem 278,11312-11319.
Briscoe. C.P .et al., (2003) J.Biol.Chem 278, 11303-11311.
Itoh, Y. et al., (2003) Nature 422, 173-176.
Le Poul, E. et al., (2003) J.Biol.Chem 278,25481-25489.
Nilsson, N.E. et al., (2003) Biochem. Biophys. Res. Commun. 314(3), 805-809.