Fig. 8.

4-CMTB requires the second extracellular loop of hFFA2 for allosteric modulation of propionate. The 2nd extracellular loop of hFFA2 (Val¹⁴⁴–Glu¹⁸²) was replaced with ECL2 from the closely related hFFA3 receptor (Val¹⁵⁰–Glu¹⁸⁷) to generate an hFFA2(ECL2 FFA3)-eYFP chimeric protein and stable Flp-In TREx 293 cells generated as before. A, extracellular loop 2 alignment between hFFA2 and hFFA3 using ClustalX (accession numbers: hFFA2, NP005297; hFFA3, NP005295). B, propionate activates hFFA2(ECL2 FFA3)-eYFP with equivalent potency to hFFA2-eYFP WT (pEC₅₀ = 3.96 ± 0.09 and 4.03 ± 0.14, respectively; n = 4) but with marginally reduced efficacy (p = 0.0501 according to an unpaired t test). C, 4-CMTB has significantly reduced efficacy at hFFA2(ECL2 FFA3) compared with hFFA2-eYFP WT (p < 0.001 according to an unpaired t test). Potency is equivalent between WT and chimeric receptors according to a t test (pEC₅₀ = 6.50 ± 0.15 and 6.75 ± 0.11, respectively; n = 4). D, allosterism between 4-CMTB and propionate is lost at the hFFA2(ECL2 FFA3)-eYFP chimera. The arrow indicates potency; n.s. represents no significant difference in potency values according to one-way ANOVA (n = 3). E, reciprocal loss of allosterism at hFFA2(ECL2 FFA3)-eYFP according to one-way ANOVA and indicated by the arrow (n = 3).