Figure 2. Responsiveness of T1R and T2R taste receptors is enhanced by REEP2. A, Maximum calcium mobilization in response to ligand application was measured in control transfected (−) and REEP2-transfected (+) cells. HEK293E and GLUTag cells were transiently transfected with plasmids encoding the fluorescent calcium sensor YC3.60 (Nagai et al., 2004), either with (solid lines) or without REEP2 (dashed lines), along with either: mT1R2 + mT1R3 + the Gα16gus44 reporter G-protein (Jiang et al., 2005); hT2R16 + Gα16gus44, hT2R44 + Gα16gus44; 5-HT1AR + Gα16i3; 5-HT3R for HEK293E cells, or with Gα16gus44 for GLUTag cells. Calcium mobilization was undetectable in GLUTag cells not overexpressing REEP2. Preincubation of GLUTag cells for 15 min with the sweet taste receptor inhibitor gurmarin (3 μg/ml) (Ninomiya and Imoto, 1995; Margolskee et al., 2007) abolished the cellular response to sucralose. Calcium mobilization traces of representative experiments are presented above the graphs, normalized to the highest intracellular calcium increase. Arrows on traces indicate ligand application, horizontal time frame bar represents 20 s. B–D, Endogenously expressed REEP2 promotes activity of endogenously expressed sweet taste receptors in GLUTag cells. B, Western blot using anti-REEP2 antibody shows that REEP2 is endogenously expressed in GLUTag cells. C, siRNA transfection downregulates REEP2 expression in GLUTag cells. PCR (25 cycles) of REEP2 and GAPDH (control) showed a strong downregulation of REEP2 mRNA in GLUTag cells transfected with siRNA targeting REEP2 (compare with nontransfected or control siRNA-transfected cells). D, The sweet tastant sucralose induces enhanced secretion of GLP-1 from nontransfected and control transfected GLUTag cells, as measured by ELISA after 1 h incubation at 37°C. REEP2-targeted siRNA transfection in GLUTag cells eliminated the sweetener-stimulated increase in GLP-1 secretion above baseline. Gastric inhibitory peptide (GIP) is a potent trigger of GLP-1 release from GLUTag cells (Brubaker et al., 1998) and was used as a positive control: GLP-1 secretion in response to GIP was similar in REEP2 siRNA-treated cells compared with nontransfected or control siRNA-transfected cells, demonstrating that siRNA transfection does not alter secretion of GLP-1 per se. Baseline, sucralose-stimulated, and GIP-stimulated GLP-1 secretions are represented by white, gray, and black bars, respectively. Ligand concentrations were as follows: 30 mm glucose, 20 mm sucralose, 10 mm phenyl-β-d-glucopyranoside (PheβGlc), 10 mm saccharin, 10 μm serotonin, 100 μm serotonin, 100 nm GIP. Averaged values of experiments done three times in triplicate are shown; values are means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.