Generation of chimeric pTAS2R20 receptors
The coding region of pTAS2R20 was cloned into the pcDNA3.1 vector (Invitrogen, USA) and amino terminally tagged with 45 amino acids of rat SSTR3 as a cell surface targeting signal (Masataka et al., 2018) followed by FLAG tag (DYKDDDDK) (Fig. 1A), which dose not interfere with the signaling of heptahelical receptors and can be used for immunocytochemistry analysis (Coin et al., 2013). The two directionally selected nonsynonymous sites, A52V and Q296H, of pTAS2R20 were indicated red (Fig. 1B). The four pTAS2R20 variants were functionally expressed in HEK-293T cells (Pronin et al., 2007). We reconstituted the GPCR reaction system, and mG15, a gift from Dr. Stephen Libeles of Harvard Medical School was used as an effector (Bufe et al., 2002) which is necessary for GPCR signal transduction. G-CaMP was generously provided by Prof. Loren L. Looger (Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA) as a fluorescent indicator (Marella et al., 2006). The changes in fluorescence were observed by fluorescence microscopy and measured with ImageJ.