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.