Zebrafish and medaka T1R (taste receptor type 1) proteins mediate highly sensitive recognition of l‐proline

Abstract

In vertebrates, nutritional tastants, such as amino acids and sugars, are recognized by G‐protein‐coupled receptors of the taste receptor type 1 (T1R) family. Previous studies have shown that fish T1Rs are functionally distinct from mammalian T1Rs in certain regards. Here, we report the existence of oral receptors with high sensitivity to amino acids in zebrafish and medaka fish. We describe the construction of multiple cell lines stably expressing functional T1Rs (from medaka fish or zebrafish) with a chimeric G‐protein (G16gust44) using the Flp‐In system. Through functional assays with these cell lines, medaka fish and zebrafish were confirmed to possess particular T1Rs highly sensitive to l‐proline, possibly reflecting the physiological importance of l‐proline in teleosts, in line with previous studies.

Allosteric modulation of the fish taste receptor type 1 (T1R) family by the extracellular chloride ion

“…In our previous study, mfT1R2c/mfT1R3 and zfT1R2a/zfT1R3 were identified as highly sensitive receptors for L-Pro 34 . In this study, their EC 50 values for L-Pro were lower in 10 mM Cl − buffer than in high Cl − buffer (Fig.…”

“…It should be noted that although the fish T1Rs commonly respond to L-amino acids, there are differences in their ligand profiles. While mfT1R2a/mfT1R3 and mfT1R2b/mfT1R3 are broadly tuned receptors, mfT1R2c/mfT1R3 is a narrowly tuned receptor highly sensitive and specific to L-Pro 6 , 8 , 34 . Both zfT1R2a/zfT1R3 and zfT1R2b/zfT1R3 respond to several L-amino acids, though the sensitivity of zfT1R2a/zfT1R3 is higher than zfT1R2b/zfT1R3 6 , 34 .…”

“…Because of unstable cellular responses to agonists when T1R-expressing cells were maintained in buffers with 5 mM Cl − or lower, in our experimental conditions, the lowest concentration of Cl − in the buffer was set at 10 mM. The agonists applied to each receptor were selected according to our previous publications 6 , 9 , 34 . It should be noted that although the fish T1Rs commonly respond to L-amino acids, there are differences in their ligand profiles.…”

“…Measurements of the responses of the cells by cell-based assay based on the intracellular Ca 2+ mobilization were carried out as described previously with some modifications 20 , 34 , 46 , 47 . Cell lines stably expressing T1R2, T1R3 and chimeric G-protein α-subunit, G16gust44 48 , were constructed using the Flp-In pcDNA5/FRT complete system (Thermo Fisher Scientific, Waltham, MA, USA) described in our previous studies 34 , 46 . The cells were seeded in 96-well plates (clear-bottomed CellBIND surface plate, Corning, Glendale, AZ, USA) coated with ε-poly-L-lysine (Cosmo Bio, Tokyo, Japan) at approximately 70,000 cells per well.…”

Chemical range recognized by the ligand-binding domain in a representative amino acid-sensing taste receptor, T1r2a/T1r3, from medaka fish

“…Another exception is medaka T1r2c, which contains an alanine at the same residue. This may be related to the fact that medakaT1r2c/T1r3 specifically responds to L-proline, a secondary amino acid, but not to other L-amino acids with a primary amino group [5,28]. Conversely, hummingbird T1r1 and gecko T1r2 share "SSO" residues, whereas hummingbird T1r1/T1r3 and gecko T1r2/T1r3 exhibit sugar responses [26,27].…”