Biomolecular and biophysical AFM probing reveals distinct binding of bitter peptide VAPFPEVF to TAS2R16 without inducing an intracellular calcium response

The casein-derived bitter peptide VAPFPEVF has been shown to stimulate proton secretion in human parietal cells (HGT-1) via bitter taste receptor TAS2R16, confirmed by siRNA knockdown. Since literature evidence is inconclusive, we hypothized that VAPFPEVF binds to TAS2R16, and investigated its effects on G protein-coupled signaling pathways. Exposure of HGT-1 cells to VAPFPEVF altered cAMP signaling without inducing a calcium response. An atomic force microscopy (AFM)-based approach was employed to demonstrate peptide binding to TAS2R16 in cellular and cell-free environments using TAS2R16-reconstituted proteoliposomes. Increased binding events were observed, reduced by the addition of salicin and TAS2R16 antagonist probenecid. AlphaFold multimer and molecular dynamics simulations suggest VAPFPEVF binds the orthosteric site of TAS2R16. These findings reveal (i) VAPFPEVF interacts with TAS2R16 to modulate cAMP levels without triggering calcium mobilization and (ii) the AFM approach as a valuable tool for studying peptide binding to TAS2R16 and possibly other G-protein coupled transmembrane receptors.