Expression and biophysical characterization of the human umami taste receptor subunits
Résumé
Umami taste perception is mediated by a heterodimeric receptor composed of the two distinct subunits, T1R1 and T1R3. T1R1, and T1R3 subunits are members of the small family of class C GPCRs. Class C GPCRs share a large N-terminal domain (NTD) linked to the transmembrane domain by a cysteine-rich region. The human T1R1/T1R3 receptor responds specifically to L-Glutamate (L-Glu) and L-Aspartate (L-Asp) with a response potentiated by 5’ ribonucleotides (IMP, GMP), which also elicit umami taste by themselves. It has been shown that whereas L-Glu binds to the hinge region of the T1R1-NTD and induce its closure, 5’ ribonucleotides bind to an adjacent site close to the opening and stabilise the T1R1-NTD in closed conformation. In contrast the functional role of T1R3-NTD in the umami compound detection remains largely unknown. Here we report the ligand binding properties of the recombinantly expressed T1R1- and T1R3-NTDs. The proteins overexpressed in Escherichia coli as insoluble aggregated protein (inclusion bodies) were solubilised, in vitro refolded and purified by two-step chromatography procedure. Circular dichroism and SEC-MALS analyses demonstrated that purified proteins are correctly refolded as monomeric form. Fluorescence spectroscopy demonstrated that T1R1- and T1R3-NTDs are both able to bind L-Glu and IMP with micromolar affinity. In summary, our results demonstrate the contribution of each subunit to the heterodimeric receptor function for the detection of these umami compounds.