The structure–function role of C-terminus in human bitter taste receptor T2R4 signaling

Abstract

Bitter taste, in humans, is sensed by 25 G protein-coupled receptors, referred to as bitter taste receptors (T2Rs). The diverse roles of T2Rs in various extraoral tissues have implicated them as a potential target for therapeutic intervention. Structure-function studies have provided insights into the role of transmembrane and loop regions in the activation mechanism of T2Rs. However, studies aimed at deciphering the role of their carboxyl-terminus (C-terminus) are limited. In this study, we identified a KLK/R motif in the C-terminus that is conserved in 19 of the 25 T2Rs. Using site-directed mutagenesis we studied the role of 16 residues in the C-terminus of T2R4. The C-terminus of T2R4 is polybasic with 6 of the 16 residues consisting of lysines, constituting two separate KK motifs. We analyzed the effect of the C-terminus mutations on plasma membrane trafficking, and characterized their function in response to the T2R4 agonist quinine. The majority of the mutants showed defective receptor trafficking with ≤50% expression on the cell surface. Interestingly, mutation of the distal Lys296 of the KLK motif in T2R4 resulted in constitutive activity. The K296A mutant displayed five-fold basal activity over wild type T2R4, while the conservative substitution K296R showed wild type characteristics. The Lys294, Leu295 and Lys296 of the KLK motif in T2R4 were found to perform crucial roles, both, in receptor trafficking and function. Results from this study provide unique mechanistic insights into the structure-function role of the C-terminus in T2R signaling.

The Pharmacochaperone Activity of Quinine on Bitter Taste Receptors

“…Desensitization of GPCRs is initiated when their cytosolic residues, containing either serine or threonine, are phosphorylated by selected kinases [ 12 ]. In T2R4, there are 13 serine and threonine residues on the intracellular side which are not well conserved among the 25 human T2Rs [ 16 , 18 ]. Among these, a few threonines in the third intracellular loop and the C-terminus are involved in T2R4 expression and/or function [ 16 , 18 ].…”

“…In T2R4, there are 13 serine and threonine residues on the intracellular side which are not well conserved among the 25 human T2Rs [ 16 , 18 ]. Among these, a few threonines in the third intracellular loop and the C-terminus are involved in T2R4 expression and/or function [ 16 , 18 ]. It is well documented that most GPCRs in response to agonist undergo phosphorylation in the third intracellular loop or the cytoplasmic tail.…”

“…It is well documented that most GPCRs in response to agonist undergo phosphorylation in the third intracellular loop or the cytoplasmic tail. Given that a number of the non-conserved serine and threonine residues in T2R4 can be potentially phosphorylated and might also be involved in receptor expression and function [ 16 , 18 ], currently it is not feasible for us to delineate all these effects. Therefore, studies directed at elucidating the T2R4 residues involved in phosphorylation could not be pursued at this time.…”

“…Viable cells at a concentration of 1×10 6 cells/ml were collected and washed with ice cold FACS buffer (PBS containing 0.5% BSA) and incubated with mouse monoclonal anti-FLAG M2 primary antibody (1:500) or rabbit polyclonal anti-T2R4 antibody (1:300, for detection of endogenous T2R4 in hASMCs) in FACS buffer for 1 h on ice. The cells were then washed 2 to 3 times with FACS buffer and incubated with Alexa 488 goat anti-mouse (1:1000) or goat anti-rabbit secondary antibody (1:1000) as shown previously [ 18 , 23 ]. For analyzing chaperone activity of quinine, FLAG-tagged T2Rs were incubated with APC-conjugated anti-FLAG monoclonal antibody (1:500) for 1 h on ice in the dark.…”

Cholesterol modulates bitter taste receptor function

“…Given the importance of cholesterol in human diet, cholesterol sensitivity of bitter taste receptors assumes physiological relevance. genes were carried out as described earlier [21][22][23]. CHO cells were transiently transfected with respective plasmid constructs using lipofectamine 2000 reagent as per the manufacturer's instructions.…”