Investigating the Impact of Altered Subunit Expression on Sweet Taste Receptor Surface Trafficking and Signaling

Abstract

The sweet taste receptor is a heterodimeric G protein‐coupled receptor (GPCR), consisting of the subunits Tas1R2 (T1R2) and Tas1R3 (T1R3). Expression of this receptor has been demonstrated in various tissues throughout the body, including the pancreas, small intestine and adipose tissue. This receptor is thought to play a role in maintaining the body's energy homeostasis, and may potentially be a target in the treatment of metabolic disorders such as diabetes and obesity. Some studies show that the two sweet taste receptor subunits are expressed in unequal quantities in pancreatic β cells and adipocytes, however the impact of this unequal expression on receptor function is not fully understood. This study aims to determine the impact of varying the degree of expression of the two subunits on receptor surface trafficking and downstream signaling pathways. To address this aim, sweet taste receptor‐expressing stable cell lines were generated in AD293 cells by sequential selection of vectors, and in HEK293 TRex cells using bicistronic vectors and the FlpIn system. Receptor expression was characterized through real‐time PCR and biotinylation pull‐down experiments, while receptor signaling was determined in live cells using a genetically encoded Fluorescence Resonance Energy Transfer (FRET) sensor and a Bioluminescence Resonance Energy Transfer (BRET) sensor. Our results show that expression of both receptor subunits is predominantly intracellular, and that co‐expression of both subunits does not improve surface expression. Co‐expression of both subunits was, however, found to be necessary for signaling, as cell lines expressing only one subunit did not show functional responses to aspartame. The magnitude of these functional responses was found to be greatest when subunit expression was closest to 1:1, suggesting that the sweet taste receptor is most efficacious as a 1:1 heterodimer. Cell lines expressing both subunits were found to signal through Gi, as well as undergo extracellular signal‐regulated kinase (ERK) phosphorylation, demonstrating the ability of this receptor to couple to multiple signaling pathways. While some studies using mouse sweet taste receptors have reported Gs signaling at high concentrations of sweeteners, this could not be replicated in this study using human receptors, suggesting that signaling pathways may be species‐specific. Overall, this study shows that co‐expression of both sweet taste receptor subunits is necessary for signaling but not for surface trafficking and that this receptor is most efficacious when both subunits are expressed at equal levels.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.