Selection of the dimeric sweet taste receptor for surface expression and intersubunit interactions

The sweet taste receptor is a heterodimer of two class C G protein-coupled receptors (GPCRs), T1R2 and T1R3. While homologous structures of individual domains have been determined, the architecture of the full length T1R2-T1R3 dimer is unknown. Using random mutagenesis and cell sorting, we identify single point mutations and a C-terminal intracellular retention motif in human T1R2 that modulate surface expression and co-trafficking with T1R3 in a HEK293-derivative cell line. A comprehensive mutational scan of T1R2 based on surface expression of both subunits revealed conserved sites for T1R3 interactions, including surfaces on lobe 1 of the ligand binding domain, cysteine rich domain, and transmembrane helix 6. Using the mutational scan to guide modeling of the T1R2-T1R3 dimer predicts a twisted architecture that can explain how outwards motion of lobes 2 in the ligand-binding domains is translated into reorganization of transmembrane regions, and further predicts that extracellular loops 2 form continuous folded structures with the cysteine-rich domains near the dimer axis. These insights into the putative structural organization of the human sweet taste receptor have general implications for the mechanism of class C GPCRs. FLAG-tagged three site-saturation mutagenesis libraries of human T1R2 (covering N-terminal, central, and C-terminal regions) were transfected into Expi293F cells stably expressing myc-tagged human T1R3. Twenty-four hours post-transfection, surface expression of T1R2 and T1R3 was detected by anti-FLAG-Cy3 and anti-myc-Alexa647 staining, respectively. To determine the effects of T1R2 mutations on T1R2 surface expression, 100% or 55% of Cy3-positive cells were collected by fluorescence-activated cell sorting. For determining the effects of T1R2 mutations on co-trafficking with T1R3 to the plasma membrane, the top 20% of Alexa647-positive cells were sorted after gating for 100% or 50% of Cy3-positive cells. RNA was extracted from the sorted cells, and cDNA was synthesized and deep sequenced. Enrichment ratios for all single amino acid substitutions of T1R2 were calculated by comparing mutation frequencies between the naïve and sorted libraries.