|Number of samples:||15|
|Release date:||Dec 1 2018|
|Last update date:||Feb 15 2019|
|Dataset link||Selection of the dimeric sweet taste receptor for surface expression and intersubunit interactions|
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.