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. 2014 May 15;289(28):19778–19788. doi: 10.1074/jbc.M114.573162

FIGURE 1.

FIGURE 1.

Activation of ORA receptors by amino acids. A, ORA receptors localize to the plasma membrane. HEK 293T cells were transfected as indicated, and receptor protein was detected with an antibody against the C-terminal HSV epitope and Alexa 488-coupled secondary antibody (green, upper row). Cell surfaces are visualized using biotinylated concanavalin A in combination with streptavidin/Alexa 633 (red label, middle row). Yellow color in the overlay (bottom row) indicates close proximity of receptors with the plasma membrane. Insets show groups of cells magnified by a factor of three. Note the absence of green signals in cells transfected with empty vector (third column). Cells expressing the ora genes (green) were counted and expressed as percentage of total cells (red) ± S.D. Scale bar, 75 μm. B, ORA1 but not ORA2 is activated by an amino acid mixture. HEK 293T cells were transiently transfected with ORA1, ORA2, TAS2R16, or empty vector, loaded with Fluo-4 AM, and stimulated with a mixture of all 20 proteinogenic amino acids (final concentration 1 mm each). Changes in calcium levels were analyzed in a fluorometric imaging plate reader. Only ORA1-transfected cells show a response, comparable in size to that of the positive control (TAS2R16 and 10 mm of the agonist d-(−)-salicin (9)). Thus, ORA1 appears to be specifically activated by one or several amino acids present in the amino acid mixture. C, ORA1-expressing cells only respond to tyrosine. HEK 293T cells were transiently transfected with ORA1 or empty vector, loaded with the calcium-sensitive dye fluo-4 AM, and analyzed in a fluorometric imaging plate reader before and after application of single amino acids at 1 mm concentration each. Duplicate wells, one of which is shown here, were tested for each experimental condition. No change in fluorescence was seen for untransfected cells (middle column) or cells transfected with empty vector (right column). The ORA1 response to tyrosine is comparable in size and time course to that of the bitter taste receptor TAS2R16 to a strong agonist (d-(−)-salicin, 10 mm (9)).