Figure 3.

5-ht_5b specifically interacts with 5-HT_1A to change the latter’s subcellular localization. (A) We transfected a representative member of each 5-HT receptor subgroup tagged with mCherry against the same set of 5-HT receptors tagged with GFP. Each image of the matrix shows N1E-115 cells co-expressing two 5-HT receptors. The images are pseudo-colored purple for the receptor indicated horizontally and pseudo-colored green for the receptor indicated vertically. Co-localization is shown in yellow. 5-ht_5b specifically interacts with 5-HT_1A (04) with the normally strictly membrane-bound 5-HT_1A receptors partly retained intracellularly. In addition, 5-ht_5a seems to interact with 5-HT₄ (12) and 5-HT₆ seems to weakly interact with 5-ht_5a (17) and 5-ht_5b (19), although 5-HT₆’s surface expression remains strong. No other 5-HT receptors seem to interact with each other in a way that changes subcellular localization. Scale bars in all images are 10 μm. (B) For quantification, we separately calculated Pearson’s correlation coefficients for the fluorescence signals at the membrane and at intracellular organelles by manually applying regions of interest (ROI). Plotting the organelle correlation coefficient over the membrane correlation coefficient for each receptor pair resulted in three populations of receptors. One population displays a high correlation at the membrane, but not at intracellular organelles. The second population displays correlation in neither compartment while the third population displays high correlation at intracellular compartments. (C) To verify interaction of 5-HT_1A and 5-ht_5b, we transfected primary hippocampal neurons with expression constructs of both receptors. 5-HT_1A (green) localizes to the cell membrane, while 5-ht_5b (purple) localizes to intracellular organelles, while co-expression of both receptors shows intracellular co-localization with no discernable membrane staining by 5-HT_1A. Scale bars in all images are 20 μm.