The family of 5-HT4 receptors comprises 16 putative splice variants. We have previously shown that there are differences in signal transduction of the h5-HT4a and h5-HT4b receptors. In the present study, the internalization of these two splice variants following receptor stimulation was investigated with confocal microscopy on living cells. Chimeric receptors, h5-HT4a-GFP and h5-HT4b-GFP were generated by fusing the coding sequence of the 5-HT4 receptor with the coding sequence of the GFP. The agonist stimulation of fluorescent receptors resulted in a time-dependent internalization of the h5-HT4b-GFP receptor, but not of the h5-HT4a-GFP receptor. The h5-HT4b receptor displays a dual coupling to Gαi,o and Gαs proteins, in contrast to the h5-HT4a receptor, which couples to Gαs proteins only. We investigated whether the difference in internalization of the two splice variant receptors was related to their differential coupling. Therefore, we performed agonist-stimulation of the receptor following inhibition of the Gαi,o protein coupling using PTX. The h5-HT4b receptor internalization is PTX insensitive. We co-transfected the fluorescent chimeric receptors with other wild-type variants, which did not produce an alteration of the receptor trafficking. These findings provide the first evidence of differential internalization between the two splice variants, 5-HT4a and 5-HT4b receptors.

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