Internalization of Human 5-HT_(4a) and 5-HT_(4b) Receptors is Splice Variant Dependent

摘要

The family of 5-HT_4 receptors comprises 16 putative splice variants. We have previously shown that there are differences in signal transduction of the h5-HT_(4a) and h5-HT_(4b) receptors. In the present study, the internalization of these two splicevariants following receptor stimulation was investigated with confocal microscopy on living cells. Chimeric receptors, h5-HT_(4a)-GFP and h5-HT_(4b)-GFP were generated by fusing the coding sequence of the 5-HT_4 receptor with the coding sequence of the GFP. The agonist stimulation of fluorescent receptors resulted in a time-dependent internalization of the h5-HT_(4b)-GFP receptor, but not of the h5-HT_(4a)-GFP receptor. The h5-HT_(4b) receptor displays a dual coupling to Galphai,o and Galphas proteins,in contrast to the h5-HT_(4a) receptor, which couples to Galphas 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 Galphai,o protein coupling using PTX. The h5-HT_(4b) 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-HT_(4a) and 5-HT_(4b) receptors.