Characterization of human 5-HT 4(d) receptor desensitization in CHO cells

Abstract

1. Serotonin 5-HT 4 receptor isoforms differ in their C-terminal tail and yet little is known about their regulation. In this study, we investigated the desensitization of two human 5-HT 4 receptors stably expressed in CHO cells, with a special emphasis on the h5-HT 4(d) isoform. 2. Exposure of h5-HT 4(d) and h5-HT 4(e) receptors to 1 μM 5-HT induced a rapid desensitization of the adenylyl cyclase response. The h5-HT 4(d) receptor desensitized with a faster rate (t 1/2 < 5 min) than the h5-HT 4(e) receptor (t 1/2 = 15 min) and after 10 min 5-HT treatment cAMP production was reduced by ∼70%. 3. 5-HT-induced h5-HT 4(d) receptor desensitization was mimicked by 8-Bromo-cAMP, a cAMP analogue, and was inhibited by [n.[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide, 2HCl] (H-89), an inhibitor of cAMP-dependent protein kinase (PKA). Inhibitors of endocytosis (sucrose, 0.45 M and concanavaline A, 0.25 mg ml -1) partially reversed the h5-HT 4(d) receptor desensitization process. 4. Given the prominent role of PKA in agonist-induced desensitization, we mutated the four putative PKA phosphorylation sites present in the third intracellular loop (Ser242, Thr253, Thr255) and the C terminal tail (Ser338) of the h5-HT 4(d) receptor. Surprisingly, mutated receptors in which either one or all four putative phosphorylation sites were substituted to alanine did not impair receptor desensitization suggesting that PKA might act on nonconsensus sites. 5. Altogether, our data demonstrate that the C-terminal tail of h5-HT 4 receptors may influence the rate of agonist-induced desensitization and we provide evidence for a major role of PKA in h5-HT 4(d) receptor desensitization.