title: Characterization of human 5-HT4(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 micro M 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 approximately 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.
authors.affiliation: Laboratoire de Cardiologie Cellulaire et Moléculaire, INSERM U-446, Université de Paris-Sud, Faculté de Pharmacie, F-92296 Châtenay-Malabry, France.
authors.forename: Jeanne
authors.initials: J
authors.lastname: Mialet
authors.forename: Rodolphe
authors.initials: R
authors.lastname: Fischmeister
authors.forename: Frank
authors.initials: F
authors.lastname: Lezoualc'h
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D016297:Mutagenesis, Site-Directed; D020033:Protein Isoforms / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D011863:Radioimmunoassay; D011985:Receptors, Serotonin / Q000187:drug effects* / Q000235:genetics / Q000502:physiology*; D044422:Receptors, Serotonin, 5-HT4; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D020033:Protein Isoforms; D011985:Receptors, Serotonin; D017366:Serotonin Receptor Agonists; D044422:Receptors, Serotonin, 5-HT4; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases