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Characterization of human 5-HT4(d) receptor desensitization in CHO cells
Jeanne Mialet1, Rodolphe Fischmeister, Frank Lezoualc'h
1Laboratoire de Cardiologie Cellulaire et Moléculaire, INSERM U-446, Université de Paris-Sud, Faculté de Pharmacie, F-92296 Châtenay-Malabry, France.
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.
Insights
The study reveals that the C-terminal tail of serotonin 5-HT(4) receptor isoforms influences desensitization rates. Protein kinase A (PKA) plays a key role in h5-HT(4(d)) receptor desensitization, potentially through non-consensus phosphorylation sites.
Area of Science:
- Pharmacology
- Molecular Biology
- Cell Signaling
Background:
- Serotonin 5-HT(4) receptor isoforms exhibit variations in their C-terminal tails, with limited understanding of their regulatory mechanisms.
- Receptor desensitization is a critical process influencing cellular responses to agonists.
Purpose of the Study:
- To investigate the desensitization of human 5-HT(4) receptor isoforms, specifically the h5-HT(4(d)) isoform.
- To elucidate the role of protein kinase A (PKA) and potential phosphorylation sites in 5-HT(4) receptor desensitization.
Main Methods:
- Stable expression of human 5-HT(4) receptor isoforms (h5-HT(4(d)) and h5-HT(4(e))) in Chinese Hamster Ovary (CHO) cells.
- Assessment of adenylyl cyclase response to 5-HT stimulation.
- Utilized cAMP analogs, PKA inhibitors (H-89), and endocytosis inhibitors (sucrose, concanavalin A).
- Site-directed mutagenesis of putative PKA phosphorylation sites on the h5-HT(4(d)) receptor.
Main Results:
- Both h5-HT(4(d)) and h5-HT(4(e)) receptors exhibited rapid desensitization upon 5-HT exposure, with h5-HT(4(d)) desensitizing faster.
- 5-HT-induced desensitization of h5-HT(4(d)) was mimicked by 8-Bromo-cAMP and inhibited by H-89, indicating PKA involvement.
- Inhibitors of endocytosis partially reversed desensitization, suggesting a role for internalization.
- Mutating putative PKA phosphorylation sites did not impair desensitization, suggesting PKA acts on non-consensus sites.
Conclusions:
- The C-terminal tail of 5-HT(4) receptors influences the rate of agonist-induced desensitization.
- PKA plays a significant role in the desensitization of the h5-HT(4(d)) receptor, potentially through phosphorylation at non-canonical sites.