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Agonist-induced Phosphorylation of the Serotonin 5-HT2C Receptor Regulates Its Interaction with Multiple PDZ Protein 1
- Source :
- Journal of Biological Chemistry. 278:21576-21583
- Publication Year :
- 2003
- Publisher :
- Elsevier BV, 2003.
-
Abstract
- Multiple PDZ domain protein 1 (MUPP1), a putative scaffolding protein containing 13 PSD-95, Dlg, ZO-1 (PDZ) domains, was identified by a yeast two-hybrid screen as a serotonin2C receptor (5-HT2C R)-interacting protein (Ullmer, C., Schmuck, K., Figge, A., and Lubbert, H. (1998) FEBS Lett. 424, 63-68). MUPP1 PDZ domain 10 (PDZ 10) associates with Ser458-Ser-Val at the carboxyl-terminal tail of the 5-HT2C R. Both Ser458 and Ser459 are phosphorylated upon serotonin stimulation of the receptor (Backstrom, J. R., Price, R. D., Reasoner, D. T., and Sanders-Bush, E. (2000) J. Biol. Chem. 275, 23620-23626). To investigate whether phosphorylation of these serines in the receptor regulates MUPP1 interaction, we used several approaches. First, we substituted the serines in the receptor carboxyl tail with aspartates to mimic phosphorylation (S458D, S459D, or S458D/S459D). Pull-down assays demonstrated that Asp mutations at Ser458 significantly decreased receptor tail interaction with PDZ 10. Next, serotonin treatment of 5-HT2C R/3T3 cells resulted in a dose-dependent reduction of receptor interaction with PDZ 10. Effects of serotonin on receptor-PDZ 10 binding could be blocked by pretreatment with a receptor antagonist. Alkaline phosphatase treatment reverses the effect of serotonin, indicating that agonist-induced phosphorylation at Ser458 resulted in a loss of MUPP1 association and also revealed a significant amount of basal phosphorylation of the receptor. We conclude that 5-HT2C R interaction with MUPP1 is dynamically regulated by phosphorylation at Ser458.
- Subjects :
- Agonist
Protein Folding
Serotonin
Glycosylation
medicine.drug_class
Recombinant Fusion Proteins
Blotting, Western
PDZ domain
Biology
Biochemistry
Serine
Mice
Receptor, Serotonin, 5-HT2C
medicine
Animals
5-HT5A receptor
Phosphorylation
Receptor
Molecular Biology
Glutathione Transferase
Dose-Response Relationship, Drug
Membrane Proteins
Valine
3T3 Cells
DNA
Cell Biology
Alkaline Phosphatase
Receptor antagonist
Molecular biology
Phosphoric Monoester Hydrolases
Protein Structure, Tertiary
5-HT2C receptor
Protein Biosynthesis
Receptors, Serotonin
Mutation
Electrophoresis, Polyacrylamide Gel
Carrier Proteins
Protein Binding
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 278
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....4d47cc38d514769d1c354d84e732cebd