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.
Highlights
A growing number of proteins containing PDZ1 domains have been shown to play important roles in the organization and/or regulation of signaling events in cells
The 5-HT2C Receptor Selectively Interacts with Multiple PDZ domain protein 1 (MUPP1) PDZ domain 10 (PDZ 10) —The carboxyl region of MUPP1 containing the last four PDZ domains (PDZ 10 to PDZ 13) was originally identified by yeast two-hybrid screening for 5-HT2C R-interacting proteins [1]
A weak interaction of the receptor tail over glutathione S-transferase (GST) alone was observed with PDZ 12 and 13, suggesting that PDZ 9 –11 is the primary 5-HT2C R interacting region
Summary
Polyclonal anti-peptide antibodies against amino acids 419 – 435 (amino acids RHTNERVARKANDPEPG) of the rat 5-HT2C R were generated as described previously [19]. Anti-glutathione S-transferase (GST) antibody was purchased from Upstate Biotechnology, Inc.
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