Agonist-induced Phosphorylation of the Serotonin 5-HT2C Receptor Regulates Its Interaction with Multiple PDZ Protein 1

Abstract

A growing number of proteins containing PDZ 1 domains have been shown to play important roles in the organization and/or regulation of signaling events in cells. PDZ domains (or GLGF repeats) were named after three proteins identified over a decade ago: postsynaptic density-95, Drosophila Discs large, and zonula occludens-1 (3-5). These three proteins belong to the membrane-associated guanylate kinase (MAGUK) family of proteins. Most MAGUK proteins contain three PDZ domains, an Src homology 2 domain, and a guanylate kinase-like domain, each having different cellular roles. PDZ domains range from 80 to 100 amino acids in length and typically bind to the carboxyl-terminal sequence of target proteins including receptors, channels, and various signaling molecules to regulate subcellular localization, trafficking, recycling, and/or signaling (6 -10).MUPP1, a protein containing 13 putative PDZ domains, was isolated in a yeast two-hybrid screening for proteins that bound to the carboxyl-terminal tail of the 5-HT 2C R (1). MUPP1 is expressed in many tissues, whereas the 5-HT 2C R is a brainspecific protein (1, 11). The 5-HT 2C R has classically been thought to couple to G q activation; however, additional G protein families have been implicated, leading to the activation of different downstream signaling pathways including phospholipase A 2 , C, or D, and various cation channels (12-17). Since PDZ-containing proteins can scaffold many signaling molecules together into a signal transduction complex, the interaction between MUPP1 and the 5-HT 2C R was further investigated. The 5-HT 2C R contains a PDZ binding motif, Ser 458 -Ser-Val, at its extreme carboxyl terminus, which is critical for interaction with PDZ 10 of MUPP1 (18). In an alternate approach to the yeast two-hybrid system, we independently show that PDZ 10 of MUPP1 is the primary site of interaction for the 5-HT 2C R.Serotonin stimulation has previously been shown to promote phosphorylation of the two serine residues of the 5-HT 2C R PDZ binding motif, Ser 458 and Ser 459 (2). We therefore hypothesize that phosphorylation of the carboxyl-terminal serines of the 5-HT 2C R regulates receptor interaction with MUPP1. To test this hypothesis, we investigated whether a modification of Ser 458 and/or Ser 459 of the 5-HT 2C R carboxyl-terminal tail would alter PDZ 10 interaction. Ser 458 and/or Ser 459 of the receptor tail were mutated to aspartate to mimic phosphorylation (i.e. introduction of a negative charge). Next, cells expressing 5-HT 2C Rs were treated with agonist or antagonist to assess the interaction of the 5-HT 2C R with MUPP1. The results of these experiments support our hypothesis that phosphorylation is a key regulator of 5-HT 2C R interaction with MUPP1. Furthermore, the results indicated that a significant amount of basal phosphorylation of the receptor may also play a yet undetermined role in regulating PDZ-protein interactions. MATERIALS AND METHODS AntibodiesPolyclonal anti-peptide antibodies against amino acids 419 -435 (amino acids RHTNERVARKANDPE...

GABAB Receptor Association with the PDZ Scaffold Mupp1 Alters Receptor Stability and Function

“…Such interactions may serve to facilitate well known examples of cross-talk between GABA B and serotonin receptors (40 -42). Interestingly, the Mupp1 interaction with the 5-HT 2C receptor is known to be regulated by agonist-mediated receptor phosphorylation (43). Similar regulation of the Mupp1-GABA B receptor interaction by agonist-promoted phosphorylation is possible and could further add to the complexity of GABA B receptor regulation.…”

Decreased Serotonin 5-HT2A Receptor-Stimulated Phosphoinositide Signaling in Fibroblasts from Melancholic Depressed Patients

“…Given that the reduction in 5-HT 2A receptor signal was retained after many cell passages, a genetic alteration is likely, either in the receptor itself or key regulatory factors, such as multidomain scaffolding proteins. For example, recent evidence suggests that the 5-HT 2A receptor (Xia et al, 2003) and its close relative, the 5-HT 2C receptor (Parker et al, 2003), are regulated by interaction with a multifunctional PDZ domain protein. In addition, genetic studies have suggested that polymorphisms of the 5-HT 2A receptor are associated with mood disorders, but the results have been mixed (Zhang et al, 1997;Spurlock et al, 1998;Turecki et al, 1999;Du et al, 1999;Hrdina and Du, 2001;Crawford et al, 2000;Arias et al, 2001).…”

5-HT2C and GABAB receptors influence handling-induced convulsion severity in chromosome 4 congenic and DBA/2J background strain mice

“…Some PDZ domain proteins possess enzymatic activity, but the MPDZ protein (encoded for by Mpdz) apparently has no such intrinsic activity, so it is plausible that it affects neuronal excitability by altering the rate or fidelity of signal transduction mediated by one or more of the proteins with which it interacts. MPDZ's interaction with 5-HT 2C receptors has been confirmed in vivo (Becamel et al, 2001), and is mediated via a PDZ recognition motif (X-S/T-X-V) at the intracellular C-terminus of the 5-HT 2C receptor that binds to the tenth PDZ domain in MPDZ (Becamel et al, 2001;Parker et al, 2003). Here, we demonstrate that chromosome 4 congenic mice show significantly less severe convulsions in response to SB242084, a selective 5-HT 2C receptor antagonist, than background strain mice.…”