The Serotonin 5-HT2A and 5-HT2C Receptors Interact with Specific Sets of PDZ Proteins

Abstract

The 5-hydroxytryptamine type 2A (5-HT 2A ) receptor and the 5-HT 2C receptor are closely related members of the G-protein-coupled receptors activated by serotonin that share very similar pharmacological profiles and cellular signaling pathways. These receptors express a canonical class I PDZ ligand (SXV) at their C-terminal extremity. Here, we have identified proteins that interact with the PDZ ligand of the 5-HT 2A and 5-HT 2C receptors by a proteomic approach associating affinity chromatography using immobilized synthetic peptides encompassing the PDZ ligand and mass spectrometry. We report that both receptor C termini interact with specific sets of PDZ proteins in vitro. ) is a major neurotransmitter that is involved in numerous functions of the mammalian central nervous system. These functions are mediated by a large number of receptors. Except for the 5-HT 3 receptor, which is a ligand-gated channel, all 5-HT receptors belong to the Gprotein-coupled receptor (GPCR) superfamily. Among the GPCRs activated by 5-HT, the 5-HT 2 receptor family, namely the 5-HT 2A , the 5-HT 2B , and the 5-HT 2C receptors, continues to raise particular interest. Indeed, they are involved in multiple physiological functions such as the control of endocrine secretion, motor behavior, mood, pain, sleep, thermoregulation, and appetite (1). Moreover, a large number of psychoactive drugs, including non-classical antipsychotic drugs, hallucinogens, anxiolytics, and anti-depressants, mediate their action at least in part through activation of 5-HT 2 receptors (1-4).Among the 5-HT 2 receptor family, the 5-HT 2A and the 5-HT 2C receptor are widely distributed in the central nervous system, whereas the 5-HT 2B receptor is sparse. The 5-HT 2A and the 5-HT 2C receptors share the highest degree of sequence homology (about 50% overall sequence identity). Thus, it is not surprising that these receptors have very similar pharmacological profiles and that only a few selective ligands are available. Initial studies of 5-HT 2A and 5-HT 2C receptor signaling showed that both receptors activate phosphatidyl inositol hydrolysis. However, some differences in signal transduction characteristics of these receptors have been reported (5, 6). In NIH3T3 cells expressing the 5-HT 2C receptor, agonist-independent activity was much more elevated than that measured in cells expressing the same density of 5-HT 2A receptors (7). This indicates that the 5-HT 2A receptor has lower intrinsic ability to adopt an active conformation than does the 5-HT 2C receptor. Different mechanisms of desensitization for the 5-HT 2A and 5-HT 2C receptor systems have also been described. In Chinese hamster ovary cells, agonist-induced desensitization of the 5-HT 2A receptor-mediated phospholipase C activation is inhibited by inhibitors of protein kinase C and Ca 2ϩ -calmodulin-dependent protein kinase II (8). In contrast, the 5-HT 2C receptormediated response is insensitive to these inhibitors. Moreover, the desensitization of the 5-HT 2C receptor system but not that of the 5-HT 2A r...

Physical interaction between the serotonin transporter and neuronal nitric oxide synthase underlies reciprocal modulation of their activity

“…These proteins were identified by MALDI-TOF MS as nNOS (or NOS-1), the only one of the three NOS isoforms that possesses a PDZ domain at its N terminus (23), and InaD-like protein [also designated as a CIPP; see supporting information (SI) Table 1]. The latter protein contains four PDZ domains and was previously identified as a binding partner of Kir4.0 potassium channel family members, N-methyl-D-aspartate receptor NR2 subunits, neurexins, neuroligins, acid-sensing ionic channels, and the 5-HT 2A receptor (22,24,25). Specific binding of these proteins to the C-terminal peptide of SERT but not to the modified peptide was confirmed by immunoblotting (Fig.…”

“…To identify proteins that interact with the putative PDZ binding motif located at the extreme C terminus of SERT, we used a proteomic approach that has already proved efficiency and sensitivity for characterizing specific PDZ binding partners of serotonin receptors (22). This approach was based on peptide-affinity chromatography by using a synthetic peptide encompassing the 15 C-terminal amino acids of SERT as bait, followed by separation of affinity-purified proteins by 2D electrophoresis.…”

Physical Interaction of Calmodulin with the 5-Hydroxytryptamine2C Receptor C-Terminus Is Essential for G Protein-independent, Arrestin-dependent Receptor Signaling

“…To date, the 5-HT 2C receptor is certainly the GPCR for which interacting proteins have been the most extensively characterized by mean of proteomic approaches (Becamel et al, 2002(Becamel et al, , 2004. The majority of these interactions take place within the receptor C-terminus.…”

Intracellular complexes of the β2 subunit of the nicotinic acetylcholine receptor in brain identified by proteomics

“…Receptor proteomics has thus emerged as a primary approach in the molecular analysis of receptor signaling and regulation (16)(17)(18). Multiprotein complexes (termed signaling complexes or signalplexes) have been found to associate with receptors such as glutamate N-methyl-D-aspartate (NMDA) and mGluR5 (19,20), glucocorticoid (21), serotonin 5-hydroxytryptamine type 2A and 2C (22), dopamine D1 and D2 (23), and the ATP-gated channel (P2X7) (24). Earlier studies based on yeast two-hybrid screens indicate that the ␣4-nAChR binds the scaffold molecule 14-3-3 (8) and the calcium sensor visinin-like protein 1 (25).…”