Résumé
G protein-coupled receptors (GPCRs) not only interact with heterotrimeric G proteins but also with accessory proteins, called GPCR-interacting proteins (GIPs). GIPs are implicated in GPCR targeting to specific cellular compartments, in their assembling into large functional complexes called “receptosomes,” in their trafficking to and from the plasma membrane, as well as in the fine-tuning of their signaling properties. Here, we describe “receptosomes“ associated with the C-terminal tails of 5-hydroxytryptamines 5-HT2A, 5-HT2C, as well as 5-HT4a and 5-HT4e receptors. The three last residues of these receptor C-termini are canonical PDZ ligands interacting with type I PDZ domain-containing proteins (5-HT2A, 5-HT2C, 5-HT4A tails) and type II (5-HT4e). The entire C-terminal tails fused to glutathione-S-transferase or synthetic peptides encompassing the last 14 C-terminal residues of the receptors were used as baits to fish out GIPs from mouse brain. Controls were made with mutant bait (mutated in the PDZ ligand). Proteins, which were specifically retained on native PDZ ligand-containing peptides, were separated on two-dimensional gels and identified by MALDI-TOF mass spectrometry or immunoblotting. Ten and seven PDZ domain-containing proteins were found to bind to the 5-HT2C and 5-HT2A receptors, respectively. The sequences of the C-terminal PDZ ligands of 5-HT2C and 5-HT2A receptors are very similar (SSV and SCV, respectively). If some of the PDZ domain-containing proteins associated with these receptors were identical (ARIP-1/MAGI-2, SAP97, PSD-95, Dlgh3/MPP3), others were clearly different. The 5-HT2C but not the 5-HT2A receptor interacted with the SAP102 and Veli3/CASK/Mint1 ternary complex, whereas the 5-HT2A but not the 5-HT2C receptors interacted with CIPP. MUPP1, which was found to interact with the 5-HT2C receptor in a two-hybrid screen, was also fished out by the 5-HT2C and 5-HT2A C-termini. A few other non-PDZ domain-containing proteins were found in these “receptosomes.” Electron microscopy (EM) studies of 5-HT2A and 5-HT2C receptors and some of their interacting proteins led to the proposition of a presynaptic and postsynaptic localization of 5-HT2C receptors and a preferential postsynaptic localization of 5-HT2A receptors. In a similar manner, we identified 10 and 3 proteins that interacted specifically with the 5-HT4a and 5-HT4e receptor splice variants, respectively. Most of them are PDZ proteins. Among them, NHERF recruited 5-HT4a receptors in microvilli, where they localized with activated ezrin, consistent with a role of 5-HT4a receptors in cytoskeleton remodeling. The same variant interacted with both the constitutive and the inducible (upon metham-phetamine treatment) forms of SNX27 (SNX27a and SNX27b, respectively). SNX27a redirected part of the 5-HT4a receptors to early endosomes.