). Most of them are scaffolding proteins that contain several structural interaction domains such as Src homology 2 (SH2) or 3 (SH3) domains, post-synapticdensity-95/disc-large/zonula-occludens-1 (PDZ) domains and Drosophila enabled and vasodilator-stimulated phosphoprotein homologous (EVH) domains. These proteins participate in the building of large submembrane protein signaling networks. We have recently isolated a complex of at least 15 proteins interacting with the C-terminal tail of the 5-HT2C receptor, using a proteomic approach combining peptide-affinity chromatography and mass spectrometry. This further supports the interaction of the GPCR C-terminus with large protein networks. Moreover, functional studies have established that the proteins that interact with the GPCR C-terminus are implicated in various GPCR functions that do not involve Gproteins. These functions include trafficking, targeting to specific subcellular compartments, clustering with effectors, fine tuning of G-protein activation and desensitization.

Serotonin 5-HT 4(a) receptor, a G-protein-coupled receptor (GPCR), was produced as a functional isolated protein using Escherichia coli as an expression system. The isolated receptor was characterized at the molecular level by circular dichroism (CD) and steady-state fluorescence. A specific change in the near-UV CD band associated with the GPCR disulfide bond connecting the third transmembrane domain to the second extracellular loop (e2) was observed upon agonist binding to the purified receptor. This is a direct experimental evidence for a change in the conformation of the e2 loop upon receptor activation. Different variations were obtained depending whether the ligand was an agonist (partial or full) or an inverse agonist. In contrast, antagonist binding did not induce any variation. These observations provide a first direct evidence for the fact that free (or antagonist-occupied), active (partial-or full agonist-occupied) and silent (inverse agonist-occupied) states of the receptor involve different arrangements of the e2 loop. Finally, ligand-induced changes in the fluorescence emission profile of the purified receptor confirmed that the partial agonist stabilized a single, welldefined, conformational state and not a mixture of different states. This result is of particular interest in a pharmacological perspective since it directly demonstrates that the efficacy of a drug is likely due to the stabilization of a ligand-specific state rather than selection of a mixture of different conformational states of the receptor.G-protein-coupled receptors are versatile biological sensors. They are responsible for the majority of cellular responses to hormones and neurotransmitters, as well as sight, smell, and taste senses (1, 2). Signal transduction is specifically associated with GPCR 1 activation. Although significant progress has been made within the last few years in dissecting GPCR-mediated signal transduction pathways, understanding of the mechanisms underlying receptor activation is still hampered by the lack of information at the molecular level (3,4). This is largely due to the fact that very few expression systems have proven satisfactory for producing these receptors in a functional state and with sufficient yields for biophysical studies to be carried out (5-7). Most of the systems that have been developed to elucidate the mechanism of GPCR activation therefore essentially rely on the use of purified rhodopsin and ␤ 2 -adrenergic receptor (3,4,8). Interestingly, most of the results obtained so far report on the conformational events occurring at the level of the cytoplasmic side of the receptors. In contrast, few reports give indications on the possible conformational rearrangements certainly occurring in the extracellular part of the receptor, in particular in the extracellular loops.Several models have been developed to conceptualize the mechanisms of activation (9, 10). The two-state model and the extended ternary model assumes that the receptor exists in an equilibrium between a resting state (R) and...