Bice Chini

The Binding Site of Neuropeptide Vasopressin V1a Receptor EVIDENCE FOR A MAJOR LOCALIZATION WITHIN TRANSMEMBRANE REGIONS

To identify receptor functional domains underlying binding of the neurohypophysial hormones vasopressin (AVP) and oxytocin (OT), we have constructed a three-dimensional (3D) model of the V1a vasopressin receptor subtype and docked the endogenous ligand AVP. To verify and to refine the 3D model, residues likely to be involved in agonist binding were selected for site-directed mutagenesis. Our experimental results suggest that AVP, which is characterized by a cyclic structure, could be completely buried into a 15-20-Å deep cleft defined by the transmembrane helices of the receptor and interact with amino acids located within this region. Moreover, the AVP-binding site is situated in a position equivalent to that described for the cationic neurotransmitters. Since all mutated residues are highly conserved in AVP and OT receptors, we propose that the same agonist-binding site is shared by all members of this receptor family. In contrast, the affinity for the antagonists tested, including those with a structure closely related to AVP, is not affected by mutations. This indicates a different binding mode for agonists and antagonists in the vasopressin receptor.

Functional architecture of vasopressin/oxytocin receptors.

Three-dimensional models of G protein-coupled receptors (GPCR) have been defined using most experimental data available and protein modeling techniques. The endogenous ligand binding sites have been qualitatively described and putative receptor activation mechanisms have been proposed. The model has been recently refined to take into account recent crystallographic data. Most experimental results published are in excellent qualitative agreement with the initial model. We have undertaken to study more systematically by site directed mutagenesis the vasopressin/oxytocin receptor binding domain as a prototype of neuropeptide receptors. The experimental results are in very good agreement with the models. The residues responsible for the neuropeptide binding have been identified and confirm the predicted localization of the neuromediator in the transmembrane domain of the receptors. The side chain of the 8th residue of vasopressin interacts with a non-conserved receptor residue located in the first extracellular loop. As predicted from the model, this interaction is completely responsible for the selectivity of the ligand-receptor interaction. Finally, aromatic residues which allow the modulation of the efficacy of agonists have been identified.

Towards understanding the role of the first extracellular loop for the binding of peptide hormones to G-protein coupled receptors

By a combination of molecular modelling and site-directed mutagenesis studies, we have recently identified a key residue in the first extracellular loop which determines agonist selectivity and high-affinity binding in the V1a vasopressin receptor. Based on primary sequence analysis and structure-activity relationship studies of other neuropeptides and their receptors, the corresponding amino acid in the first extracellular loop is proposed to play a homologous role in conferring affinity and selectivity. This would seem to be the case notably for angiotensin, cholecystokinin, neuropeptide Y and neurokinin receptors.

Three-dimensional structure of G protein-coupled receptors: from speculations to facts

Publisher Summary This chapter discusses the speculations and facts regarding the three-dimensional structure of G protein-coupled receptors with the help of an experimental validation. G protein-coupled receptors (GPCR) represent very important targets for drug design in most therapeutic areas. The chapter briefly reviews the biochemical features of GPCRs and the three-dimensional model of their complex with different ligands. The experimental validation of these models and their putative usefulness for drug design is also discussed. G protein-coupled receptors belong to the same functional and structural protein family. These receptors are found in cell membrane. The G protein is a heterotrimer which dissociates into alpha and beta-gamma subunits upon activation. GPCR endogenous ligands present a striking structural diversity. The challenge for the medicinal chemist is to understand how molecules as different as small cationic neorotransmitters, small cyclic or non-cyclic peptides, proteins and glycoproteins can interact with structurally related receptors.