G protein-coupled receptors (GPCRs) are the largest superfamily of cell surface signaling membrane proteins comprising up to 800 molecules in human. They are among the most important targets for drugs for many human diseases, with up to 30% of prescribed drugs on the market. GPCRs are complex allosteric machines with high flexibility and comprise a bundle of 7 transmembrane domains (TM). Their ability to adopt different conformations allow GPCRs to respond to various extracellular stimuli (light, peptides, lipids, and proteins) by transmitting the signal across the membrane to activate diverse intracellular signaling pathways. The high conformational flexibility of GPCRs is thus a prerequisite to sense a variety of stimuli and link them to different signaling partners such as G proteins and arrestins. The transmembrane proteins are capable of high plasticity to interact with an important range of signaling partners, a characteristic that represents a challenge for structure determination.
The V2 receptor (V2R) is considered as an archetype of GPCRs responding to small peptides and hormones. It is a typical class A (rhodopsin-like) GPCR which preferentially couples to Gs protein resulting in the activation of adenylyl cyclase. It is also the most used model in the literature to study molecular pharmacology of the arrestin recruitment to GPCRs. As most GPCRs, V2R follows a classical desensitization mechanism being phosphorylated by G protein-coupled receptor kinases and interacting with arrestin2 (also known as β-arrestin1, Arr2) and arrestin3 (β-arrestin2, Arr3). Interacting with V2R, the arginine-vasopressin (AVP), a nine-amino acid peptide hormone, mainly acts in the mammalian kidneys by regulating our body water balance and solute transport, but also possessing important physiological effects in the whole body. Dysfunctions of this GPCR result in clinical disorders from dysregulation of the water balance resulting in syndrome of inappropriate antidiuretic hormone secretion (SIADH, as a consequence of many forms of cancer), congestive heart failure, hepatic cirrhosis, to urine disorders (incontinence, nocturia). Thus, this transmembrane protein plays a key role as a therapeutic target linked to unmet medical needs. In addition, loss-of-function or constitutively active mutations of the V2R lead to two rare genetic diseases, respectively: 1) the congenital Nephrogenic Diabetes Insipidus (cNDI) characterized by excessive urine voiding, 2) the nephrogenic syndrome of inappropriate antidiuresis (NSIAD) characterized by excessive water loading and hyponatremia. Finally, V2R is also a target for treating autosomal dominant polycystic kidney disease (PKD), the most frequent Mendelian inherited disorder affecting million people worldwide. Half of PKD patients over sixty require dialysis or kidney transplantation. Unfortunately, as of today, there is no safe prescribed drug on the market to treat disorders linked to V2R. The nonpeptide antagonist Tolvaptan, has been approved for treating hyponatremia (SAMSCA®) and recently for treating PKD (JINARC®), but with many concerns due to hepatotoxicity meaning that people cannot use it for long periods. Thus, there is an urgent need to define structural determinants of the V2R responsible for its activation and inhibition, in order to be able to rationally design, on a structural basis, new selective ligands with potential therapeutic value (greater safety, better efficacy).
To understand the molecular mechanisms of this complex machinery, it is necessary to obtain atomic resolution. Moreover, it will be of importance to solve a minimum of 3 different states to get the whole drawing of the activation of a given receptor. This means to determine the atomic structure of (a) the active agonist-receptor-G protein complex, (b) the active agonist-receptor arrestin complex, and (c) an inactive state (bound to an inverse agonist or an antagonist). Following the characterization of the active state of the V2R coupled to Gs protein, the 3D-V2R project aimed at determining the active agonist-bound receptor-arrestin complex conformation and its antagonist-bound inactive state of the V2R and finally at comparing the different structures of the V2R to better understand the molecular events involved in its activation to trigger associated signaling pathways.
