Periodic Reporting for period 1 - 3D-V2R (Structural and functional characterization of different states of the arginine vasopressin V2 receptor)
Reporting period: 2023-02-01 to 2025-01-31
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.
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.
We started first to characterized the V2R-arrestin2-scFv30 complex. We produced each partner of the complex independently before to mix them in order to form the complex and used it for cryo-electron microscopy (cryo-EM) analysis. We collected a large dataset of 14,000 movies at the EMBL of Heildeberg from where we analyzed the data using cryoSPARC. We successfully ended up with a cryo-EM map at 4.7 Å resolution allowing us to reconstruct a model at high-resolution. The structure revealed (a) an atypical position of the arrestin2 compared to previously described GPCR-arrestin assemblies, (b) an original interface between the receptor and the arrestin2 involving all the intracellular loops of the V2R, and (c) all the V2R carboxyl terminus portion are fully phosphorylated to bind in a furrow of the arrestin2. Those results highlight a notable variability among GPCR-arrestin complexes. Finally, we also compared the two V2R active structures (G-protein versus arrestin2 coupled to V2R) obtained. We observed that the receptor is in the overall same position when coupled to both signaling proteins. Interestingly, the helix5 of the G-protein and the finger loop of the arrestin bind in the exact same position into the intracellular cavity of the receptor, even implicating the same V2R residue (R137). This may explain the desensitization mechanism where the arrestin take the place of the G-protein stopping its signalization. Also, the large binding interface between the arrestin2 and the receptor explain the so-called “long-standing” interaction of the arrestin with the V2R. All the data from that work were published in the Science Advances journal in 2022. The data were also used to write two reviews, one in Vitamins and Hormones in 2022, and one in Membranes in 2023. Since the publication of the article, it was cited 69 times (on Feb 2025) showing its importance in the field.
We then started to characterized the inactive structure of the V2R bound to antagonist. This a truly challenging aim, as the size of the receptor (45 kDa) is beyond the limit of cryo-EM (100 kDa), and due to its intrinsic dynamic. To allow its structural characterization, we replaced one of the intracellular loops of the receptor by a BRIL domain, on which we added a Fab anti-BRIL, and finally a nanobody anti-Fab, allowing to obtain a complex at 115 kDa. Such addition has been previously used to determine the inactive structure of some GPCRs. As antagonist of the V2R, we choose to work with either the tolvaptan, a well-known non-peptidic antagonist of the receptor, or the green mamba snake mambaquaretin (MQ) toxin. Both fully antagonize the receptor and bind with a low nanomolar affinity. Always using the cryo-EM approach, we successfully determined the two inactive structures at a resolution of 2.5 Å. Interestingly, the two antagonists inactivate the receptor by different binding mode. The tolvaptan, which is a small molecule, go in the binding pocket with a direct interaction with the toggle switch, while the MQ, which is larger (6.2 kDa) have a large interaction at the extracellular side implicating all the TM (except TM1), and have an indirect interaction with the toggle-switch. Overall, the two inactive states have the same TM positioning except at the top of the receptor with slight difference, mainly due to a difference in size between the two ligands. When comparing to the active state, we can see the repositioning of several residues belonging to the motif known for the activation of the receptor, and of the well-known opening of the TM6 that allow the access of the intracellular partners. All those results are in a manuscript, which is freely accessible in BioRXiv, and also under review in the Nature Communications journal (Feb 2025).
We then started to characterized the inactive structure of the V2R bound to antagonist. This a truly challenging aim, as the size of the receptor (45 kDa) is beyond the limit of cryo-EM (100 kDa), and due to its intrinsic dynamic. To allow its structural characterization, we replaced one of the intracellular loops of the receptor by a BRIL domain, on which we added a Fab anti-BRIL, and finally a nanobody anti-Fab, allowing to obtain a complex at 115 kDa. Such addition has been previously used to determine the inactive structure of some GPCRs. As antagonist of the V2R, we choose to work with either the tolvaptan, a well-known non-peptidic antagonist of the receptor, or the green mamba snake mambaquaretin (MQ) toxin. Both fully antagonize the receptor and bind with a low nanomolar affinity. Always using the cryo-EM approach, we successfully determined the two inactive structures at a resolution of 2.5 Å. Interestingly, the two antagonists inactivate the receptor by different binding mode. The tolvaptan, which is a small molecule, go in the binding pocket with a direct interaction with the toggle switch, while the MQ, which is larger (6.2 kDa) have a large interaction at the extracellular side implicating all the TM (except TM1), and have an indirect interaction with the toggle-switch. Overall, the two inactive states have the same TM positioning except at the top of the receptor with slight difference, mainly due to a difference in size between the two ligands. When comparing to the active state, we can see the repositioning of several residues belonging to the motif known for the activation of the receptor, and of the well-known opening of the TM6 that allow the access of the intracellular partners. All those results are in a manuscript, which is freely accessible in BioRXiv, and also under review in the Nature Communications journal (Feb 2025).
Based on the results collected, we are actually screening multiple peptide libraries to find hit that can interact with the V2R, followed by pharmacological assays to find potential on-site candidates that will open doors to future drug design initiatives.
To resume, the objectives of the 3D-V2R were completely achieve resulting in several cryo-EM structures (available through the PDB) of both active and inactive states, in the publication of several articles in high impact journal that are freely available allowing open access, and in the dissemination of the data in multiple national and international congress related to the cryo-EM and/or GPCR fields. As there is an urgent need to define structural determinants of the V2R responsible for its activation and inhibition, our structural data will give additional information to rationally design new selective ligands with potential therapeutic value. We will pursue on that goal in addition to continue to add more structures to ameliorate the structure based-drug design approach.
To resume, the objectives of the 3D-V2R were completely achieve resulting in several cryo-EM structures (available through the PDB) of both active and inactive states, in the publication of several articles in high impact journal that are freely available allowing open access, and in the dissemination of the data in multiple national and international congress related to the cryo-EM and/or GPCR fields. As there is an urgent need to define structural determinants of the V2R responsible for its activation and inhibition, our structural data will give additional information to rationally design new selective ligands with potential therapeutic value. We will pursue on that goal in addition to continue to add more structures to ameliorate the structure based-drug design approach.