Récepteurs membranaires : structure, dynamique et pathologie
par- 20 mai 2012
Le projet est actuellement disponible en anglais mais il sera traduit prochainement.
Our program will aim at unraveling the molecular and structural bases of functional selectivity/differential coupling of class A GPCRs. To this end, we will investigate : 1°) their structure and dynamics, 2°) the regulation of their function by interacting proteins and by new potential therapeutic molecules.
GPCRs are the largest family of integral membrane proteins, participate in the regulation of most physiological functions and are the targets of most currently marketed drugs. GPCRs are consequently key regulators of signal transduction by which cells respond to variations in their environment.
GPCR signaling is regulated by both extracellular and intracellular stimuli that are known to induce/modulate receptor conformational states (see scheme 1). Three types of partners, ligands, other GPCRs (leading to the formation of homo or heterodimers) and coupling proteins (G protein, arrestins) have been reported to play major roles in the receptor functioning and therefore in the fine-tuning of cell responses to external stimuli. The knowledge of the molecular mechanisms allowing a receptor to undergo different conformational changes and being responsible for inducing different cell responses is of fundamental interest.
The interactions of GPCR with ligands, GPCRs and signaling proteins will be investigated at structural, pharmacological and physiological levels. Depending on the objectives, various experimental approaches will be used. Dynamics and structural analyses will be performed on purified receptors, while pharmacological and physiological questions will be developed on receptor expressed in heterologous cell systems or in native tissues.
We consider the Class A receptors for the neurohypophysial hormones arginine-vasopressin (AVP) and oxytocin (OT) as an ideal model for understanding mechanisms of GPCR activation. Thanks to our collaborations (M. Manning, (Toledo, USA and M. Hibert, Strasbourg, France), a broad spectrum of analog ligands, including peptide and small molecular weight molecules, is available. The existence of different receptor conformational states, stabilized with different classes of ligands (agonists, antagonists, inverse agonists, biased agonists), has been proposed. AVP/OT receptors have been shown to homo- and heterodimerize and this phenomenon plays a role in ligand binding cooperativity. These GPCRs have also been shown to undergo differential coupling. Lastly, AVP/OT receptors possess a high clinical importance, being involved in neuropsychiatric, cardiovascular or kidney diseases as well as in dysfunction of the reproductive system.
Aim 1 : Dynamics and structural analysis of purified receptors. Supported by the 2011-2013 ANR grant “Progamme blanc” Architect.
The goal of the first aim is to analyse the consequence of ligand binding, receptor dimerization and interaction with signaling pathway proteins (G proteins, β-arrestins, etc…) on purified receptor conformations. Functional receptors will be overexpressed in bacterial or insect cell systems (the strategies are already developed in the lab, collaboration with JL Banères, Montpellier, France) and investigation of receptor structure and dynamics will be based on spectroscopic techniques. Two types of investigations will be performed.
1°) Intramolecular FRET (fluorescence resonance energy transfer) experiments will be performed on receptors labeled at various positions with fluorophores (cysteine or FlAsH (Fluorescein Arsenical hairpin binder) labeling). Modifications of receptor conformation will be followed by analyses of FRET signal modifications consecutive to labeled receptor interaction with ligands, receptors or intracellular proteins.
2°) Intermolecular FRET experiments will also be performed between receptors, or receptors and intracellular proteins in order to study protein-protein interactions. Using endogenous agonist (AVP, OT), inverse agonist (SR121463) or functionally-selective pharmacochaperone (MCF 57) compounds with the purified AVP V2 receptor will be studied first. The effects of protein partners (G proteins, arrestins) onto these ligand/V2 complexes will be studied. These studies will also be performed using naturally-occuring mutants of the V2 (see below).
In parallel, besides the study of the V2 organization in lipid bilayers by atomic force microscopy which is currently ongoing (collaboration with PE Milhiet, Montpellier), a more direct structural approach (X-ray cristallography) will start in order to solve the three-dimensional structure of the AVP V2 receptor. We will develop new strategies to stabilize the purified receptor, such as co-crystallization with gC1q-R, a high-affinity V2-interacting chaperone protein. Crystallogenesis will be tried in bicelles or lipid cubic phases (collaborations with B. Kobilka, Stanford, USA and G. Schertler, Cambridge, England). Stabilizing inverse agonists of the V2, selective V2 antibodies as well as the availability of natural mutants (see below) of the receptor will also be helpful. NMR studies of functional domains of the V2 receptor will be investigated as well (collaboration with H. Déméné, Montpellier, France).
AIM 2 : Pharmacological analysis with receptors expressed in heterologous cell systems. Supported by CisBio International and by the 2010-2013 ANR grant “Programme blanc” InnovGABAB.
Differential coupling of AVP/OT receptors can be explained by variations in the ligand/receptor dimer stoichiometry. Preliminary results indicate that a single or a double occupation of the binding sites of a dimer can result in the activation of different signaling pathways. The ligand/receptor stoichiometry will be analyzed using time-resolved FRET experiments performed with fluorescent ligands (which have been already characterized) and/or SNAP-tag labeled receptors (collaborations with the team of Dr. JP Pin and the CisBio biotech). Differential coupling can also be explained by receptor compartmentalization in specialized membrane domains. The modification of the membrane compartmentalization (with methyl-beta-cyclodextrine) on receptor-associated signalling pathways will be analyzed. In addition, whether the properties of our newly developed biased V2 ligands depend on ligand/receptor stoichiometry or are related to receptor compartmentalization, will be established.
AIM 3 : Physiological analysis on native models. Supported by CisBio International and by the 2010-2013 ANR grant “Programme blanc” InnovGABAB.
Two aspects will be analyzed. 1°) GPCR oligomerization has been conclusively demonstrated in heterologous expression systems but its existence in native tissues remains elusive. Because classic FRET experiments performed with labeled receptors cannot be performed in native tissues, we have developed a strategy of time-resolved FRET between fluorescent ligands bound onto the receptors and we have demonstrated the existence of OT receptor dimers in mammary gland preparations. Moreover, we provided evidence for establishment of asymmetric functioning of OT receptor dimers. The data suggest that a single agonist binds with high affinity per receptor dimer, in agreement with negative binding cooperativity. This approach will be extended to other receptors. The development of new labeled ligands in collaboration with CisBio International will allow us to analyze homodimerization of the V2 receptor, as well as heterodimerization between AVP/OT receptors and other families of GPCRs. We propose to study AVP/OT receptor interactions with urotensin, apelin receptors for which potential heterodimerization in kidney is suggested.
2°) Dysfunctions of AVP and OT receptors have pathophysiological impacts. Two aspects will be analysed regarding the AVP V2 receptor. First, many natural different point mutations of the V2 induce receptor sequestration into intracellular compartments, altering its function and leading to congenital nephrogenic diabetes insipidus (cNDI) genetic disease. Besides the non-peptide antagonists acting as pharmacological chaperones able to rescue most of these mutant receptors, we developed agonist pharmacochaperones which exhibit a preferential affinity for the AVP V2 receptor and which are efficient on few receptor mutants. We expect to develop other agonist pharmacochaperones with high affinity and selectivity, and to rescue a larger panel of cNDI mutants (this will be done in collaboration with the team of M. Hibert, Strasbourg). In parallel, proteins responsible for the sequestration of NDI mutants will be searched and characterized. Second, activation of the V2 receptor is also involved in the establishment of polycystic kidney disease. It is not clear whether the activation of cAMP signaling or MAP kinase pathway, respectively, is responsible for the cyst growth. However, V2 antagonists inhibit cystogenesis in animal models, presumably by downregulating cAMP signaling or cell proliferation. The discovery of new V2 biased agonist pharmacochaperones (able to stimulate cAMP signaling and inhibit receptor internalization and MAP kinase activity) will help us to decipher cellular signals involved in cyst establishment and to propose new potential therapeutic ligands. The effects of agonist pharmacochaperones on animal models will first be studied (collaboration with Dr. Ralph Witzgall, University of Regensburg, Germany).
The research plan is technically challenging and very ambitious. Our team combines many expertises and preliminary studies have already been realized, significantly increasing the feasibility of the project. Moreover, we also have developed precious collaborations with different groups of chemists in order to develop/characterize new ligands. The participation of all members of the team to all aspects of the scientific program constitutes our strength. The project should help us in better understanding functioning of GPCRs and in the development of new potential therapeutic compounds, especially in V2-related kidney diseases.