Study of signal transduction mechanisms through investigation of receptor conformational changes – ARCHITECT

In order to study conformational changes of receptors upon binding of their selective ligands, it is necessary to develop several methodological approaches. First, receptors have to be produced and purified in quantities compatible with their biophysical and structural analysis. Then receptors are reconstituted in artificial systems able to maintain them as water-soluble entities to be manipulated in vitro. To measure their activity, receptors are labeled with two different compatible fluorophores at different precise positions. Upon interaction with their ligands, the conformational changes are recorded through variations of fluorescence signals, more particularly through measurement of fluorescence energy transfer between the two fluorophores. In parallel, the receptor cell protein partners, G proteins and arrestins, have to be produced and purified as well. Then, receptor / receptor, receptor / G protein and receptor / arrestin complexes are in vitro reconstituted and their structural organization studied by fluorescence approaches. Finally, receptor and protein partner activities and their ligand-induced responses, is established using in vitro functional assays. These assays are adapted to purified receptor systems, easy to set up. Altogether, these approaches have to be developed in the laboratory and results must be reproducible.

We focused our study on the vasopressin V2 receptor subtype. It is mainly localized in kidney where it is responsible for the antidiuretic activity of the hormone. This effect is of major physiological importance for mammals and humans since the regulation of the body water balance is crucial for terrestrial life. We have been able to demonstrate that upon specific ligand binding, conformational changes of the V2 involved in the coupling and activation of G proteins (more particularly Gs subunit) are different from those responsible for the arrestin recruitment. We correlated biased properties of several ligands (those which are able to activate or inhibit only a set of the signal transduction pathways usually induced by the endogenous hormone or neurotransmitter) to specific receptor conformational states. The functional selectivity of a ligand results from the combination of coordonated movements of different functional domains of its receptor, allowing or not the different transduction pathways generally associated to the considered receptor to be activated. Each ligand induces or stabilizes a set of specific receptor changes which is responsible for its efficacy (therapeutic effect) and its functional selectivity (capacity to only increase beneficial effects).

Establishing relationships/correlations between a given ligand-activated receptor structural conformation and activation of a G protein-dependent or arrestin-dependent specific biological response should allow in the future to rationally develop novel therapeutic compounds with an increased efficiency and less side effects. These results should help in designing better, more « intelligent » disease therapies.

An article has been published in an internationally well-known journal having a high impact factor.
Rahmeh R. et al., Structural insights into biased G protein-coupled receptor signaling revealed by fluorescence spectroscopy. Proc. Natl. Acad. Sci. USA, 2012, 109, 6733-6738.

G protein-coupled receptors (GPCRs) constitute the largest family of integral membrane proteins, respond to a wide variety of extracellular signals (proteins, peptides, amino-acids, lipids, catecholamines, ions, light), 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. A fine-tuning of the cellular responses is related to different aspects of GPCR functioning. These receptors are remarkably versatile, being able to activate G protein-dependent and -independent signaling pathways, simultaneously or not, often in a ligand-specific manner. In addition, their interactions with many membrane (like other GPCRs) or cytoplamic (G proteins, arrestins) partners, influence their pharmacological and their functional properties. This plasticity can be attributed to structural flexibility of GPCRs and the ability of ligands to induce or stabilize ligand-specific conformations.
The knowledge of the molecular mechanisms allowing a receptor to undergo different conformational changes and being responsible for activating different cell responses is of fundamental interest. Moreover, the dynamic character of GPCRs is likely to be essential for their physiological functions and understanding this molecular plasticity might facilitate structure-based drug discovery.
X-ray crystallography has proved to define GPCR 3D structure. Today, the available GPCR crystal structures are those of inactive conformations, the receptors being complexed with inverse agonists. These structures provide only a static view of dynamic signaling molecules. It is thus necessary to develop other approaches like fluorescence spectroscopy, that can characterize the dynamic nature of these conformationally complex receptors. Such pioneering studies have been undertaken using the beta2-adrenergic receptor and are of primary importance. The data suggest a transduction model for GPCRs in which ligand binding occurs through a series of conformational intermediates, each able to activate different signaling pathways.
These outstanding results show that it is necessary to put major efforts on these signaling molecules and to get new data with other GPCR families.
Our goal is to address the dynamics and conformational changes of the vasopressin/ocytocin (AVP/OT) receptor family and to demonstrate the feasibility of using biophysical approaches to study their structure. We will explore the use of fluorescence spectroscopy to measure ligand-induced conformational changes. We consider AVP/OT receptors as a valuable model for understanding mechanisms of GPCR activation. A broad spectrum of analog ligands including peptide and small molecular weight agonists and antagonist is available. Diifferent receptor conformational states, stabilized with different classes of ligands, have been proposed. AVP/OT receptors possess a high clinical importance and have a high value as a model system for G protein-coupled receptors in general, and they are considered as prototypes of receptors activated by peptide ligands.
This project will follow several steps : 1) overexpression and functional purification of AVP/OT receptors, 2) production of purified active G proteins and arrestins, 3) development of chromatographic columns to achieve ligand-based functional purification, 4) stabilization of receptors in detergent or phospholipids, 5) labeling of purified receptors, G proteins and arrestins at specific positions with fluorophores, 6) recording of ligand-induced receptor and protein partner conformational changes by fluorescence.
This project will provide the first direct observation of structural changes in a peptide receptor family and will allow us to study the structural basis of efficacy, functional selectivity and multiple signaling properties of GPCR/ligand complexes.