GPCR CONFORMATIONS

Structural studies of ligand-induced conformational changes in G protein-coupled receptors

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 Obiettivo del progetto (Objective)

'G protein-coupled receptors (GPCRs) are eukaryotic seven-alpha-helix transmembrane proteins which are involved in extracellular signal transduction across the cellular membrane. Aim of the proposed research is to structurally characterise ligand-induced conformational changes in GPCRs, to identify the number of alternative conformations the GPCR can adopt, and to engineer conformationally stabilised GPCR variants for subsequent biophysical and crystallographic studies. The research will focus on cannabinoid receptor type 2 and beta-1 adrenergic receptor. Both GPCRs can be expressed and purified from recombinant Escherichia coli as well as insect and mammalian cells. Degree of ligand-induce conformational change will be determined for each amino acid residue by using a novel protein-engineering approach that combines alanine scanning mutagenesis and conformational phi-value analysis. Transition temperatures of denaturation for native and each mutated protein will be determined in presence and absence of particular ligand by high-throughput differential scanning fluorimetry or radioligand-binding assays. These results will be used in engineering of GPCR mutants that are more stable and locked in the specific (eg, activated or inactive) conformational state. Ligand-binding to the stabilised mutants and their functionality will be monitored by fluorescence anisotropy titrations, as well as by radioactive assays using the corresponding G proteins. The stable and functional variants will be used in crystallisation trials. The diffraction data from the expected microcrystals will be collected on the microfocus X-ray beamline. Crystal structures will be determined by molecular replacement. The proposed research will give insights into the activation mechanisms of GPCRs. Detailed understanding of the conformational changes accompanying receptor activation is a prerequisite for rational design of therapeutic drugs affecting GPCRs.'

Introduzione (Teaser)

The technically demanding X-ray crystallography method can be used to delineate the three-dimensional structure of molecules. A European research team set out to determine the molecular structure of G-protein coupled receptors (GPCRs).

Descrizione progetto (Article)

GPCRs are transmembrane proteins capable of translating ligand binding into a specific cellular response. There are approximately 800 different GPCRs encoded in the human DNA, of which 400 are non-sensory receptors with potential medical relevance.

Despite their functional diversity, GPCRs undergo a common conformational change upon stimulation which allows them to interact with different protein partners inside the cell. G proteins are the most prominent GPCR partners and when activated they dissociate into G-? and G-?-? subunits. In turn, these subunits modulate distinct cellular signalling pathways and cause a change downstream in the cell.

The EU-funded 'Structural studies of ligand-induced conformational changes in G protein-coupled receptors' (GPCR CONFORMATIONS) project worked on understanding how different ligands modulate receptor signalling outcome. Researchers focused on the human cannabinoid CB2 receptor and the human vasopressin V2 receptor.

CB2 mainly functions in the immune and peripheral nervous systems while V2 is present in the kidneys. Both constitute important drug targets for treating inflammatory diseases, osteoporosis, and diabetes insipidus.

The experimental outline entailed the determination of the molecular structure of these receptors when bound to different molecules. In this context, researchers used a series of molecules capable of activating (agonists), suppressing (antagonists) or inversing (inverse agonists) the function of the receptor. Following extensive optimisation of the different crystallisation conditions and techniques, researchers were able to suitably modify and express the V2 and CB2 receptors for X-ray crystallography.

Project activities should help shed light on the mechanism of action of these GPCRs. This, in turn, should facilitate the pharmaceutical development of targeted drugs with higher specificity and greater avidity.