Watching activation and signalling in individual GPCRs

G-protein coupled receptors (GPCRs) represent one of the most important families of drug targets in pharmaceutical development. They are 7-transmembrane domain (7TMD) membrane receptors that control most neurological functions and comprise the largest family (~1%; >1,000 in number) of mammalian proteins. They are therefore at the focus of intense efforts in drug design and functional studies, not least because ~75% of drugs address only 5% of known GPCRs. We now (as of October 2007) have a structure of a ligand-binding Class A GPCR, the beta2-adrenergic receptor, to complement the 3D structures of rhodopsin. In the beta2-ADR structures, putative cholesterol binding sites and dimerisation interfaces were identified. Dimerisation of GPCRs may be fundamental to their signal activation mechanism, and lipid interactions crucial to ligand binding and/or the signalling process, both of which are areas of significant controversy and have only been partially addressed and not fully resolved. 3D structures are a very good start in such studies, but dynamics, the membrane involvement and kinetic mesurements are all essential in functional descriptions, even ahead of 3D structures. In particular, questions about ligand stoichiometry, interface interactions and lipid involvement in signalling are all important issues for understanding GPCR activation and signalling. The hurdle of obtaining a purified, reconstituted and functionally competent GPCR for such studies has been overcome for the chosen GPCR (neurotensin receptor 1). Studies now published by us (Harding, Biophys. J., 2008) demonstrate dimerisation in lipid bilayer of a specific composition. Here we will capitalize on this investment and use single molecule methods to dissect details about GPCR function using novel in vitro approaches for the first time on a GPCR.

In the majority of signaling events initiated by extra-cellular ligands such as hormones, neurotransmitters, growth factors or drugs, binding of these signaling molecules to seven trans-membrane (7TM) G-protein-coupled receptors (GPCRs) results in the activation of G-proteins localized at the cytoplasmic surface of the plasma membrane. Agonist activation of a GPCR also results in feedback regulation of G-protein coupling, receptor endocytosis and signaling through G-protein independent pathways. GPCR activity is the result of the coordinated balance between inter-related processes which govern receptor signaling, desensitisation and resensitisation. Agonist-induced GPCR phosphorylation results in receptor desensitisation and internalisation, and consequent receptor trafficking in cells. Therefore, to understand the early stages of the response of a GPCR to ligand (agonist, antagonist or inverse agonist) binding, quantitative in vitro studies with purified receptor are needed. In addition, the lipid environment in which a membrane protein is embedded is crucial to function, as shown for many other examples, but not yet for purified, ligand binding GPCRs. Dimerisation of GPCRs is thought to be a crucial initial step in signaling process but significant controversy still exists about oligomerisation in GPRC signaling, and whether this is a generic or variable phenomenon depending on type. This is all in no small part due to the fact that few GPCRs are in a suitable form for detailed biophysical studies, and we have a well characterised, ligand binding recombinant and reconstituted GPCR, the neurotensin receptor 1 (NTR1). Here we will study initial signaling events from ligand (agonist, antagonist) binding through conformational changes and oligomerization to G-protein activation in various compositionally defined bilayers. The kinetics, stoichiometry of ligand binding and lipid requirements will be explored using novel single molecule methods.