Watching activation and signalling in individual GPCRs

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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.