Recent Progress in the Structure Determination of GPCRs, a Membrane Protein Family with High Potential as Pharmaceutical
G protein-coupled receptors (GPCRs) constitute a highly diverse and ubiquitous family of integral membrane proteins, transmitting signals inside the cells in response to an assortment of disparate extracellular stimuli. Their strategic location on the cel
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1. Introduction G protein-coupled receptors (GPCRs) comprise the largest family of integral membrane proteins in the human genome, with ~800 GPCR family members identified (1). These receptors communicate Jean-Jacques Lacapère (ed.), Membrane Protein Structure Determination: Methods and Protocols, Methods in Molecular Biology, vol. 654, DOI 10.1007/978-1-60761-762-4_8, © Springer Science+Business Media, LLC 2010
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signals across the plasma membrane in response to a variety of extracellular stimuli, ranging from photons, ions, and small molecules, to peptides and proteins. The signals are amplified and transmitted to downstream effectors inside the cells primarily through coupling to heterotrimeric guanidine nucleotide binding proteins (G proteins). Upon activation, receptors trigger complex cascades of reactions controlling crucial physiological and cellular processes. As such, GPCRs are implicated in multitude of diseases, making them important pharmaceutical targets. In fact, about 40% of drugs on the market act on the G protein-coupled receptors (2). GPCR family members share common architecture of a seven transmembrane a-helical bundle (3). It remains intriguing how such a relatively simple scaffold has evolved to selectively bind to thousands of diverse ligands transmitting signals to dozens of different effectors. The primary questions in the GPCR field include understanding mechanisms of signal transduction and aspects of ligand selectivity and specificity. These and other essential questions can only be answered when a variety of biophysical and biochemical data are combined with high-resolution structures of family members stabilized in different functional states. Despite the great importance of GPCRs and the immense worldwide efforts aimed at obtaining their structures, very little progress has been achieved until recently. Indeed, until late 2007, a high resolution structure of only one highly atypical GPCR member, visual rhodopsin, was known (4). Rhodopsin is unusual in that it is highly abundant in the eye’s retina and contains a covalently bound ligand, 11-cis retinal, keeping the receptor conformationally stable in a fully inactive state. However, all other receptors are expressed in miniscule amounts in native tissues and activated by diffusible ligands. For structural studies, it has therefore become necessary that these proteins be heterologously overexpressed, solubilized, and purified. Human integral membrane proteins, including GPCRs, are notoriously difficult to express and they are highly unstable when solubilized by detergents. What makes GPCRs even more challenging is their observed conformational heterogeneity highlighting dynamic equilibrium between multiple functional states (5), obviously vital for signal transduction; however, a formidable roadblock for structural studies. For example, most GPCRs possess a certain level of basal activity in the apo, or unliganded state. Ligands that increase activity are called agonists, those that bind to the r
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