The Family of G Protein-Coupled Receptors: An Example of Membrane Proteins
The G protein coupled receptors belong to the largest group of membrane proteins that regulates many essential physiological properties and represents an important class of drug targets. In this chapter, we show how the synergy between a laboratory experi
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1. Introduction The G protein-coupled receptors (GPCRs) are the largest group of cell surface membrane proteins, which form a transmembrane bundle composed of seven membrane-spanning alpha helices connected by loop regions. Binding with endogenous mediators causes conformational changes in GPCRs (1, 2) that lead to high affinity interaction of GPCRs with the cognate G protein and in turn initiate numerous downstream signaling pathways in cells (3, 4). GPCRs are signal transmitters for hormones, neuromediators, cytokines, lipids, peptides, small molecules, and various sensory exogenous stimuli, such as light, odors, and taste. Consequently, GPCRs are regulators of many life important cell processes and universal drug targets against various diseases. Because of structure-based drug design is a rational way to design novel small molecule ligands and to improve binding and 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_23, © Springer Science+Business Media, LLC 2010
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selectivity of old drugs, many laboratories have been working on structural delineation of the ligand binding site of GPCRs. As membrane proteins, GPCRs have difficulties to be expressed, purified and crystallized in a large scale. Therefore, there is a limited number of high-resolution GPCR structures today: bovine and squid rhodopsin (5–9) opsin (10), b2 and b1 adrenergic (11–14), and A2A adenosine (15) receptors. For decades, the structural insight of GPCRs ligand binding site has being mainly gained using indirect methods such as receptor mutagenesis, ligand structure– activity relationships (SAR), and receptor modeling. Thus, till 2007, only the crystal structure of bovine rhodopsin was available and used to construct the low-resolution homology-based models of GPCRs. Although, the sequence homology between light-activated rhodopsin and ligand-activated GPCRs is low (about 11–17%) and the second extracellular loop (EL2) of rhodopsin structure buries deeply into the helical bundle and closes a putative ligand binding cavity, several modeling strategies have been developed to delineate the ligand binding site of ligand-activated GPCRs based on the rhodopsin structure. In one approach, the EL2 was removed before the docking of known ligands and added back when the ligand interactions with transmembrane helices were defined by molecular docking and receptor mutagenesis (16, 17). In another approach, forced molecular dynamics simulations were applied straight away to drive the ligand–protein interactions in the initial homology model according to experimental data (18–20). In addition, molecular simulations of the rhodopsin-based homology model in water– lipid environment were used for the formation of a binding cavity by initially added spheres with a flexible Van der Waals radius, and the formed cavity then were employed for docking of ligands (21). In all these approaches, the iterative application of compu
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