Our knowledge about GPCR structures has advanced considerably since the first GPCR structure (bovine rhodopsin) was published in 2000 by Palczewski and colleagues. Currently, there are only a few GPCRs for which inactive state structures are known. For crystallization trials, receptors must be purified in the presence of detergents;the choice of detergent becomes critical to maintain the GPCR in a functional, correctly folded form. Rhodopsin shows remarkable stability in detergent solution as long as it is kept in the dark to maintain its inactive state;this detergent tolerance allowed extensive crystallization screens and led to diffraction-quality crystals. The beta-1 adrenergic receptor is much less stable in detergent solution;an extensive alanine/leucine scanning mutagenesis approach was used to identify a mutant receptor suitable for crystallization. The beta-2 adrenergic and adenosine A2a receptors were engineered with T4 lysozyme replacing most of the flexible intracellular loop 3. The receptors were crystallized with an inverse agonist or antagonist bound to promote the inactive state. The transmembrane cores look similar but not identical in these receptor structures. Differences are seen in the N- and C-terminal receptor regions and in the loops connecting the helices. The interpretation of these differences remains uncertain since they may originate from the respective receptor sequences, disorder in flexible regions, or possibly from protein engineering. Recently, the structures of active GPCRs, and a complex of receptor with G-protein have been solved. NTS1 is very stable in an optimized detergent mixture for purification, but has reduced stability in detergents preferred for crystallization. Mutational approaches must be applied to obtain a mutant receptor which stays in a biologically relevant, single conformation, long enough for crystallization to occur. A structure of NTS1 alone and in complex with an antagonist will show whether different inactive conformations exist and how these relate to the currently known receptor structures with emphasis on Gq-coupled receptors. A structure of NTS1 in complex with neurotensin in its active form will show the conformation of such a key signaling state and how agonist binding effects the changes on the intracellular receptor surface needed for G-protein engagement, and allows the comparison with other receptor structures thought to represent active state conformations.

Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2011
Total Cost
$787,802
Indirect Cost
City
State
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Zip Code
Vaidehi, Nagarajan; Grisshammer, Reinhard; Tate, Christopher G (2016) How Can Mutations Thermostabilize G-Protein-Coupled Receptors? Trends Pharmacol Sci 37:37-46
Xiao, Su; Shiloach, Joseph; Grisshammer, Reinhard (2015) Construction of recombinant HEK293 cell lines for the expression of the neurotensin receptor NTSR1. Methods Mol Biol 1272:51-64
Krumm, Brian E; Grisshammer, Reinhard (2015) Peptide ligand recognition by G protein-coupled receptors. Front Pharmacol 6:48
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Bhattacharya, Supriyo; Lee, Sangbae; Grisshammer, Reinhard et al. (2014) Rapid Computational Prediction of Thermostabilizing Mutations for G Protein-Coupled Receptors. J Chem Theory Comput 10:5149-5160
Lee, Sangbae; Bhattacharya, Supriyo; Grisshammer, Reinhard et al. (2014) Dynamic behavior of the active and inactive states of the adenosine A(2A) receptor. J Phys Chem B 118:3355-65
Xiao, Su; White, Jim F; Betenbaugh, Michael J et al. (2013) Transient and stable expression of the neurotensin receptor NTS1: a comparison of the baculovirus-insect cell and the T-REx-293 expression systems. PLoS One 8:e63679
Shibata, Yoko; Gvozdenovic-Jeremic, Jelena; Love, James et al. (2013) Optimising the combination of thermostabilising mutations in the neurotensin receptor for structure determination. Biochim Biophys Acta 1828:1293-301
Niesen, Michiel J M; Bhattacharya, Supriyo; Grisshammer, Reinhard et al. (2013) Thermostabilization of the β1-adrenergic receptor correlates with increased entropy of the inactive state. J Phys Chem B 117:7283-91

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