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 five GPCRs (bovine and squid rhodopsin, beta-1 and beta-2 adrenergic receptors, adenosine A2a receptor) 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. All five receptors were crystallized with an inverse agonist or antagonist bound to promote the inactive state. The transmembrane cores look similar but not identical in all five 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. 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 opsin structures thought to represent an active state conformation.
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| Grisshammer, Reinhard (2017) New approaches towards the understanding of integral membrane proteins: A structural perspective on G protein-coupled receptors. Protein Sci 26:1493-1504 |
| Lee, Sangbae; Mao, Allen; Bhattacharya, Supriyo et al. (2016) How Do Short Chain Nonionic Detergents Destabilize G-Protein-Coupled Receptors? J Am Chem Soc 138:15425-15433 |
| Vaidehi, Nagarajan; Grisshammer, Reinhard; Tate, Christopher G (2016) How Can Mutations Thermostabilize G-Protein-Coupled Receptors? Trends Pharmacol Sci 37:37-46 |
| Krumm, Brian E; Lee, Sangbae; Bhattacharya, Supriyo et al. (2016) Structure and dynamics of a constitutively active neurotensin receptor. Sci Rep 6:38564 |
| Krumm, Brian E; Grisshammer, Reinhard (2015) Peptide ligand recognition by G protein-coupled receptors. Front Pharmacol 6:48 |
| 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 |
| Lee, Sangbae; Bhattacharya, Supriyo; Tate, Christopher G et al. (2015) Structural dynamics and thermostabilization of neurotensin receptor 1. J Phys Chem B 119:4917-28 |
| Krumm, Brian E; White, Jim F; Shah, Priyanka et al. (2015) Structural prerequisites for G-protein activation by the neurotensin receptor. Nat Commun 6:7895 |
| 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 |
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