Neurotensin is a 13-residue peptide that acts as a neuromodulator of classical neurotransmitters such as dopamine and glutamate in the mammalian central nervous system, mainly by activating the G protein-coupled receptor (GPCR), neurotensin receptor 1 (NTS). Agonist binding to GPCRs shifts the conformational equilibrium of the transmembrane helices towards distinct, thermodynamically favorable conformations that favor effector protein interactions and promotes cell signaling. The introduction of site specific labels for NMR spectroscopy has proven useful for investigating this dynamic process, but the low expression levels and poor stability of GPCRs is a hindrance to solution NMR experiments. Several thermostabilized mutants of NTS have been engineered to circumvent this, with the crystal structures of four of these published. The conformational dynamics of NTS however, has not been thoroughly investigated with NMR. It is generally accepted that stabilized GPCRs exhibit attenuated signaling, thus we thoroughly characterized the signaling characteristics of several thermostabilized NTS variants to identify an optimal variant for protein NMR studies. A variant termed enNTS exhibited the best combination of signaling capability and stability upon solubilization with detergents. enNTS was subsequently labeled with CH-methionine in E. coli and purified to homogeneity in the absence of bound ligands. Using solution NMR spectroscopy we observed several well dispersed CH-methionine resonances, many of which exhibited chemical shift changes upon the addition of the high affinity agonist peptide, NT8-13. Thus, enNTS represents a novel tool for investigating ligand induced conformational changes in NTS to gain insights into the molecular mechanisms underlying neurotensin signaling.

By analyzing and simulating inactive conformations of the highly homologous dopamine D2 and D3 receptors (D2R and D3R), we find that eticlopride binds D2R in a pose very similar to that in the D3R/eticlopride structure but incompatible with the D2R/risperidone structure. In addition, risperidone occupies a sub-pocket near the Na+ binding site, whereas eticlopride does not. Based on these findings and our experimental results, we propose that the divergent receptor conformations stabilized by Na+-sensitive eticlopride and Na+-insensitive risperidone correspond to different degrees of inverse agonism. Moreover, our simulations reveal that the extracellular loops are highly dynamic, with spontaneous transitions of extracellular loop 2 from the helical conformation in the D2R/risperidone structure to an extended conformation similar to that in the D3R/eticlopride structure. Our results reveal previously unappreciated diversity and dynamics in the inactive conformations of D2R. These findings are critical for rational drug discovery, as limiting a virtual screen to a single conformation will miss relevant ligands.