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.

Partial agonists of the dopamine D 2 receptor 18 (D 2 R) have been developed to treat the symptoms of schizophrenia without causing the side effects elicited by antagonists. The receptor-ligand interactions that determine the intrinsic efficacy of such drugs, however, are poorly understood. Aripiprazole has an extended structure comprising a phenylpiperazine primary pharmacophore and a 1,2,3,4-tetrahydroquinolin-2-one secondary pharmacophore. We combined site-directed mutagenesis, analytical pharmacology, ligand fragments, and molecular dynamics simulations to *

The dopamine D2 receptor (D2R) is the target of drugs used to treat the symptoms of Parkinson’s disease and schizophrenia. The D2R is regulated through its interaction with and phosphorylation by G protein receptor kinases (GRKs) and interaction with arrestins. More recently, D2R arrestin-mediated signaling has been shown to have distinct physiological functions to those of G protein signalling. Relatively little is known regarding the patterns of D2R phosphorylation that might control these processes. We aimed to generate antibodies specific for intracellular D2R phosphorylation sites to facilitate the investigation of these mechanisms. We synthesised double phosphorylated peptides corresponding to regions within intracellular loop 3 of the hD2R and used them to raise phosphosite-specific antibodies to capture a broad screen of GRK-mediated phosphorylation. We identify an antibody specific to a GRK2/3 phosphorylation site in intracellular loop 3 of the D2R. We compared measurements of D2R phosphorylation with other measurements of D2R signalling to profile selected D2R agonists including previously described biased agonists. These studies demonstrate the utility of novel phosphosite-specific antibodies to investigate D2R regulation and signalling.

Idiopathic pulmonary fibrosis is a chronic and progressive fibrotic lung disease, and current treatments are limited by their side effects. Proliferation of human lung fibroblasts in the pulmonary interstitial tissue is a hallmark of this disease and is driven by prolonged ERK signalling in the nucleus in response to growth factors such as platelet-derived growth factor (PDGF). Agents that increase cAMP have been suggested as alternative therapies, as this second messenger can inhibit the ERK cascade. We previously examined a panel of eight Gαs-cAMP-coupled G protein-coupled receptors (GPCRs) endogenously expressed in human lung fibroblasts. Although the cAMP response was important for the anti-fibrotic effects of GPCR agonists, the magnitude of the acute cAMP response was not predictive of anti-fibrotic efficacy. Here we examined the reason for this apparent disconnect by stimulating the Gαs-coupled prostacyclin receptor and measuring downstream signalling at a sub-cellular level. MRE-269 and treprostinil caused sustained cAMP signalling in the nucleus and complete inhibition of PDGF-induced nuclear ERK and fibroblast proliferation. In contrast, iloprost caused a transient increase in nuclear cAMP, there was no effect of iloprost on PDGF-induced ERK in the nucleus, and this agonist was much less effective at reversing PDGF-induced proliferation. This suggests that sustained elevation of cAMP in the nucleus is necessary for efficient inhibition of PDGF-induced nuclear ERK and fibroblast proliferation. This is an important first step towards understanding of the signalling events that drive GPCR inhibition of fibrosis.

Heterotrimeric G proteins are the main signalling effectors for G protein-coupled receptors. Understanding the distinct functions of different G proteins is key to understanding how their signalling modulates physiological responses. Pertussis toxin, a bacterial AB5 toxin, inhibits Gαi/o G proteins and has proven useful for interrogating inhibitory G protein signalling. Pertussis toxin, however, does not inhibit one member of the inhibitory G protein family, Gαz. The role of Gαz signalling has been neglected largely due to a lack of inhibitors. Recently, the identification of another Pertussis-like AB5 toxin was described. Here we show that this toxin, that we call OZITX, specifically inhibits Gαi/o and Gαz G proteins and that expression of the catalytic S1 subunit is sufficient for this inhibition. We identify mutations that render Gα subunits insensitive to the toxin that, in combination with the toxin, can be used to interrogate the signalling of each inhibitory Gα G protein.

P-Rex (PI(3,4,5)P3-dependent Rac exchanger) guanine nucleotide exchange factors potently activate Rho GTPases. P-Rex guanine nucleotide exchange factors are autoinhibited, synergistically activated by Gβγ and PI(3,4,5)P3 binding and dysregulated in cancer. Here, we use X-ray crystallography, cryogenic electron microscopy and crosslinking mass spectrometry to determine the structural basis of human P-Rex1 autoinhibition. P-Rex1 has a bipartite structure of N- and C-terminal modules connected by a C-terminal four-helix bundle that binds the N-terminal Pleckstrin homology (PH) domain. In the N-terminal module, the Dbl homology (DH) domain catalytic surface is occluded by the compact arrangement of the DH-PH-DEP1 domains. Structural analysis reveals a remarkable conformational transition to release autoinhibition, requiring a 126° opening of the DH domain hinge helix. The off-axis position of Gβγ and PI(3,4,5)P3 binding sites further suggests a counter-rotation of the P-Rex1 halves by 90° facilitates PH domain uncoupling from the four-helix bundle, releasing the autoinhibited DH domain to drive Rho GTPase signaling.

Background-The dopamine D2 receptor (D2R) is a therapeutic target for the treatment of several neuropsychiatric disorders. The D2R can couple to all members of the heterotrimeric Galpha-i/o/z protein family but the physiological importance of coupling to distinct G protein subtypes remains unclear. It has been demonstrated that slow-dissociating agonists display time-dependent biased agonism between endpoints at different levels in the D2R signalling cascade(1). The aim of this study was to explore biased agonism at the level of coupling to different G proteins and how this might be influenced by agonist binding kinetics. Methods-D2R expressing FLPIN HEK293 cells were stimulated by a panel of agonists with varied intrinsic efficacy and binding kinetics. Galpha-i/o/z activation was measured in live cells by BRET-based biosensors which detect dissociation of the Gbeta-gamma subunit upon activation of the G protein(2). Biased agonism was quantified by analysing concentration response curves of agonists at different timepoints using an operational model of agonism(3). Results-The D2R showed a coupling preference for GoA/B and Gz over Gi1/2/3 as indicated by the relative potency of high efficacy agonists. Time-dependent biased agonism was observed for some agonists relative to ropinirole primarily between Gi1/2/3 and Gz activation. Interestingly, all agonists, independent of their receptor binding kinetics, displayed a large increase in potency over time for Gz activation while the potency of agonists at all other G proteins either did not change, or changed in manner consistent with their dissociation rate from the D2R. We provide evidence that the large increase in potency over time at Gz is dependent on a slow rate of GTP hydrolysis. Conclusion-We observed no correlation between association or dissociation rate of selected agonists and their efficacy to activate different G proteins. Subtle but statistically significant bias was detected between Gz and Gi1/2/3 activation for some agonists, and this pattern of bias changes over time. Finally, we have provided evidence that rates of G protein activation/deactivation in combination with ligand binding kinetics may underlie some observations of biased agonism.