The Effect of Ligands and Transducers on the Neurotensin Recep-tor 1 (NTS1) Conformational Ensemble

Using a discrete, intracellular 19F-NMR probe on Neurotensin receptor 1 (NTS1) transmembrane helix (TM) 6, we aim to understand how ligands and transducers modulate the receptors structural ensemble in solution. For apo NTS1, 19F-NMR spectra reveal an ensemble of at least three states (one inactive and two active-like) in equilibrium that exchange on the ms-s timescale. Dynamic NMR experiments reveal that these substates follow a linear three-site exchange process that is both thermodynamically and kinetically remodeled by orthosteric ligands. As previously observed in other GPCRs, the full agonist is insufficient to completely stabilize the active state. Receptor coupling to b-arrestin-1 or the C-terminal helix of Gaq, which comprises >60% of the GPCR/G protein interface surface area, abolishes the inactive substate. But whereas b-arrestin-1 selects for preexisting active-like substates, the Gaq peptide induces two new substates. Both transducer molecules promote substantial line-broadening of active states suggesting contributions from additional us-ms exchange processes. Together, our study suggests i) the NTS1 allosteric activation mechanism is alternatively dominated by induced fit or conformational selection depending on the coupled transducer, and ii) the available static structures do not represent the entire conformational ensemble observed in solution.

Expression of mini-G proteins specifically halt cognate GPCR trafficking and intracellular signalling

Mini-G proteins are engineered thermostable variants of Gα subunits designed to specifically stabilise G protein-coupled receptors (GPCRs) in their active conformation for structural analyses. Due to their smaller size and ease of use, they have become popular tools in recent years to assess specific GPCR behaviours in cells, both as reporters of receptor coupling to each G protein subtype and for in-cell assays designed to quantify compartmentalised receptor signalling from a range of subcellular locations. Here, we describe a previously unappreciated consequence of the co-expression of mini-G proteins with their cognate GPCRs, namely a profound disruption in GPCR trafficking and intracellular signalling caused by the co-expression of the specific mini-G subtype coupled to the affected receptor. We studied the Gαs-coupled pancreatic beta cell class B GPCR glucagon-like peptide-1 receptor (GLP-1R) as a model to describe in detail the molecular consequences derived from this effect, including a complete halt in β-arrestin-2 recruitment and receptor internalisation, despite near-normal levels of receptor GRK2 recruitment and lipid nanodomain segregation, as well as the disruption of endosomal GLP-1R signalling by mini-Gs co-expression. We also extend our analysis to a range of other prototypical GPCRs covering the spectrum of Gα subtype coupling preferences, to unveil a widely conserved phenomenon of GPCR internalisation blockage by specific mini-G proteins coupled to a particular receptor. Our results have important implications for the design of methods to assess intracellular GPCR signalling. We also present an alternative adapted bystander intracellular signalling assay for the GLP-1R in which we substitute the mini-Gs by a nanobody, Nb37, with specificity for active Gαs:GPCR complexes and no deleterious effect on the capacity for GLP-1R internalisation.

Decoupling the ATP-binding Domain in the CheA Kinase by Increasing Linker Flexibility Dramatically Alters Kinase Activity

In bacterial chemotaxis chemoreceptors regulate the cytosolic dimeric histidine kinase CheA. To test the role that interdomain linkers play in CheA regulation the linkers that connect the P4 kinase domain to the P3 dimerization domain (L3) and the P5 regulatory domain (L4) were extended and altered in variants of Thermotoga maritima (Tm) CheA. Flexible extensions of the L3 and L4 linkers in CheA-LV1 (linker variant 1) allow for a well-folded kinase domain that retains WT-like binding affinities for nucleotide and normal interactions with the receptor-coupling protein CheW. However, CheA-LV1 autophosphorylation activity registers ~50-fold lower compared to wild-type. Formation of the CheA-LV1 / CheA WT heterodimer fails to rescue CheA-LV1 autophosphorylation and instead reduces the activity of the WT subunit. Neither CheA WT nor CheA-LV1 can phosphorylate P1 in a CheA dimer that contains a single P4 domain. Rescue of autophosphorylation activity in variants with a poly-alanine L3 or an L3 that maintains a heptad repeat suggest that positioning and conformational transitions of P4 depend on L3 assuming helical structure. Pulse dipolar ESR measurements indicate that the CheA-LV1 P4 domains are in close proximity whereas broader distributions in other variants correlate with increased activity. CheA-LV1 has a substantially larger hydrodynamic radius than does CheA WT by SAXS, despite the P4 domains assuming a closed, inhibited conformation. These results explain negative cooperativity in CheA nucleotide binding, demonstrate coupling between P4 disposition and P1 / P2 dynamics and underscore the importance of P4-P4 interactions and an L3 a- helix in CheA activity and regulation.

Characterization of dopamine D2 receptor coupling to G proteins in postmortem brain of subjects with schizophrenia

Background Alterations of dopamine D1 (D1R) and D2 receptor (D2R) are proposed in schizophrenia but brain neuroimaging and postmortem studies have shown controversial results in relation to D1R and D2R density. Besides, scarce information on the functionality of brain D1R and D2R is available. The present study characterized G-protein activation by D1R and D2R agonists in postmortem human brain. Furthermore, D2R functional status was compared between schizophrenia and control subjects. Methods G-protein receptor coupling was assessed in control caudate nucleus and frontal cortex by [35S]GTPγS-binding stimulation induced by increasing concentrations (10–10–10–3 M) of dopamine, and the selective dopaminergic agonists SKF38393 (D1R) and NPA (D2R). Concentration–response curves to NPA stimulation of [35S]GTPγS binding were analyzed in antipsychotic-free (n = 10) and antipsychotic-treated (n = 7) schizophrenia subjects and matched controls (n = 17). Results In caudate, [35S]GTPγS-binding responses to agonists were compatible with the existence of functional D2R. In contrast, stimulations in cortex showed responses that did not correspond to D1R or D2R. [35S]GTPγS-binding activation by NPA in caudate displayed biphasic curves with similar profile in schizophrenia (EC50H = 7.94 nM; EC50L = 7.08 μM) and control (EC50H = 7.24 nM; EC50L = 15.14 μM) subjects. The presence or absence of antipsychotic medication did not influence the pharmacological parameters. Conclusions Feasibility of functional evaluation of dopamine receptors in postmortem human brain by conventional [35S]GTPγS-binding assays appears to be restricted to signalling through inhibitory Gi/o proteins. These findings provide functional information about brain D2R status in subjects with schizophrenia and do not support the existence of D2R supersensitive in this mental disorder.

Competitive binding of STATs to receptor phospho-Tyr motifs accounts for altered cytokine responses

Cytokines elicit pleiotropic and non-redundant activities despite strong overlap in their usage of receptors, JAKs and STATs molecules. We use IL-6 and IL-27 to ask how two cytokines activating the same signaling pathway have different biological roles. We found that IL-27 induces more sustained STAT1 phosphorylation than IL-6, with the two cytokines inducing comparable levels of STAT3 phosphorylation. Mathematical and statistical modelling of IL-6 and IL-27 signaling identified STAT3 binding to GP130, and STAT1 binding to IL-27Rα, as the main dynamical processes contributing to sustained pSTAT1 levels by IL-27. Mutation of Tyr613 on IL-27Rα decreased IL-27-induced STAT1 phosphorylation by 80% but had limited effect on STAT3 phosphorylation. Strong receptor/STAT coupling by IL-27 initiated a unique gene expression program, which required sustained STAT1 phosphorylation and IRF1 expression and was enriched in classical Interferon Stimulated Genes. Interestingly, the STAT/receptor coupling exhibited by IL-6/IL-27 was altered in patients with systemic lupus erythematosus (SLE). IL-6/IL-27 induced a more potent STAT1 activation in SLE patients than in healthy controls, which correlated with higher STAT1 expression in these patients. Partial inhibition of JAK activation by sub-saturating doses of Tofacitinib specifically lowered the levels of STAT1 activation by IL-6. Our data show that receptor and STATs concentrations critically contribute to shape cytokine responses and generate functional pleiotropy in health and disease.

Conformational specificity of opioid receptors is determined by subcellular location irrespective of agonist

The prevailing model for the variety in drug responses is that they stabilize distinct active states of their G protein-coupled receptor (GPCR) targets, allowing coupling to different effectors. However, whether the same ligand can produce different GPCR active states based on the environment of receptors in cells is a fundamental unanswered question. Here we address this question using live cell imaging of conformational biosensors that read out distinct active conformations of the δ-opioid receptor (DOR), a physiologically relevant GPCR localized to Golgi and the surface in neurons. We show that, although Golgi and surface pools of DOR regulated cAMP, the two pools engaged distinct conformational biosensors in response to the same ligand. Further, DOR recruited arrestin on the plasma membrane but not the Golgi. Our results suggest that the same agonist drives different conformations of a GPCR at different locations, allowing receptor coupling to distinct effectors at different locations.

Receptor-Arrestin Interactions: The GPCR Perspective

Arrestins are a small family of four proteins in most vertebrates that bind hundreds of different G protein-coupled receptors (GPCRs). Arrestin binding to a GPCR has at least three functions: precluding further receptor coupling to G proteins, facilitating receptor internalization, and initiating distinct arrestin-mediated signaling. The molecular mechanism of arrestin–GPCR interactions has been extensively studied and discussed from the “arrestin perspective”, focusing on the roles of arrestin elements in receptor binding. Here, we discuss this phenomenon from the “receptor perspective”, focusing on the receptor elements involved in arrestin binding and emphasizing existing gaps in our knowledge that need to be filled. It is vitally important to understand the role of receptor elements in arrestin activation and how the interaction of each of these elements with arrestin contributes to the latter’s transition to the high-affinity binding state. A more precise knowledge of the molecular mechanisms of arrestin activation is needed to enable the construction of arrestin mutants with desired functional characteristics.

Mu-opioids suppress GABAergic synaptic transmission onto orbitofrontal cortex pyramidal neurons with subregional selectivity

The orbitofrontal cortex (OFC) plays a critical role in evaluating outcomes in a changing environment. Administering opioids to the OFC can alter the hedonic reaction to food rewards and increase their consumption in a subregion specific manner. However, it is unknown how mu-opioid signalling influences synaptic transmission in the OFC. Thus, we investigated the cellular actions of mu-opioids within distinct subregions of the OFC. Using in-vitro patch clamp electrophysiology in brain slices containing the OFC, we found that the mu-opioid agonist, DAMGO produced a concentration-dependant inhibition of GABAergic synaptic transmission onto medial OFC (mOFC), but not lateral OFC (lOFC) neurons. This effect was mediated by presynaptic mu-opioid receptor activation of local parvalbumin (PV+)-expressing interneurons. The DAMGO-induced suppression of inhibition was long-lasting and not reversed upon washout of DAMGO, or by application of the mu-opioid receptor antagonist, CTAP, suggesting an inhibitory long-term depression (iLTD) induced by an exogenous mu-opioid. We show that LTD at inhibitory synapses is dependent on downstream cAMP/PKA signaling, which differs between the mOFC and lOFC. Finally, we demonstrate that endogenous opioid release triggered via moderate physiological stimulation can induce LTD. Taken together, these results suggest that presynaptic mu-opioid stimulation of local PV+ interneurons induces a long-lasting suppression of GABAergic synaptic transmission, which depends on subregional differences in mu-opioid receptor coupling to the downstream cAMP/PKA intracellular cascade. These findings provide mechanistic insight into the opposing functional effects produced by mu-opioids within the OFC.Significance StatementConsidering that both the OFC and the opioid system regulate reward, motivation, and food intake; understanding the role of opioid signaling within the OFC is fundamental for a mechanistic understanding of the sequelae for several psychiatric disorders. This study makes several novel observations. First, mu-opioids induce a long-lasting suppression of inhibitory synaptic transmission onto OFC pyramidal neurons in a regionally selective manner. Secondly, mu-opioids recruit PV+ inputs to suppress inhibitory synaptic transmission in the mOFC. Thirdly, the regional selectivity of mu-opioid action of endogenous opioids is due to the efficacy of mu-opioid receptor coupling to the downstream cAMP/PKA intracellular cascades. These experiments are the first to reveal a cellular mechanism of opioid action within the OFC.

Further characterization of the synergistic activation mechanism of cationic channels by M2 and M3 muscarinic receptors in mouse intestinal smooth muscle cells

In mouse ileal myocytes, muscarinic receptor-mediated cationic current ( mIcat) occurs mainly through synergism of M2 and M3 subtypes involving Gi/o-type GTP-binding proteins and phospholipase C (PLC). We have further studied the M2/M3 synergistic pathway. Carbachol-induced mIcat was markedly depressed by YM-254890, a Gq/11 protein inhibitor. However, the mIcat was unaffected by heparin, calphostin C, or chelerythrine, suggesting that mIcat activation does not involve signaling molecules downstream of phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown. M2-knockout (KO) mice displayed a reduced mIcat (~10% of wild-type mIcat) because of the lack of M2-Gi/o signaling. The impaired mIcat was insensitive to neuropeptide Y possessing a Gi/o-stimulating activity. M3-KO mice also displayed a reduced mIcat (~6% of wild-type mIcat) because of the lack of M3-Gq/11 signaling, and the mIcat was insensitive to prostaglandin F2α possessing a Gq/11-stimulating activity. These results suggest the importance of Gq/11/PLC-hydrolyzed PIP2 breakdown itself in mIcat activation and also support the idea that the M2/M3 synergistic pathway represents a signaling complex consisting of M2-Gi/o and M3-Gq/11-PLC systems in which both G proteins are special for this pathway but not general in receptor coupling.