Search Results (9)

Adolescent binge drinking has lasting behavioral consequences by disrupting the endocannabinoid system (ECS) and depleting brain omega-3. The natural accumulation of omega-3 fatty acids in cell membranes is crucial for maintaining the membrane structure, supporting interactions with the ECS, and restoring synaptic plasticity and cognition impaired by prenatal ethanol (EtOH) exposure. However, it remains unclear whether omega-3 supplementation can mitigate the long-term effects on the ECS, endocannabinoid-dependent synaptic plasticity, and cognition following adolescent binge drinking. Here, we demonstrated that omega-3 supplementation during EtOH withdrawal increases CB1 receptors in hippocampal presynaptic terminals of male mice, along with the recovery of receptor-stimulated [³⁵S]GTPγS binding to Gαi/o proteins. These changes are associated with long-term potentiation (LTP) at excitatory medial perforant path (MPP) synapses in the dentate gyrus (DG), which depends on anandamide (AEA), transient receptor potential vanilloid 1 (TRPV1), and N-methyl-D-aspartate (NMDA) receptors. Finally, omega-3 intake following binge drinking reduced the time and number of errors required to locate the escape box in the Barnes maze test. Collectively, these findings suggest that omega-3 supplementation restores Barnes maze performance to levels comparable to those of control mice after adolescent binge drinking. This recovery is likely mediated by modulation of the hippocampal ECS, enhancing endocannabinoid-dependent excitatory synaptic plasticity. Full article

Most α₂-AR agonists derived from dexmedetomidine have few structural differences between them and have no selectivity for α_2A/2B-AR or Gi/Gs, which can lead to side effects in drugs. To obtain novel and potent α_2A-AR agonists, we performed virtual screening for human α_2A-AR and α_2B-AR to find α_2A-AR agonists with higher selectivity. Compound P300–2342 and its three analogs significantly decreased the locomotor activity of mice (p < 0.05). Furthermore, P300–2342 and its three analogs inhibited the binding of [³H] Rauwolscine with IC₅₀ values of 7.72 ± 0.76 and 12.23 ± 0.11 μM, respectively, to α_2A-AR and α_2B-AR. In α_2A-AR-HEK293 cells, P300–2342 decreased forskolin-stimulated cAMP production without increasing cAMP production, which indicated that P300–2342 activated α_2A-AR with coupling to the Gαi/o pathway but without Gαs coupling. P300–2342 exhibited no agonist but slight antagonist activities in α_2B-AR. Similar results were obtained for the analogs of P300–2342. The docking results showed that P300–2342 formed π-hydrogen bonds with Y394, V114 in α_2A-AR, and V93 in α_2B-AR. Three analogs of P300–2342 formed several π-hydrogen bonds with V114, Y196, F390 in α_2A-AR, and V93 in α_2B-AR. We believe that these molecules can serve as leads for the further optimization of α_2A-AR agonists with potentially few side effects. Full article

Despite G protein-coupled receptors (GPCRs) being important theapeutic targets, the signaling properties of many GPCRs remain poorly characterized. GPCR activation primarily initiates heterotrimeric G protein signaling. To detect ligand-induced G protein activation, Bioluminescence Resonance Energy Transfer (BRET)-based biosensors were previously developed. Here, we designed a novel set of Nanoluciferase (NLuc) BRET-based biosensors (REGA-SIGN) that covers all Gα protein families (i.e., Gα_i/o, Gα_Ss/L, Gα_12/13 and Gα_q/15). REGA-SIGN uses NLuc as a bioluminescent donor and LSS-mKATE2, a red-shifted fluorophore, as an acceptor. Due to the enhanced spectral separation between donor and acceptor emission and the availability of a stable substrate for NLuc, this donor–acceptor pair enables sensitive kinetic assessment of G protein activity. After optimization, the NLuc integration sites into the Gα subunit largely corresponded with previously reported integration sites, except for Gα_Ss/L for which we describe an alternative NLuc insertion site. G protein rescue experiments validated the biological activity of these Gα donor proteins. Direct comparison between EGFP and LSS-mKATE2 as acceptor fluorophores revealed improved sensitivity for nearly all G protein subtypes when using the latter one. Hence, REGA-SIGN can be used as a panel of kinetic G protein biosensors with high sensitivity. Full article

Numerous studies have investigated the roles of the type 1 cannabinoid receptor (CB1) in glutamatergic and GABAergic neurons. Here, we used the cell-type-specific CB1 rescue model in mice to gain insight into the organizational principles of plasma membrane targeting and Gαi/o protein signalling of the CB1 receptor at excitatory and inhibitory terminals of the frontal cortex and hippocampus. By applying biochemical fractionation techniques and Western blot analyses to synaptosomal membranes, we explored the subsynaptic distribution (pre-, post-, and extra-synaptic) and CB1 receptor compartmentalization into lipid and non-lipid raft plasma membrane microdomains and the signalling properties. These data infer that the plasma membrane partitioning of the CB1 receptor and its functional coupling to Gαi/o proteins are not biased towards the cell type of CB1 receptor rescue. The extent of the canonical Gαi/o protein-dependent CB1 receptor signalling correlated with the abundance of CB1 receptor in the respective cell type (glutamatergic versus GABAergic neurons) both in frontal cortical and hippocampal synaptosomes. In summary, our results provide an updated view of the functional coupling of the CB1 receptor to Gαi/o proteins at excitatory and inhibitory terminals and substantiate the utility of the CB1 rescue model in studying endocannabinoid physiology at the subcellular level. Full article

G protein activation represents an early key event in the complex GPCR signal transduction process and is usually studied by label-dependent methods targeting specific molecular events. However, the constrained environment of such “invasive” techniques could interfere with biological processes. Although histamine receptors (HRs) represent (evolving) drug targets, their signal transduction is not fully understood. To address this issue, we established a non-invasive dynamic mass redistribution (DMR) assay for the human H_1–4Rs expressed in HEK cells, showing excellent signal-to-background ratios above 100 for histamine (HIS) and higher than 24 for inverse agonists with pEC₅₀ values consistent with literature. Taking advantage of the integrative nature of the DMR assay, the involvement of endogenous Gα_q/11, Gα_s, Gα_12/13 and Gβγ proteins was explored, pursuing a two-pronged approach, namely that of classical pharmacology (G protein modulators) and that of molecular biology (Gα knock-out HEK cells). We showed that signal transduction of hH_1–4Rs occurred mainly, but not exclusively, via their canonical Gα proteins. For example, in addition to Gα_i/o, the Gα_q/11 protein was proven to contribute to the DMR response of hH_3,4Rs. Moreover, the Gα_12/13 was identified to be involved in the hH₂R mediated signaling pathway. These results are considered as a basis for future investigations on the (patho)physiological role and the pharmacological potential of H_1–4Rs. Full article

The regulator of G-protein signaling 5 (RGS5) acts as an inhibitor of Gα_q/11 and Gα_i/o activity in vascular smooth muscle cells (VSMCs), which regulate arterial tone and blood pressure. While RGS5 has been described as a crucial determinant regulating the VSMC responses during various vascular remodeling processes, its regulatory features in resting VSMCs and its impact on their phenotype are still under debate and were subject of this study. While Rgs5 shows a variable expression in mouse arteries, neither global nor SMC-specific genetic ablation of Rgs5 affected the baseline blood pressure yet elevated the phosphorylation level of the MAP kinase ERK1/2. Comparable results were obtained with 3D cultured resting VSMCs. In contrast, overexpression of RGS5 in 2D-cultured proliferating VSMCs promoted their resting state as evidenced by microarray-based expression profiling and attenuated the activity of Akt- and MAP kinase-related signaling cascades. Moreover, RGS5 overexpression attenuated ERK1/2 phosphorylation, VSMC proliferation, and migration, which was mimicked by selectively inhibiting Gα_i/o but not Gα_q/11 activity. Collectively, the heterogeneous expression of Rgs5 suggests arterial blood vessel type-specific functions in mouse VSMCs. This comprises inhibition of acute agonist-induced Gα_q/11/calcium release as well as the support of a resting VSMC phenotype with low ERK1/2 activity by suppressing the activity of Gα_i/o. Full article

The neurotransmitter molecule acetylcholine is capable of activating five muscarinic acetylcholine receptors, M1 through M5, which belong to the superfamily of G-protein-coupled receptors (GPCRs). These five receptors share high sequence and structure homology; however, the M1, M3, and M5 receptor subtypes signal preferentially through the Gαq/11 subset of G proteins, whereas the M2 and M4 receptor subtypes signal through the Gαi/o subset of G proteins, resulting in very different intracellular signaling cascades and physiological effects. The structural basis for this innate ability of the M1/M3/M5 set of receptors and the highly homologous M2/M4 set of receptors to couple to different G proteins is poorly understood. In this study, we used molecular dynamics (MD) simulations coupled with thermodynamic analyses of M1 and M2 receptors coupled to both Gαi and Gαq to understand the structural basis of the M1 receptor’s preference for the Gαq protein and the M2 receptor’s preference for the Gαi protein. The MD studies showed that the M1 and M2 receptors can couple to both Gα proteins such that the M1 receptor engages with the two Gα proteins in slightly different orientations and the M2 receptor engages with the two Gα proteins in the same orientation. Thermodynamic studies of the free energy of binding of the receptors to the Gα proteins showed that the M1 and M2 receptors bind more strongly to their cognate Gα proteins compared to their non-cognate ones, which is in line with previous experimental studies on the M3 receptor. A detailed analysis of receptor–G protein interactions showed some cognate-complex-specific interactions for the M2:Gαi complex; however, G protein selectivity determinants are spread over a large overlapping subset of residues. Conserved interaction between transmembrane helices 5 and 6 far away from the G-protein-binding receptor interface was found only in the two cognate complexes and not in the non-cognate complexes. An analysis of residues implicated previously in G protein selectivity, in light of the cognate and non-cognate structures, shaded a more nuanced role of those residues in affecting G protein selectivity. The simulation of both cognate and non-cognate receptor–G protein complexes fills a structural gap due to difficulties in determining non-cognate complex structures and provides an enhanced framework to probe the mechanisms of G protein selectivity exhibited by most GPCRs. Full article

A hallmark of G-protein-coupled receptors (GPCRs) is their ability to recognize and respond to chemically diverse ligands. Lysophospholipids constitute a relatively recent addition to these ligands and carry out their biological functions by activating G-proteins coupled to a large family of cell-surface receptors. This review aims to highlight salient features of cell signaling by one class of these receptors, known as lysophosphatidic acid (LPA) receptors, in the context of phosphatidylinositol 3-kinase (PI3K)–AKT pathway activation. LPA moieties efficiently activate AKT phosphorylation and activation in a multitude of cell types. The interplay between LPA, its receptors, the associated Gαi/o subunits, PI3K and AKT contributes to the regulation of cell survival, migration, proliferation and confers chemotherapy-resistance in certain cancers. However, detailed information on the regulation of PI3K–AKT signals induced by LPA receptors is missing from the literature. Here, some urgent issues for investigation are highlighted. Full article

Pertussis toxin (PTX) is a typical A-B toxin. The A-protomer (S1 subunit) exhibits ADP-ribosyltransferase activity. The B-oligomer consists of four subunits (S2 to S5) and binds extracellular molecules that allow the toxin to enter the cells. The A-protomer ADP-ribosylates the α subunits of heterotrimeric G_i/o proteins, resulting in the receptors being uncoupled from the G_i/o proteins. The B-oligomer binds proteins expressed on the cell surface, such as Toll-like receptor 4, and activates an intracellular signal transduction cascade. Thus, PTX modifies cellular responses by at least two different signaling pathways; ADP-ribosylation of the Gα_i/o proteins by the A-protomer (G_i/o protein-dependent action) and the interaction of the B-oligomer with cell surface proteins (G_i/o protein-independent action). Full article