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Adenosine is a constituent of many molecules of life; increased free extracellular adenosine indicates cell damage or metabolic stress. The importance of adenosine signaling in basal physiology, as opposed to adaptive responses to danger/damage situations, is unclear. We generated mice lacking all four adenosine receptors (ARs), Adora1-/-;Adora2a-/-;Adora2b-/-;Adora3-/- (quad knockout [QKO]), to enable investigation of the AR dependence of physiologic processes, focusing on body temperature. The QKO mice demonstrate that ARs are not required for growth, metabolism, breeding, and body temperature regulation (diurnal variation, response to stress, and torpor). However, the mice showed decreased survival starting at about 15 weeks of age. While adenosine agonists cause profound hypothermia via each AR, adenosine did not cause hypothermia (or bradycardia or hypotension) in QKO mice, indicating that AR-independent signals do not contribute to adenosine-induced hypothermia. The hypothermia elicited by adenosine kinase inhibition (with A134974), inosine, or uridine also required ARs, as each was abolished in the QKO mice. The proposed mechanism for uridine-induced hypothermia is inhibition of adenosine transport by uridine, increasing local extracellular adenosine levels. In contrast, adenosine 5'-monophosphate (AMP)-induced hypothermia was attenuated in QKO mice, demonstrating roles for both AR-dependent and AR-independent mechanisms in this process. The physiology of the QKO mice appears to be the sum of the individual knockout mice, without clear evidence for synergy, indicating that the actions of the four ARs are generally complementary. The phenotype of the QKO mice suggests that, while extracellular adenosine is a signal of stress, damage, and/or danger, it is less important for baseline regulation of body temperature.

The neurotransmitter dopamine has central roles in mood, appetite, arousal and movement 1 . Despite its importance in brain physiology and function, and as a target for illicit and therapeutic drugs, the human dopamine transporter (hDAT) and mechanisms by which it is inhibited by small molecules and Zn 2+ are without a high-resolution structural context. Here we determine the structure of hDAT in a tripartite complex with the competitive inhibitor and cocaine analogue, (–)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane 2 (β-CFT), the non-competitive inhibitor MRS7292 3 and Zn 2 + (ref. 4 ). We show how β-CFT occupies the central site, approximately halfway across the membrane, stabilizing the transporter in an outward-open conformation. MRS7292 binds to a structurally uncharacterized allosteric site, adjacent to the extracellular vestibule, sequestered underneath the extracellular loop 4 (EL4) and adjacent to transmembrane helix 1b (TM1b), acting as a wedge, precluding movement of TM1b and closure of the extracellular gate. A Zn 2+ ion further stabilizes the outward-facing conformation by coupling EL4 to EL2, TM7 and TM8, thus providing specific insights into how Zn 2+ restrains the movement of EL4 relative to EL2 and inhibits transport activity. Cryo-electron microscopy structure of the human dopamine transporter in complex with multiple inhibitors illuminates mechanisms of allosteric inhibition.

G protein-coupled receptors (GPCRs) are embedded in phospholipids that strongly influence drug-stimulated signaling. Anionic lipids are particularly important for GPCR signaling complex formation, but a mechanism for this role is not understood. Using NMR spectroscopy, we explore the impact of anionic lipids on the function-related conformational equilibria of the human A 2A adenosine receptor (A 2A AR) in bilayers containing defined mixtures of zwitterionic and anionic phospholipids. Anionic lipids prime the receptor to form complexes with G proteins through a conformational selection process. Without anionic lipids, signaling complex formation proceeds through a less favorable induced fit mechanism. In computational models, anionic lipids mimic interactions between a G protein and positively charged residues in A 2A AR at the receptor intracellular surface, stabilizing a pre-activated receptor conformation. Replacing these residues strikingly alters the receptor response to anionic lipids in experiments. High sequence conservation of the same residues among all GPCRs supports a general role for lipid-receptor charge complementarity in signaling. In cell membranes, lipids are ubiquitous regulators of protein function. Here, Thakur et al. observe anionic phospholipids impact the conformational dynamics of a class A human GPCR.

Adenosine is an endogenous modulator exerting its functions through the activation of four adenosine receptor (AR) subtypes, termed A1, A2A, A2B and A3, which belong to the G protein-coupled receptor (GPCR) superfamily. The human A3AR (hA3AR) subtype is implicated in several cytoprotective functions. Therefore, hA3AR modulators, and in particular agonists, are sought for their potential application as anti-inflammatory, anticancer, and cardioprotective agents. Structure-based molecular modeling techniques have been applied over the years to rationalize the structure–activity relationships (SARs) of newly emerged A3AR ligands, guide the subsequent lead optimization, and interpret site-directed mutagenesis (SDM) data from a molecular perspective. In this review, we showcase selected modeling-based and guided strategies that were applied to elucidate the binding of agonists to the A3AR and discuss the challenges associated with an accurate prediction of the receptor extracellular vestibule through homology modeling from the available X-ray templates.

There are four subtypes of adenosine receptors (ARs), named A1, A2A, A2B and A3, all of which are G protein-coupled receptors (GPCRs). Locally produced adenosine is a suppressant in anti-tumor immune surveillance. The A2BAR, coupled to both Gαs and Gαi G proteins, is one of the several GPCRs that are expressed in a significantly higher level in certain cancer tissues, in comparison to adjacent normal tissues. There is growing evidence that the A2BAR plays an important role in tumor cell proliferation, angiogenesis, metastasis, and immune suppression. Thus, A2BAR antagonists are novel, potentially attractive anticancer agents. Several antagonists targeting A2BAR are currently in clinical trials for various types of cancers. In this review, we first describe the signaling, agonists, and antagonists of the A2BAR. We further discuss the role of the A2BAR in the progression of various cancers, and the rationale of using A2BAR antagonists in cancer therapy.

Adenosine receptors (ARs) function in the body's response to conditions of pathology and stress associated with a functional imbalance, such as in the supply and demand of energy/oxygen/nutrients. Extracellular adenosine concentrations vary widely to raise or lower the basal activation of four subtypes of ARs. Endogenous adenosine can correct an energy imbalance during hypoxia and other stress, for example, by slowing the heart rate by A AR activation or increasing the blood supply to heart muscle by the A AR. Moreover, exogenous AR agonists, antagonists, or allosteric modulators can be applied for therapeutic benefit, and medicinal chemists working toward that goal have reported thousands of such agents. Thus, numerous clinical trials have ensued, using promising agents to modulate adenosinergic signaling, most of which have not succeeded. Currently, short-acting, parenteral agonists, adenosine and Regadenoson, are the only AR agonists approved for human use. However, new concepts and compounds are currently being developed and applied toward preclinical and clinical evaluation, and initial results are encouraging. This review focuses on key compounds as AR agonists and positive allosteric modulators (PAMs) for disease treatment or diagnosis. AR agonists for treating inflammation, pain, cancer, non-alcoholic steatohepatitis, angina, sickle cell disease, ischemic conditions and diabetes have been under development. Multiple clinical trials with two A AR agonists are ongoing.

Uncontrolled inflammation is one of the leading causes of kidney failure. Pro-inflammatory responses can occur in the absence of infection, a process called sterile inflammation. Here we show that the purinergic receptor P2Y14 (GPR105) is specifically and highly expressed in collecting duct intercalated cells (ICs) and mediates sterile inflammation in the kidney. P2Y14 is activated by UDP-glucose, a damage-associated molecular pattern molecule (DAMP) released by injured cells. We found that UDP-glucose increases pro-inflammatory chemokine expression in ICs as well as MDCK-C11 cells, and UDP-glucose activates the MEK1/2-ERK1/2 pathway in MDCK-C11 cells. These effects were prevented following inhibition of P2Y14 with the small molecule PPTN. Tail vein injection of mice with UDP-glucose induced the recruitment of neutrophils to the renal medulla. This study identifies ICs as novel sensors, mediators and effectors of inflammation in the kidney via P2Y14.

In response to adenosine 5′-diphosphate, the P2Y 1 receptor (P2Y 1 R) facilitates platelet aggregation, and thus serves as an important antithrombotic drug target. Here we report the crystal structures of the human P2Y 1 R in complex with a nucleotide antagonist MRS2500 at 2.7 Å resolution, and with a non-nucleotide antagonist BPTU at 2.2 Å resolution. The structures reveal two distinct ligand-binding sites, providing atomic details of P2Y 1 R's unique ligand-binding modes. MRS2500 recognizes a binding site within the seven transmembrane bundle of P2Y 1 R, which is different in shape and location from the nucleotide binding site in the previously determined structure of P2Y 12 R, representative of another P2YR subfamily. BPTU binds to an allosteric pocket on the external receptor interface with the lipid bilayer, making it the first structurally characterized selective G-protein-coupled receptor (GPCR) ligand located entirely outside of the helical bundle. These high-resolution insights into P2Y 1 R should enable discovery of new orthosteric and allosteric antithrombotic drugs with reduced adverse effects. Two X-ray crystal structures are presented of the human P2Y 1 G-protein-coupled receptor, which is an important target for anti-thrombotic drugs; the structures unexpectedly reveal two ligand-binding sites. Human P2Y 1 receptor structure In this manuscript, Beili Wu and colleagues report X-ray crystal structures of the human P2Y 1 receptor, a G-protein-coupled receptor (GPCR). Like the P2Y 12 receptor, this membrane protein regulates platelet activation and thrombus formation. Both GPCRs are important targets for the development of new antithrombotic drugs. Comparison of this structure to a previously published P2Y 12 receptor structure indicates that the orthosteric ligand-binding sites of these two GPRCs are quite different: the binding site of the P2Y 1 receptor is much shallower than the binding site of the P2Y 12 receptor. The authors solved structures of the protein in the presence of the nucleotide antagonist MRS2500 and the non-nucleotide antagonist BPTU. MRS2500 binds in the orthosteric site, but BPTU binds to an unusual pocket at the GPCR/lipid bilayer interface.

Glucose uptake by peripheral tissues such as skeletal muscles and adipocytes is important in the maintenance of glucose homeostasis. We previously demonstrated that P2Y6 receptor (P2Y6R) agonists protect pancreatic islet cells from apoptosis and stimulate glucose-dependent insulin release. Here, we investigated the effects of P2Y6R activation on glucose uptake in insulin target tissues. An agonist of the P2Y6R, P1-(5'-uridine)-P3-(5'-N4-methoxycytidine)-triphosphate (MRS2957), significantly increased the uptake of [3H]2-deoxyglucose in mouse C2C12 myotubes and 3T3-L1 adipocytes, and this stimulation was significantly decreased by a selective P2Y6R antagonist N,N″-1,4-butanediyl-bis[N'-(3-isothiocyanatophenyl)thiourea] (MRS2578). Pre-incubation with Compound C (an inhibitor of 5'-AMP-activated protein kinase, AMPK), or AMPK siRNA abolished the stimulatory effect of MRS2957 on glucose uptake. Also, MRS2957 (60 min incubation) increased recruitment of the facilitated glucose transporter-4 (GLUT4) to the cell membrane, which was blocked by MRS2578. Treatment of C2C12 myotubes with MRS2957 induced significant phosphorylation of AMPK, which increase GLUT4 expression through histone deacetylase (HDAC)5 signaling. Glucose uptake in primary mouse adipocytes from wild-type mice was stimulated upon P2Y6R activation by either MRS2957 or native agonist UDP, and the P2Y6R effect was antagonized by MRS2578. However, in adipocytes from P2Y6R-knockout mice P2Y6R agonists had no effect on glucose uptake, and there was no change in the glucose uptake by insulin. Our results indicate that the P2Y6R promotes glucose metabolism in peripheral tissues, which may be mediated through AMPK signaling.

An X-ray structure of human P2Y 12 receptor, a clinical drug target for platelet aggregation inhibitors, is presented in complex with an agonist, providing insight into the δ-group of class A G-protein-coupled receptors. Key platelet aggregation GPCR structures Two papers in this issue of Nature present the crystal structures of the human P2Y 12 receptor, first in complex with the antithrombotic drug AZD1283, and second, bound to a full agonist (a close analogue of endogenous agonist ADP) and to a partial agonist. P2Y receptors are a family of purinergic G-protein-coupled receptors (GPCRs) that are activated by extracellular nucleotides. The P2Y 12 receptor is found mainly on the surface of platelets, where it regulates platelet activation and thrombus formation, and it is the target of several important antithrombotic drugs. In overall structure, P2Y 12 receptor is found to be similar to other GPCRs, although both the shape and location of the ligand-binding pocket are unusual. Comparisons of the three newly determined structures reveal that agonist binding induces a large-scale rearrangement of the extracellular domains of the GPCR. The P2Y 12 receptor (P2Y 12 R), one of eight members of the P2YR family expressed in humans, is one of the most prominent clinical drug targets for inhibition of platelet aggregation. Although mutagenesis and modelling studies of the P2Y 12 R provided useful insights into ligand binding 1 , 2 , 3 , 4 , the agonist and antagonist recognition and function at the P2Y 12 R remain poorly understood at the molecular level. Here we report the structures of the human P2Y 12 R in complex with the full agonist 2-methylthio-adenosine-5′-diphosphate (2MeSADP, a close analogue of endogenous agonist ADP) at 2.5 Å resolution, and the corresponding ATP derivative 2-methylthio-adenosine-5′-triphosphate (2MeSATP) at 3.1 Å resolution. These structures, together with the structure of the P2Y 12 R with antagonist ethyl 6-(4-((benzylsulfonyl)carbamoyl)piperidin-1-yl)-5-cyano-2-methylnicotinate (AZD1283) 5 , reveal striking conformational changes between nucleotide and non-nucleotide ligand complexes in the extracellular regions. Further analysis of these changes provides insight into a distinct ligand binding landscape in the δ-group of class A G-protein-coupled receptors (GPCRs). Agonist and non-nucleotide antagonist adopt different orientations in the P2Y 12 R, with only partially overlapped binding pockets. The agonist-bound P2Y 12 R structure answers long-standing questions surrounding P2Y 12 R–agonist recognition, and reveals interactions with several residues that had not been reported to be involved in agonist binding. As a first example, to our knowledge, of a GPCR in which agonist access to the binding pocket requires large-scale rearrangements in the highly malleable extracellular region, the structural and docking studies will therefore provide invaluable insight into the pharmacology and mechanisms of action of agonists and different classes of antagonists for the P2Y 12 R and potentially for other closely related P2YRs.