GPR17: Molecular modeling and dynamics studies of the 3-D structure and purinergic ligand binding features in comparison with P2Y receptors

<p>Abstract</p> <p>Background</p> <p>GPR17 is a G-protein-coupled receptor located at intermediate phylogenetic position between two distinct receptor families: the P2Y and CysLT receptors for extracellular nucleotides and cysteinyl-LTs, respectively. We previously showed that GPR17 can indeed respond to both classes of endogenous ligands and to synthetic compounds active at the above receptor families, thus representing the first fully characterized non-peptide "hybrid" GPCR. In a rat brain focal ischemia model, the selective <it>in vivo </it>knock down of GPR17 by anti-sense technology or P2Y/CysLT antagonists reduced progression of ischemic damage, thus highlighting GPR17 as a novel therapeutic target for stroke. Elucidation of the structure of GPR17 and of ligand binding mechanisms are the necessary steps to obtain selective and potent drugs for this new potential target. On this basis, a 3-D molecular model of GPR17 embedded in a solvated phospholipid bilayer and refined by molecular dynamics simulations has been the first aim of this study. To explore the binding mode of the "purinergic" component of the receptor, the endogenous agonist UDP and two P2Y receptor antagonists demonstrated to be active on GPR17 (MRS2179 and cangrelor) were then modeled on the receptor.</p> <p>Results</p> <p>Molecular dynamics simulations suggest that GPR17 nucleotide binding pocket is similar to that described for the other P2Y receptors, although only one of the three basic residues that have been typically involved in ligand recognition is conserved (Arg255). The binding pocket is enclosed between the helical bundle and covered at the top by EL2. Driving interactions are H-bonds and salt bridges between the 6.55 and 6.52 residues and the phosphate moieties of the ligands. An "accessory" binding site in a region formed by the EL2, EL3 and the Nt was also found.</p> <p>Conclusion</p> <p>Nucleotide binding to GPR17 occurs on the same receptor regions identified for already known P2Y receptors. Agonist/antagonist binding mode are similar, but not identical. An accessory external binding site could guide small ligands to the deeper principal binding site in a multi-step mechanism of activation. The nucleotide binding pocket appears to be unable to allocate the leukotrienic type ligands in the same effective way.</p

Steps towards collective sustainability in biomedical research

The optimism surrounding multistakeholder research initiatives does not match the clear view of policies that are needed to exploit the potential of these collaborations. Here we propose some action items that stem from the integration between research advancements with the perspectives of patient-advocacy organizations, academia, and industry

G PROTEIN-COUPLED RECEPTOR DESENSITISATION REGULATES STEM CELL DIFFERENTIATION

G-protein coupled receptors (GPCRs) play a key role in many complex biological processes, including regulation of stem cell pluripotency and differentiation. Signal transduction pathways that are activated during stem cell renewal and differentiation are shared, cross-activated or synergistic with GPCR stimulation [1]. Regulation of GPCR responses involved the activation of desensitization machinery, which started with phosphorylation of agonist-activated receptor by second messenger-dependent and/or GPCR kinases (GRKs)[1]. Besides controlling receptor responsiveness, GRKs can also act as agonist-regulated scaffolds assembling macromolecular signalosomes in the receptor environment, thereby contributing to signal propagation from cytosol to nucleus, and controlling gene transcription machinery [2]. Recent evidence suggests that the desensitization machinery fulfils a vital role in regulating cellular responses to GPCRs, and that changes in expression/functioning of these regulatory proteins may be crucial in the control of cell differentiation program [3]. These data are consistent with the notion that GPCR responsiveness may be differentially regulated during cell differentiation. In our hands, two different cellular models (oligodendrocyte precursor cells, OPCs, and mesenchymal stem cells, MSCs) were used to investigate the role of the GPCR desensitisation machinery in stem cell differentiation. During OPC differentiation, defective control of the membrane receptor GPR17 has been suggested to block cell maturation and impairs remyelination under demyelinating conditions [4]. Here we show, for the first time, a role for Murine double minute 2 (Mdm2), a ligase previously involved in ubiquitination/degradation of p53 protein. In maturing OPCs, the inhibition of Mdm2-p53 interactions increased GRK2 sequestration by Mdm2, leading to impaired GPR17 down-regulation and OPC maturation block. In MSCs, the A2B adenosine receptor (A2BAR) has been recently emerged as the major AR involved in osteoblastogenesis [5]. Proinflammatory cytokines, such as Tumour Necrosis Factor- (TNF-, have been demonstrated to regulate MSC differentiation and bone remodelling. Herein, we show that TNF- diminished GRK2 levels in MSCs, thus blocking A2BAR desensitization. As a result, TNF- enhanced the A2BAR-mediated responses and favoured MSC differentiation to osteoblasts in response to receptor agonists. The findings get new insights for discovering of the signals at the basis of cell differentiation

Temporomandibular joint inflammation activates glial and immune cells in both the trigeminal ganglia and in the spinal trigeminal nucleus

<p>Abstract</p> <p>Background</p> <p>Glial cells have been shown to directly participate to the genesis and maintenance of chronic pain in both the sensory ganglia and the central nervous system (CNS). Indeed, glial cell activation has been reported in both the dorsal root ganglia and the spinal cord following injury or inflammation of the sciatic nerve, but no data are currently available in animal models of trigeminal sensitization. Therefore, in the present study, we evaluated glial cell activation in the trigeminal-spinal system following injection of the Complete Freund's Adjuvant (CFA) into the temporomandibular joint, which generates inflammatory pain and trigeminal hypersensitivity.</p> <p>Results</p> <p>CFA-injected animals showed ipsilateral mechanical allodynia and temporomandibular joint edema, accompanied in the trigeminal ganglion by a strong increase in the number of GFAP-positive satellite glial cells encircling neurons and by the activation of resident macrophages. Seventy-two hours after CFA injection, activated microglial cells were observed in the ipsilateral trigeminal subnucleus caudalis and in the cervical dorsal horn, with a significant up-regulation of Iba1 immunoreactivity, but no signs of reactive astrogliosis were detected in the same areas. Since the purinergic system has been implicated in the activation of microglial cells during neuropathic pain, we have also evaluated the expression of the microglial-specific P2Y₁₂receptor subtype. No upregulation of this receptor was detected following induction of TMJ inflammation, suggesting that any possible role of P2Y₁₂in this paradigm of inflammatory pain does not involve changes in receptor expression.</p> <p>Conclusions</p> <p>Our data indicate that specific glial cell populations become activated in both the trigeminal ganglia and the CNS following induction of temporomandibular joint inflammation, and suggest that they might represent innovative targets for controlling pain during trigeminal nerve sensitization.</p

Roles of P2 receptors in glial cells: focus on astrocytes

Central nervous system glial cells release and respond to nucleotides under both physiological and pathological conditions, suggesting that these molecules play key roles in both normal brain function and in repair after damage. In particular, ATP released from astrocytes activates P2 receptors on astrocytes and other brain cells, allowing a form of homotypic and heterotypic signalling, which also involves microglia, neurons and oligodendrocytes. Multiple P2X and P2Y receptors are expressed by both astrocytes and microglia; however, these receptors are differentially recruited by nucleotides, depending upon specific pathophysiological conditions, and also mediate the long-term trophic changes of these cells during inflammatory gliosis. In astrocytes, P2-receptor-induced gliosis occurs via activation of the extracellular-regulated kinases (ERK) and protein kinase B/Akt pathways and involves induction of inflammatory and anti-inflammatory genes, cyclins, adhesion and antiapoptotic molecules. While astrocytic P2Y1 and P2Y2,4 are primarily involved in short-term calcium-dependent signalling, multiple P2 receptor subtypes seem to cooperate to astrocytic long-term changes. Conversely, in microglia, exposure to inflammatory and immunological stimuli results in differential functional changes of distinct P2 receptors, suggesting highly specific roles in acquisition of the activated phenotype. We believe that nucleotide-induced activation of astrocytes and microglia may originally start as a defence mechanism to protect neurons from cytotoxic and ischaemic insults; dysregulation of this process in chronic inflammatory diseases eventually results in neuronal cell damage and loss. On this basis, full elucidation of the specific roles of P2 receptors in these cells may help exploit the beneficial neuroprotective features of activated glia while attenuating their harmful properties and thus provide the basis for novel neuroprotective strategies that specifically target the purinergic system

P2Y receptors in GtoPdb v.2021.3

P2Y receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on P2Y Receptors [3, 5, 192]) are activated by the endogenous ligands ATP, ADP, uridine triphosphate, uridine diphosphate and UDP-glucose. The relationship of many of the cloned receptors to endogenously expressed receptors is not yet established and so it might be appropriate to use wording such as 'uridine triphosphate-preferring (or ATP-, etc.) P2Y receptor' or 'P2Y1-like', etc., until further, as yet undefined, corroborative criteria can be applied [47, 110, 190, 383, 396]. Clinically used drugs acting on these receptors include the dinucleoside polyphosphate diquafosol, agonist of the P2Y2 receptor subtype, approved in Japan for the management of dry eye disease [241], and the P2Y12 receptor antagonists prasugrel, ticagrelor and cangrelor, all approved as antiplatelet drugs [53, 323]