Abstract
BackgroundGPR17 is a hybrid G-protein-coupled receptor (GPCR) activated by two unrelated ligand families, extracellular nucleotides and cysteinyl-leukotrienes (cysteinyl-LTs), and involved in brain damage and repair. Its exploitment as a target for novel neuro-reparative strategies depends on the elucidation of the molecular determinants driving binding of purinergic and leukotrienic ligands. Here, we applied docking and molecular dynamics simulations (MD) to analyse the binding and the forced unbinding of two GPR17 ligands (the endogenous purinergic agonist UDP and the leukotriene receptor antagonist pranlukast from both the wild-type (WT) receptor and a mutant model, where a basic residue hypothesized to be crucial for nucleotide binding had been mutated (R255I) to Ile.ResultsMD suggested that GPR17 nucleotide binding pocket is enclosed between the helical bundle and extracellular loop (EL) 2. The driving interaction involves R255 and the UDP phosphate moiety. To support this hypothesis, steered MD experiments showed that the energy required to unbind UDP is higher for the WT receptor than for R255I. Three potential binding sites for pranlukast where instead found and analysed. In one of its preferential docking conformations, pranlukast tetrazole group is close to R255 and phenyl rings are placed into a subpocket highly conserved among GPCRs. Pulling forces developed to break polar and aromatic interactions of pranlukast were comparable. No differences between the WT receptor and the R255I receptor were found for the unbinding of pranlukast.ConclusionsThese data thus suggest that, in contrast to which has been hypothesized for nucleotides, the lack of the R255 residue doesn't affect the binding of pranlukast a crucial role for R255 in binding of nucleotides to GPR17. Aromatic interactions are instead likely to play a predominant role in the recognition of pranlukast, suggesting that two different binding subsites are present on GPR17.
Highlights
GPR17 is a hybrid G-protein-coupled receptor (GPCR) activated by two unrelated ligand families, extracellular nucleotides and cysteinyl-leukotrienes, and involved in brain damage and repair
To assure that the topology that we found for GPR17 was not an artefact due to the template, and to assess if it was still reliable in view of the new G-protein-coupled receptors (GPCRs) structures, we compared our model with the structure of the human A2A receptor (A2AR), the three structures of the human b2AR and the structure of the turkey b1AR
We focused our subsequent experiments on CII, where the tetrazole group of pranlukast is close to the H-X-X-R motif, and the phenyl rings are placed into a hydrophobic subpocket, that is highly conserved among GPCRs: this is believed to be a common ancestral recognition target for this receptor superfamily
Summary
GPR17 is a hybrid G-protein-coupled receptor (GPCR) activated by two unrelated ligand families, extracellular nucleotides and cysteinyl-leukotrienes (cysteinyl-LTs), and involved in brain damage and repair. There exists a functional crosstalk between the P2Y and CysLT receptor families, since both nucleotides and cysteinyl-LTs massively accumulate at sites of inflammation and both types of receptors are co-expressed in the same peripheral inflammatory cells This evidence shows a cross-regulated response typical of the chemoattractant systems [8]. P2Y12 was found to be promiscuously activated by both nucleotides and LTE4 [11], further underlying the close relationship between the two families Both P2Y and CysLT receptors share the typical seven-transmembrane spanning topology of GPCRs. Both P2Y and CysLT receptors share the typical seven-transmembrane spanning topology of GPCRs Besides their heterogeneity in function and tissue distribution, P2Y and CysLT receptors share a phylogenetic relationship, given that both families, together with GPR17 and other related receptors, belong to the so called “purine receptor cluster” of GPCRs [12]. We recently cloned the human, rat and mouse GPR17 and demonstrated that they all respond to both nucleotides and cysteinyl-LTs [13,14]
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