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  • We hypothesized that the atopic phenotype may be partially

    2019-07-08

    We hypothesized that the atopic phenotype may be partially influenced by an altered binding and/or signalling due to the amino-acid substitution in the receptor protein. The principal aim of this study was to investigate the signalling mechanisms of the variants CysLT1-G300S and CysLT1-I206S induced by their natural ligands, LTD4 and LTC4, in comparison with the CysLT1-WT receptor.
    Materials and methods
    Results
    Discussion In the present work, we have shown that the natural variants CysLT1-G300S and CysLT1-I206S respond differently to cysLTs in comparison to the WT receptor. Although the cell surface expression of the receptors is equivalent, the production of IPs, phosphorylation of Erk, as well as IL-8 and IL-13 promoter transactivation are higher in the Sodium Orthovanadate expressing the variant CysLT1-G300S. In addition, the binding of LTD4 appears to be higher as well. In contrast, when the cells express the variant CysLT1-I206S, their responses to cysLTs tend to be lower or similar to those of the WT receptor. In other studies, natural mutations in the structure of receptors were shown to affect their expression. Gonadotropin-Releasing Hormone Receptor sees its expression increased [35], whereas mutant m1 muscarinic receptor and Dopamine receptor DRD2 have their expression decreased [36], [37]. However, other variants of GPCR receptors do not alter their cell surface expression nor their trafficking as is the case for α2A and α2C adrenergic receptors [38]. When we assessed whether the naturally occurring serine mutations in structure of the CysLT1 receptor would affect its constitutive expression, we found that the mutations did not affect the expression levels (Fig. 1), suggesting that the differences in the functional responses could be attributable to the signalling and not to the constitutive expression of receptors. Since the mutation to serine could potentially play a role in signalling, we used a bio-informatic server NetPhos 2.0 [39] to predict the phosphorylation sites in the receptor. It showed that the residues 300S and 206S would not create new phosphorylation sites, and that the potential differences would not be due to altered new phosphorylation-dependent desensitization. In terms of functional responses, cells expressing the receptor variant CysLT1-G300S produced more IPs than cells expressing the WT receptor following stimulation with the ligands LTD4 and LTC4. IPs are produced following GPCR activation of its associated Gαq protein that triggers the activation of PLCβ and the generation of IP-3. This activation pathway appears to be more efficient with cysLT-induced CysLT1-G300S activation (Fig. 2). It is one of the first steps in the signalling cascade upon the activation of GPCR receptors, and there is already a difference between the variants and the WT receptors at this level of the cascade. Several elements could explain this difference, such as a difference in receptor binding capacity, in recruitment of G-proteins or in phosphorylation levels. When we compared basal IP levels in the transfected cells expressing CysLT1-WT and CysLT1-G300S, there was no significant difference that would have suggested that the receptor variant was more constitutively active than the WT receptor and CysLT1-I206S variant. We further explored other levels of signalling and found that ligand activation of the variant CysLT1-G300S induced an augmented level of phosphorylation of the MAPKinase Erk. However, the proteins p65 of NF-κB pathway, the MAPKinase p38, and the protein c-Jun activated by the MAPKinase JNK, were not differentially activated by the variant CysLT1-G300S, compared to the WT receptor (Fig. 3). Although the variant induces the activation and phosphorylation of the NF-κB and the other MAPKinases as well, these results suggest that it favours the Erk pathway. Activation of the Gαq-coupled CysLT1 receptor induces indirectly the activation of PKC proteins that in turn activate the IKK complex, phosphorylate Iκ-B and release the dimeric complex NF-κB (p65 and p50/52) [24], [33]. CysLT1 receptor is also known to activate the Erk pathway [23], [40]. This led us to speculate that the point of divergence of the response of the variant CysLT1-G300S would be upstream the MAPK Erk, separate from the elements activating the NF-κB pathway. Thus, it would be of interest to further explore the signalling cascade, including the activation of Ras proteins and PKCs, to differentiate the two pathways of activation of CysLT1 receptor and its variants [20].