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Regulation of expression and trafficking of P2X4 and P2X7 receptors in immune cells
journal contributionposted on 2023-06-08, 19:52 authored by Miyyada Boumechache, Masin Masin, Anbalakan Paramasivam, Ruth Murrell-LagnadoRuth Murrell-Lagnado
P2X4 and P2X7 are the predominant purinergic receptor subtypes expressed in macrophages, microglia and epithelial cells, and they are potentially important therapeutic targets for treatment of pain and inflammation. For both subtypes, there is evidence that plasmamembrane expression is tightly regulated. P2X4 receptors are prominently localized to lysosomes and resist degradation by virtue of N-linked glycans decorating the intra-luminal loop of the receptor. P2X7 receptors are reported to be predominantly intracellular in monocytes and are upregulated at the plasma membrane upon differentiation of monocytes to macrophages. We have previously shown an interaction between P2X4 and P2X7 receptors, suggesting that they might form an association. The mechanisms that regulate their plasma membrane expression are not well understood, and we have used biochemical methods to look at the size and distribution of the native complexes in a variety of cell types in which they are co-expressed. We have compared the proportion of receptors expressed at the cell surface in cultured microglia and macrophages following exposure to modulators of microglial/macrophage activation. Surface expression was analysed by biotinylation of exposed proteins and by cross-linking proteins with membrane impermeant cross-linkers, followed by SDS-PAGE and western blotting. The modulators included lipopolysaccharide (LPS), ATP and phorbol esters. Cross-linking of surface receptors also provides a means of analysing the subunit composition of the complexes at the plasma membrane, based upon the size difference of P2X4 and P2X7 subunits. These results are compared with those obtained using blue native (BN)-PAGE analysis of the total P2X receptor population.
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- Biochemistry Publications
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