ABSTRACT
BACKGROUND: Narcolepsy with cataplexy is a neurological sleep disorder, which is believed to arise from the autoimmune destruction of hypocretin-producing neurons. The purinergic receptor P2Y11 is associated with narcolepsy in genome-wide association studies, and P2RY11 sequencing has further revealed eight rare missense mutations associated with the disease. Some of these mutations alter the signaling properties of P2Y11, but for some, no functional effects have been discovered so far. METHODS: This study examined the surface expression of the eight narcolepsy-associated P2Y11 mutations using an in vitro surface expression assay. RESULTS: The assay showed excellent discrimination between cells transfected with tagged wild type and the untagged P2Y11 receptor, proving complete specificity towards the 3HA-N-tag used for the assay. Our results show a decreased surface expression of the R307W P2Y11 mutant and a surface expression similar to wild type for the other seven mutants. CONCLUSION: Based on the present findings, alteration in surface expression is not likely to play a role in how P2Y11 influences narcolepsy pathogenesis. This is important because intact surface expression increases the usefulness of P2Y11 as a future drug target.
Subject(s)
Gene Expression , Narcolepsy/genetics , Receptors, Purinergic P2/genetics , Genetic Variation , HEK293 Cells , Humans , Mutation , Neurons/metabolism , Orexins/metabolism , TransfectionABSTRACT
Adenosine triphosphate (ATP) is known to induce cell death in T lymphocytes at high extracellular concentrations. CD4+ and CD8+ T lymphocytes have a differential response to ATP, which in mice is due to differences in the P2X7 receptor expression levels. By contrast, we observed that the difference in human CD4+ and CD8+ T lymphocyte response toward the synthetic ATP-analog BzATP is not explained by a difference in human P2X7 receptor expression. Rather, the BzATP-induced human P2X7 receptor response in naïve and immune-activated lymphocyte subtypes correlated with the expression of another ATP-binding receptor: the human P2Y11 receptor. In a recombinant expression system, the coexpression of the human P2Y11 receptor counteracted BzATP-induced human P2X7 receptor-driven lactate dehydrogenase release (a marker of cell death) and pore formation independent of calcium signaling. A mutated non-signaling human P2Y11 receptor had a similar human P2X7 receptor-inhibitory effect on pore formation, thus demonstrating that the human P2X7 receptor interference was not caused by human P2Y11 receptor signaling. In conclusion, we demonstrate an important species difference in the ATP-mediated cell death between mice and human cells and show that in human T lymphocytes, the expression of the human P2Y11 receptor correlates with human P2X7 receptor-driven cell death following BzATP stimulation.
Subject(s)
Adenosine Triphosphate/metabolism , Pore Forming Cytotoxic Proteins/metabolism , Receptors, Purinergic P2X7/metabolism , Receptors, Purinergic P2/metabolism , T-Lymphocytes/physiology , Animals , Calcium Signaling , Cell Death , Cells, Cultured , Diphosphonates/pharmacology , Humans , Mice , Naphthalenesulfonates/pharmacology , Purinergic P2Y Receptor Agonists/pharmacology , Receptor Cross-Talk , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2X7/genetics , Transgenes/geneticsABSTRACT
The P2X7 receptor is a frequently studied member of the purinergic receptor family signalling via channel opening and membrane pore formation. Fluorescent imaging is an important molecular method for studying cellular receptor expression and localization. Fusion of receptors to fluorescent proteins might cause major functional changes and requires careful functional evaluation such as has been done for the rat P2X7 receptor. This study examines fusion constructs of the human P2X7 receptor. We assessed surface expression, channel opening with calcium influx, and pore formation using YO-PRO-1 dye uptake in response to BzATP stimulation in transfected cells. We found that tagging at the N-terminal of the human P2X7 receptor with the enhanced green fluorescent protein (eGFP) disturbed channel opening and pore formation despite intact surface expression. A triple hemagglutinin (3HA) fused to the N-terminal also disrupted pore formation but not channel opening showing that even a small tag alters the normal function of the receptor. Together, this suggests that in contrast to what has been observed for the rat P2X7 receptor, the human P2X7 receptor contains N-terminal motifs important for signalling that prevent the construction of a functionally active fusion protein.