The C-Terminus of Human Nucleotide Receptor P2X7 Is Critical for Receptor Oligomerization and N-Linked Glycosylation

Background: The P2X(7) receptor binds extracellular ATP to mediate numerous inflammatory responses and is considered a potential biomarker and therapeutic target for diverse inflammatory and neurological diseases. P2X(7) contains many single nucleotide polymorphisms, including several mutations located within its intracellular C-terminal trafficking domain. Mutations within the trafficking domain result in attenuated receptor activity and cell surface presentation, but the mechanisms by which amino acid changes within this region promote altered P2X(7) function have not been elucidated. Methods and Results: We analyzed the amino acid sequence of P2X(7) for any potential trafficking signals and found that P2X(7) contains putative Arg-X-Arg ER retention sequences. Alanine substitutions near or within these sequences were constructed, and we determined that single mutation of R574 and R578 but not R576 or K579 attenuates P2X(7)-stimulated activation of ERK1/2 and induction of the transcription factors Delta FosB and DFosB. We found that mutation of R578 within the trafficking domain to the naturally occurring Gln substitution disrupts P2X(7) localization at the plasma membrane and results in R578Q displaying a higher apparent molecular weight in comparison to wild-type receptor. We used the glycosidase endoglycosidase H to determine that this difference in mass is due in part to the R578Q mutant possessing a larger mass of oligosaccharides, indicative of improper N-linked glycosylation addition and/or trimming. Chemical cross-linking experiments were also performed and suggest that the R578Q variant also does not form trimers as well as wild-type receptor, a function required for its full activity. Conclusions: These data demonstrate the distal C-terminus of P2X(7) is important for oligomerization and post-translational modification of the receptor, providing a mechanism by which mutations in the trafficking domain disrupt P2X(7) activity and localization at the plasma membrane.