Bone development and homeostasis require the interplay between several cell types, including mesenchymal osteoblasts and osteocytes, as well as hematopoietic osteoclasts. Recent evidence suggests that cell proliferation, differentiation and apoptosis of both mesenchymal and hematopoietic stem cells, which are fundamental for tissue regeneration and treatment of degenerative diseases, is controlled by P2 receptors (i.e., P2X and P2Y receptors). Both types of P2 receptors are versatile transducers of diverse signals activated by extracellular nucleotides like ATP that are released in response to tissue injury, infection or shear stress. The P2X family of receptors has been shown to mediate multiple signaling events including the influx of calcium, activation of mitogen activated protein kinases (MAPKs) and induction of AP-1 family members known to regulate bone development. Support for the significance of P2X7 in regulating bone development and homeostasis has been provided by several studies focusing on animal models and single nucleotide polymorphisms. P2 receptors are functionally expressed in both bone forming osteoblasts and bone resorbing osteoclasts, while recent findings also suggest that these receptors translate mechanical stimuli in osteocytes. Their ability to respond to external nucleotide analogs renders these cell surface proteins excellent targets for skeletal regenerative therapies. This overview summarizes mechanisms by which nucleotide receptors control skeletal cells and contribute to bone tissue development remodeling and repair.

BackgroundThe P2X7 receptor binds extracellular ATP to mediate numerous inflammatory responses and is considered a potential biomarker and therapeutic target for diverse inflammatory and neurological diseases. P2X7 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 P2X7 function have not been elucidated.Methods and ResultsWe analyzed the amino acid sequence of P2X7 for any potential trafficking signals and found that P2X7 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 P2X7-stimulated activation of ERK1/2 and induction of the transcription factors FosB and ΔFosB. We found that mutation of R578 within the trafficking domain to the naturally occurring Gln substitution disrupts P2X7 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.ConclusionsThese data demonstrate the distal C-terminus of P2X7 is important for oligomerization and post-translational modification of the receptor, providing a mechanism by which mutations in the trafficking domain disrupt P2X7 activity and localization at the plasma membrane.