Title : Mutation of putative N-linked glycosylation
sites on the human nucleotide
receptor P2X7 reveals a key
residue important for
receptor function
Abstract :
- The nucleotide receptor P2X( 7) is an immunomodulatory cation channel and a potential therapeutic target
- P2X( 7) is expressed in immune cells such as monocytes and macrophages and is activated by extracellular ATP following tissue injury or infection
- Ligand binding to P2X( 7) can stimulate ERK1/2 , the transcription factor CREB , enzymes linked to the production of reactive oxygen species and interleukin-1 isoforms , and the formation of a nonspecific pore
- However, little is known about the biochemistry of P2X( 7), including whether the receptor is N-linked glycosylated and if this modification affects receptor function
- Here we provide evidence that P2X( 7) is sensitive to the glycosidases EndoH and PNGase F and that the human receptor appears glycosylated at N187, N202, N213, N241, and N284
- Mutation of N187 results in weakened P2X( 7) agonist-induced phosphorylation of ERK1/2 , CREB , and p90 ribosomal S6 kinase , as well as a decreased level of pore formation
- In further support of a role for glycosylation in receptor function, treatment of RAW 264.7 macrophages with the N-linked glycosylation synthesis inhibitor tunicamycin attenuates P2X( 7) agonist-induced, but not phorbol ester-induced, ERK1/2 phosphorylation
- Interestingly, residue N187 belongs to an N-linked glycosylation consensus sequence found in six of the seven P2X family members, suggesting this site is fundamentally important to P2X receptor function
- To address the mechanism whereby N187 mutation attenuates receptor activity, we developed a live cell proteinase K digestion assay that demonstrated altered cell surface expression of P2X( 7) N187A
- This is the first report to map human P2X( 7) glycosylation sites and reveal residue N187 is critical for receptor trafficking and function