Human P2RX7 (P2X purinoceptor 7) ELISA Kit

P2X7 is an ATP-gated membrane ion channel that is expressed by multiple cell types. Brief exposure to ATP induces the opening of a nonselective cation channel; while repeated or prolonged exposure induces formation of a transmembrane pore. This process may be partially regulated by alternative splicing of full-length P2RX7A pre-mRNA, producing isoforms that delete or retain functional domains. Here, we report cloning and expression of a novel P2RX7 splice variant, P2RX7L, that is, characterized by skipping of exons 7 and 8. In HEK 293 cells, expression of P2RX7L produces a protein isoform, P2X7L, that forms a heteromer with P2X7A. A haplotype defined by six single nucleotide polymorphisms (SNPs) (rs208307, rs208306, rs36144485, rs208308, rs208309, and rs373655596) promotes allele-specific alternative splicing, increasing mRNA levels of P2RX7L and another isoform, P2RX7E, which in addition has a truncated C-terminus. Skipping of exons 7 and 8 is predicted to delete critical amino acids in the ATP-binding site. P2X7L-transfected HEK 293 cells have phagocytic but not channel, pore, or membrane-blebbing function, and double-transfected P2X7L and P2X7A cells have reduced pore function. Heteromeric receptor complexes of P2X7A and P2X7L are predicted to have reduced numbers of ATP-binding sites, which potentially alters receptor function compared to homomeric P2X7A complexes.

The ingrained capacity of melanoma cells to rapidly evolve toward an aggressive phenotype is manifested by their increased ability to develop drug-resistance, evident in the case of vemurafenib, a therapeuticagent targeting BRAF(V600E). Previous studies indicated a tight correlation between heightened melanomaassociated macroautophagy/autophagy and acquired Vemurafenib resistance. However, how this vesicular trafficking pathway supports Vemurafenib resistance remains unclear. Here, using isogenic human and murine melanoma cell lines of Vemurafenib-resistant and patient-derived melanoma cells with primary resistance to the BRAF(V600E) inhibitor, we found that the enhanced migration and invasion of the resistant melanoma cells correlated with an enhanced autophagic capacity and autophagosomemediated secretion of ATP. Extracellular ATP (eATP) was instrumental for the invasive phenotype and the expansion of a subset of Vemurafenib-resistant melanoma cells. Compromising the heightened autophagy in these BRAF(V600E) inhibitor-resistant melanoma cells through the knockdown of different autophagy genes (ATG5, ATG7, ULK1), reduced their invasive and eATP-secreting capacity. Furthermore, eATP promoted the aggressive nature of the BRAF(V600E) inhibitor-resistant melanoma cells by signaling through the purinergic receptor P2RX7. This autophagy-propelled eATP-dependent autocrine-paracrine pathway supported the maintenance and expansion of a drug-resistant melanoma phenotype. In conclusion, we have identified an autophagy-driven response that relies on the secretion of ATP to drive P2RX7-based migration and expansion of the Vemurafenib-resistant phenotype. This emphasizes the potential of targeting autophagy in the treatment and management of metastatic melanoma.