BACKGROUND AND PURPOSE: The P2X receptor family consists of seven subunit types - P2X1-P2X7. All but P2X6 are able to assemble as homotrimers. In addition, various subunit permutations have been reported to form heterotrimers. Evidence for heterotrimer formation includes co-localization, co-immunoprecipitation and the generation of receptors with novel functional properties; however, direct structural evidence for heteromer formation, such as chemical cross-linking and single-molecule imaging, is available in only a few cases. Here we examined the nature of the interaction between two pairs of subunits - P2X2 and P2X4, and P2X4 and P2X7. EXPERIMENTAL APPROACH: We used several experimental approaches, including in situ proximity ligation, co-immunoprecipitation, co-isolation on affinity beads, chemical cross-linking and atomic force microscopy (AFM) imaging. KEY RESULTS: Both pairs of subunits co-localize upon co-transfection, interact intimately within cells, and can be co-immunoprecipitated and co-isolated from cell extracts. Despite this, chemical cross-linking failed to show evidence for heteromer formation. AFM imaging of isolated receptors showed that all three subunits had the propensity to form receptor dimers. This self-association is likely to account for the observed close interaction between the subunit pairs, in the absence of true heteromer formation. CONCLUSIONS AND IMPLICATIONS: We conclude that both pairs of receptors interact in the form of distinct homomers. We urge caution in the interpretation of biochemical evidence indicating heteromer formation in other cases.
BACKGROUND AND PURPOSE: The P2X receptor family consists of seven subunit types - P2X1-P2X7. All but P2X6 are able to assemble as homotrimers. In addition, various subunit permutations have been reported to form heterotrimers. Evidence for heterotrimer formation includes co-localization, co-immunoprecipitation and the generation of receptors with novel functional properties; however, direct structural evidence for heteromer formation, such as chemical cross-linking and single-molecule imaging, is available in only a few cases. Here we examined the nature of the interaction between two pairs of subunits - P2X2 and P2X4, and P2X4 and P2X7. EXPERIMENTAL APPROACH: We used several experimental approaches, including in situ proximity ligation, co-immunoprecipitation, co-isolation on affinity beads, chemical cross-linking and atomic force microscopy (AFM) imaging. KEY RESULTS: Both pairs of subunits co-localize upon co-transfection, interact intimately within cells, and can be co-immunoprecipitated and co-isolated from cell extracts. Despite this, chemical cross-linking failed to show evidence for heteromer formation. AFM imaging of isolated receptors showed that all three subunits had the propensity to form receptor dimers. This self-association is likely to account for the observed close interaction between the subunit pairs, in the absence of true heteromer formation. CONCLUSIONS AND IMPLICATIONS: We conclude that both pairs of receptors interact in the form of distinct homomers. We urge caution in the interpretation of biochemical evidence indicating heteromer formation in other cases.
Authors: Christopher J Davis; Ping Taishi; Kimberly A Honn; John N Koberstein; James M Krueger Journal: Am J Physiol Regul Integr Comp Physiol Date: 2016-10-05 Impact factor: 3.619
Authors: Mónica González-Magaldi; Raúl Postigo; Beatriz G de la Torre; Yuri A Vieira; Miguel Rodríguez-Pulido; Eduardo López-Viñas; Paulino Gómez-Puertas; David Andreu; Leonor Kremer; María F Rosas; Francisco Sobrino Journal: J Virol Date: 2012-07-11 Impact factor: 5.103
Authors: Marco Bortolato; Megan M Yardley; Sheraz Khoja; Sean C Godar; Liana Asatryan; Deborah A Finn; Ronald L Alkana; Stan G Louie; Daryl L Davies Journal: Int J Neuropsychopharmacol Date: 2012-09-17 Impact factor: 5.176