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Epithelial membrane proteins induce membrane blebbing and interact with the P2X7 receptor C terminus
Heather L Wilson1, Stuart A Wilson, Annmarie Surprenant
1Institute of Molecular Physiology, University of Sheffield S10 2TN, United Kingdom. H.L.Wilson@sheffield.ac.uk
Abstract:
The binding of extracellular ATP to the P2X(7) receptor opens an integral cation-permeable channel; it also leads to membrane blebbing and, in certain immune cells, interleukin-1beta secretion and eventual death. The latter three effects are unique to the P2X(7) receptor; also unique among P2X receptors is the long intracellular C terminus of the protein. We have shown that the C-terminal domain of the P2X(7) receptor is responsible for the cell blebbing phenotype. A screen for proteins that associate with the C-terminal domain of the P2X(7) receptor and might mediate the blebbing phenotype, identified epithelial membrane protein 2 (EMP-2). The interaction between EMP-2 and P2X(7) was confirmed biochemically by co-immunoprecipitation, co-purification, and glutathione S-transferase pull-down assays, and this interaction was entirely dependent on the C-terminal domain of P2X(7). The P2X(7) receptor also interacted with the other members of the epithelial membrane protein family (EMP-1, EMP-3, and PMP-22). All four EMPs were found to be expressed in HEK-293 cells and in THP-1 monocytes, which express P2X(7) receptors. Interestingly, the constitutive overexpression of any of the EMPs in HEK-293 cells led to cell blebbing, annexin V binding, and cell death, by a caspase-dependent pathway. These findings suggest that the P2X(7) C-terminal domain associates with EMPs, and this interaction may mediate some aspects of the downstream signaling following P2X(7) receptor activation.
Insights
The P2X(7) receptor
Area of Science:
- Cell Biology
- Immunology
- Molecular Biology
Background:
- The P2X(7) receptor (P2X7R) mediates cellular responses to extracellular ATP, including ion channel opening, membrane blebbing, and immune cell activation.
- Unique features of P2X7R include its role in interleukin-1beta secretion and cell death, along with a notably long intracellular C-terminal domain.
Purpose of the Study:
- To investigate the role of the P2X7R C-terminal domain in mediating the cell blebbing phenotype.
- To identify proteins interacting with the P2X7R C-terminal domain that may be involved in downstream signaling.
Main Methods:
- Co-immunoprecipitation, co-purification, and glutathione S-transferase pull-down assays were used to confirm protein interactions.
- Expression analysis of epithelial membrane proteins (EMPs) in HEK-293 cells and THP-1 monocytes.
- Overexpression studies of EMPs in HEK-293 cells to assess their effects on cell viability and apoptosis.
Main Results:
- The C-terminal domain of P2X7R was identified as responsible for the cell blebbing phenotype.
- Epithelial membrane protein 2 (EMP-2) was found to interact with the P2X7R C-terminal domain.
- All four epithelial membrane proteins (EMP-1, EMP-3, and PMP-22) interacted with P2X7R and were expressed in relevant cell types.
- Overexpression of EMPs induced cell blebbing, annexin V binding, and caspase-dependent cell death.
Conclusions:
- The P2X7R C-terminal domain associates with epithelial membrane proteins.
- This interaction likely mediates downstream signaling events, including cell blebbing and death, following P2X7R activation.