Cardiff University | Prifysgol Caerdydd ORCA
Online Research @ Cardiff 
WelshClear Cookie - decide language by browser settings

Investigating the role of the P2X7 intracellular domains in coupling to downstream signalling

Jaradat, Ahmad 2021. Investigating the role of the P2X7 intracellular domains in coupling to downstream signalling. PhD Thesis, Cardiff University.
Item availability restricted.

[thumbnail of Jaradat AQ thesis 090821 (1).pdf] PDF - Accepted Post-Print Version
Download (8MB)
[thumbnail of Cardiff University Electronic Publication Form] PDF (Cardiff University Electronic Publication Form) - Supplemental Material
Restricted to Repository staff only

Download (122kB)

Abstract

The P2X7 receptor is a trimeric ATP-gated cation channel expressed in many mammalian cells, including immune and cancer cells. The wild-type receptor monomer is 595 amino acids in length, comprised of two transmembrane domains, a large extracellular loop, a short intracellular N-terminal domain and a 250 amino-acid long intracellular C-terminal domain. P2X7 receptor activation by extracellular ATP leads to the passage of small cations, such as Ca++ , K+ , and Na+ across the plasma membrane. It also initiates cell-dependent signalling pathways including cell blebbing, pore formation, cell proliferation and death, and ERK1/2 phosphorylation. Intracellular signalling via P2X7 activation is thought to be dependent on both the N- and C-terminal domains, but the specific sub-domains involved in distinct pathways are not known. In this study, we set out to identify which intracellular domains are important for P2X7- mediated downstream signalling and whether distinct intracellular sub-domains govern different signalling phenomena. Using whole plasmid PCR, we constructed 17 truncation and deletion mutations in a C-terminally GFP-tagged rat P2X7 receptor construct; one within the N- terminal domain and 16 within the C-terminal domain. We analysed the effects of these mutations on ATP-induced cell blebbing, calcium influx, pore formation (through the uptake of YoPro1 dye) and ERK1/2 phosphorylation in transfected mammalian cells. We found that a receptor construct lacking almost the entire ballast domain (1-380) was still capable of cell blebbing as was a construct lacking the C-Cys anchor (362-379), suggesting that these domains may not be necessary for P2X7-dependent cell blebbing. Deletion of the N-terminal domain (2-23) and many sub-regions within the ballast domain abolished cell blebbing, calcium influx, and pore formation, likely due to impaired receptor cell surface expression. The exceptions to this were receptor constructslacking amino acids 441-460 or 583-595 in the ballast domain, which were capable of cell blebbing, and pore formation. However, combining the two deletions into one construct gave rise to a non-functional receptor with no detectable cell-surface expression. Strikingly, depletion of cholesterol from the plasma membrane abolished cell blebbing in all constructs including wild-type, but potentiated pore formation in the mutant lacking the ballast domain (1-380). XV Investigation of the molecular mechanism for cell blebbing focused on the proposed role for dissociation of P2X7 from the actin-myosin cytoskeleton following receptor activation. However, we were unable to detect an interaction between P2X7 and non-muscle myosin (NMMHC-IIA) using pull-down assay from cell lysates, and while we were able to demonstrate that P2X7 receptors lie in close proximity to NMMHC-IIA in Hela cells using the DuolinkTM proximity ligation assay, we did not observe a change in proximity upon P2X7 activation with ATP. Wild-type P2X7 was capable of ATP-induced ERK1/2 phosphorylation, but nearly all mutants abolished it, although issues with data collection and analysis prevented a full statistical comparison. Strikingly, for the N-terminal deletion mutant, which displayed undetectable levels of cell-surface expression, both ERK1 and ERK2 were phosphorylated in non-treated cells, and phosphorylation was significantly reduced by ATP treatment. This suggests that some receptor may be at the cell surface, responding to ATP, but how this may lead to a reduction in phosphorylation is entirely unclear. In conclusion, our data suggests that P2X7-dependent cell blebbing and YoPro uptake do not require the intracellular C-terminal domain of the receptor. Cell blebbing has an absolute requirement for plasma membrane cholesterol, whereas cholesterol inhibits pore formation in mutants lacking the ballast domain. Finally, ERK1/2 phosphorylation may require the intracellular domains of the receptor; both the N-terminal domain and C-terminal domain may be important, but it is not possible to draw firm conclusions about their specific roles.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Biosciences
Subjects: Q Science > Q Science (General)
Date of First Compliant Deposit: 10 August 2021
Last Modified: 10 Aug 2021 13:11
URI: https://orca.cardiff.ac.uk/id/eprint/143264

Actions (repository staff only)

Edit Item Edit Item

Downloads

Downloads per month over past year

View more statistics