Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.248126
Title: The regulation of the human A₁ adenosine receptor and the sphingosine 1-phosphate receptor, EDG1
Author: Watterson, Kenneth Robert
ISNI:       0000 0001 3564 5288
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2002
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Abstract:
The adenosine A1 receptor (A1AR) and the sphingosine-1-phosphate (SSP/SIP) receptor, endothelial differentiation gene 1 (EDG1) are members of the large superfamily of cell surface G-protein-coupled receptors (GPCRs). A1AR activation by the purine, adenosine results in a number of cardio- and neuroprotective effects and has been implicated in the process of ischaemic preconditioning. S1P-mediated activation of the EDG1 receptor also elicits a range of biological effects and has recently been shown to be heavily involved in the process of new blood vessel formation called angiogenesis. Following a sustained agonist exposure, many GPCRs are desensitised such that their responses plateau and then diminish. The relative ability or inability of a GPCR to undergo agonist-mediated receptor phosphorylation and subsequent internalisation away from the cell surface is an important measure of a GPCR's ability to become desensitised. This study has characterised the phosphorylation and internalisation of the human A1AR and the human EDG1 receptor. Whole cell receptor phosphorylation assays demonstrated that A1ARs stably expressed in CHO cells were not phosphorylated in response to the agonist, R-PIA. In contrast, the A3AR, which is similar in terms of structure, G-protein coupling specificity and biological effects was rapidly phosphorylated following R-PIA exposure. Additionally, cell surface biotinylation assays showed that, whereas the A3AR was internalised rapidly following R-PIA exposure (t1/2= 10 min), the agonist-dependent loss of A1AR from the cell surface was much slower (t1/2= 90 min) and less complete. Using confocal analysis, it was shown that the mutation of Cys309, a site of palmitoylation within the C-terminal, had no visible effect on the cell surface expression of A1ARs tagged with green fluorescent protein (GFP) following a 1 hour agonist exposure. In contrast, parallel studies within the lab demonstrated that mutation of Cys302 and Cys305 within the C-terminal of the A3AR resulted in a marked increase in basal receptor phosphorylation and an increased rate of internalisation. Whole cell phosphorylation studies on hamster lung CCL-39 fibroblasts stably expressing human EDG1 receptors showed that EDG1 is phosphorylated in response to agonist (SIP) and also PMA, a phorbol ester that activates PKC subtypes. However, lysophosphatidic acid (LPA), a bioactive lipid similar in structure and biological effects to EDG1, had no effect on EDG1 phosphorylation. Phosphoamino acid analysis showed that S1P and PMA stimulate the accumulation of phosphoserine and phosphothreonine but not phosphotyrosine. Preincubation with the PKC inhibitor, GF109203X abolished EDG1 phosphorylation in the presence of PMA but failed to block S1P-mediated EDG1 phosphorylation, suggesting that these processes are mechanistically distinct. Additionally, the removal of 12 amino acids from the C-terminal tail of EDG1 significantly reduced S1P- but not PMA-stimulated phosphorylation, providing further evidence of two distinct mechanisms of receptor phosphorylation. However, incubation of S1P and PMA together did not result in a significant increase in EDG1 phosphorylation when compared to that achieved by S1P treatment alone. Therefore, although PMA- and S1P-mediated EDG1 phosphorylation were distinct, each pathway utilised overlapping phosphorylation sites within EDG1. In vitro assays implicated a role for GRK2 in S1P-induced EDG1 phosphorylation observed in intact cells. Also, the region between the last 12 and the last 32 amino acids of the EDG1 C-terminal was shown to be responsible for the in vitro S1P-dependent phosphorylation of EDG1 in the presence of GRK2.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.248126  DOI: Not available
Keywords: Endothelial differentiation gene
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