The regulation of the human sphingosine 1-phosphate receptor, S1P 3

Ord-Shrimpton, Fiona Ursula (2005) The regulation of the human sphingosine 1-phosphate receptor, S1P 3. PhD thesis, University of Glasgow.

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The S1P3 receptor is a member of the cell surface G-protein-coupled receptor superfamily (GPCR). S1P-mediated activation of the S1P3 receptor elicits an array of biological effects including angiogenesis, the process of new blood vessel formation, and may have an important role in atherosclerosis. After sustained exposure to agonist, many GPCRs undergo desensitisation, defined as the waning of receptor responsiveness in the face of persistent stimulation. The relative ability or inability of a GPCR to undergo classic agonist-mediated receptor phosphorylation and subsequent internalisation away from the cell surface is an important marker of GPCR desensitisation. This study has characterised, for the first time, the phosphorylation and internalisation of the human S1P3 receptor. Whole cell phosphorylation studies on hamster lung CCL-39 fibroblasts stably expressing human S1P3 receptors showed that S1P3 is phosphorylated in response to S1P in a time and concentration-dependent manner. In contrast activation of multiple 2nd messenger-activated kinases was without effect on S1P3 phosphorylation under the same conditions. It is well known that the C-terminal region of GPCRs often holds the primary site of interaction for receptor phosphorylation and interaction with β-arrestin. Engineering constructs that truncate the receptor by removing significant and potential sites of phosphorylation allowed the identification of a region bounded by residues Leu332 to Val352 as containing the sites of receptor phosphorylation. As GPCRs are known to be phosphorylated by GRKs a panel of GRKs were investigated, of these GRK2 appeared to phosphorylate the S1P3 receptor in vitro suggesting that GRK2 or a similar kinase is present during S1P3 phosphorylation. S1P3 receptor internalisation is not detectable in CCL-39 cells as determined by biotinylation assays. Also, sucrose density gradient experiments cound not detect a wholesale shift of receptor between lipid raft and non-lipid raft compartments after S1P exposure. Although, studies of S1P3 interaction with β-arrestin are currently preliminary, initial fundings from this research suggest that both receptor and arrestin co-localise after agonist stimulation. This hints at the possibility that internalisation may be obstructed by something other than interaction with arrestin and provokes a receptor-complex paradigm that involves both receptor, arrestin and another protein which is cell-type specific in order to allow internalisation. Experiments in HEK293 cells demonstrated that in this system, WT and phosphorylation resistant SIP3 receptors cound both internalise over identical time courses following agonist exposure. This suggests that SIP3 sensitivity to internalisation is regulated specifically depending on celltype and does not require receptor phosphorylation. This has implications in embryogenesis and also angiogenesis where cell growth and proliferation are vital to development of organs and vessels.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Tim, Dr. Palmer
Date of Award: 2005
Depositing User: Mrs Monika Milewska-Fiertek
Unique ID: glathesis:2005-31020
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 08 Nov 2018 11:15
Last Modified: 12 Aug 2021 14:46
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