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Title: Molecular analysis of rat A3 adenosine receptor regulation
Author: Ferguson, Gail
ISNI:       0000 0001 3460 0766
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2001
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The regulatory effects of adenosine are dependent upon its ability to bind four distinct adenosine receptor (AR) subtypes termed A1, A2A, A2B and A3 (Ralevic and Bumstock, 1998). Despite coupling to the same class of inhibitory guanine nucleotide-binding regulatory protein (G-protein) and binding the same physiological ligand, the A1 and A3ARS are subject to distinct regulatory events following agonist exposure. Rapid termination of G protein-coupled receptor (GPCR) signalling is typically regulated by their phosphorylation by second messenger-activated and/or G protein-coupled receptor kinases (GRKs). Upon phosphorylation by G protein- coupled receptor kinases (GRKs), many GPCRs bind arrestin proteins that serve to uncouple activated receptors from G-proteins leading to a functional desensitisation of G protein-linked signalling, cluster activated receptors to clathrin-coated vesicles (CCVs) and recruit and activate Src family tyrosine kinases ultimately resulting in the activation of the mitogen activated protein kinase (MAPK) cascade. It is thought that receptors proceed from CCVs to endosomes where they may be dephosphorylated and recycled back to the plasma membrane or degraded and down- regulated. In this study, by employing the use of epitope and fluorescently tagged receptors, we show that agonist activation is required for A3AR phosphorylation and internalisation and that this process can be blocked by an A3AR-selective antagonist MRS 1523. We have characterised rates of A3AR internalisation and recycling and shown that it is dynamically controlled by three phosphorylation sites Thr 307, 318 and 319 and two presumed palmitoylation sites Cys 302 and 305 in its carboxyl- terminal (C) tail. Mutation of the Thr sites renders the receptor resistant to phosphorylation by GRKs, ultimately resulting in a loss of receptor sequestration compared to WT A3AR. In contrast, mutation of the Cys residues produces an increase in the rate of receptor internalisation compared to wild type (WT), a phenomenon not observed by the introduction of a similar mutation in the WT A1AR (Cys309Ala, data not shown). By the use of confocal microscopy, we reveal that despite their differences in trafficking rates, both WT and (C-A)A3ARs co-localise with transferrin (Tfn) receptor-positive early endosomes. We also demonstrate that this accumulation is dependent upon receptor phosphorylation as the non-internalising A1AR can be directed through this endosomal pathway by replacing the C-terminal tail with the GRK-phosphorylatable 14 amino acids of the A3AR (A1CT3AR). The process of receptor phosphorylation also dictates the pattern of arrestin distribution following agonist stimulation. A1AR activation caused a re-distribution of arrestin3 to distinct spots at the plasma membrane whereas WT A3AR, (C-A)A3AR and A1CT3 produced an accumulation of arrestin3 at both the plasma membrane and diffusely within the cytoplasm This study also demonstrates that long-term agonist exposure causes a down- regulation in total A3AR number via an as yet undefined pathway and may be directed by the C-terminal region of the receptor (Tsao and von Zastrow 2000b). Consequently, we have presented initial confocal images to suggest that recovery from down-regulation requires newly synthesised receptor to be transported from the Golgi apparatus to the plasma membrane.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry