The role of C-terminal cysteines in regulating human proteinase-activated receptor-1 function
Thesis or dissertation
- © 2010 Xiaodan Guo. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder.
Proteinase-activated receptors (PARs) are a novel group of G-protein coupled receptors (GPCRs). The most striking evidence to distinguish them from other GPCRs is that they carry their own tethered ligand within extracellular N-terminus. To activate the receptors, the tethered ligand is exposed by proteolytic cleavage which subsequently binds to the extracellular loop 2 to trigger receptor function. Four members have been identified so far in this group—PAR1, PAR2, PAR3 and PAR4. Palmitoylation is the reversible covalent attachment of fatty acids to the cysteine residues of some GPCRs via a thioester linkage; resulting in a fourth intracellular loop. Although recent evidence has suggested that palmitoylation can have a profound effect on GPCR function such as cell surface expression and receptor signalling, the role of palmitoylation in regulating PAR function is currently unknown. This study focused on the role of putative palmitoylation region of hPAR1--C-terminal cysteines (C387and C388) in regulating receptor function.
Wild type hPAR1 (wt-hPAR1) and hPAR1 mutants (hPAR1C387A, hPAR1C388A and hPAR1C387AC388A) were constructed and permanently expressed in Kirsten virus sarcoma transformed rat kidney epithelial cells (KNRK). hPAR1C387A and hPAR1C388A displayed similar cell surface expression (~80%) to that of wt-hPAR1, but hPAR1C387AC388A displayed only ~40% cell surface expression. hPAR1C387A, hPAR1C388A and wt-hPAR1 displayed similar sensitivity in calcium signalling towards selective PAR1 agonists—thrombin and TFLLR-NH2. Surprisingly, hPAR1C387AC388A failed to generate a calcium signal to either PAR1 agonists. The reduced cell surface expression of hPAR1C387A388A was not responsible for the lack of calcium signal since a wt-hPAR1 cell line with similar cell surface expression to hPAR1C387AC388A displayed robust responses to both thrombin and TFLLR-NH2. hPAR1C387AC388A was also unable to trigger ERK1/2 phosphorylation in response to either PAR1 agonists. In agonist triggered internalisation experiments all mutant receptors internalised in response to thrombin and TFLLR, except for wt-hPAR1 which only internalised in response to thrombin. Therefore, we conclude that putative palmitoylation sites within hPAR1 regulate receptor expression, agonist triggered internalisation and are critical for hPAR1 coupling to calcium and ERK1/2.
- Postgraduate Medical Institute, The University of Hull
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