hPAR2 (human proteinase-activated receptor-2) is a member of the novel family of proteolytically activated GPCRs (G-protein-coupled receptors) termed PARs (proteinase-activated receptors). Previous pharmacological studies have found that activation of hPAR2 by mast cell tryptase can be regulated by receptor N-terminal glycosylation. In order to elucidate other post-translational modifications of hPAR2 that can regulate function, we have explored the functional role of the intracellular cysteine residue Cys361. We have demonstrated, using autoradiography, that Cys361 is the primary palmitoylation site of hPAR2. The hPAR2C361A mutant cell line displayed greater cell-surface expression compared with the wt (wild-type)-hPAR2-expressing cell line. hPAR2C361A also showed a decreased sensitivity and efficacy (intracellular calcium signalling) towards both trypsin and SLIGKV. In stark contrast, hPAR2C361A triggered greater and more prolonged ERK (extracellular-signal-regulated kinase) phosphorylation compared with that of wt-hPAR2 possibly through Gi, since pertussis toxin inhibited the ability of this receptor to activate ERK. Finally, flow cytometry was utilized to assess the rate and extent of receptor internalization following agonist challenge. hPAR2C361A displayed faster internalization kinetics following trypsin activation compared with wt-hPAR2, whereas SLIGKV had a negligible effect on internalization for either receptor. In conclusion, palmitoylation plays an important role in the regulation of PAR2 expression, agonist sensitivity, desensitization and internalization.
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Research Article|
August 12 2011
Palmitoylation of human proteinase-activated receptor-2 differentially regulates receptor-triggered ERK1/2 activation, calcium signalling and endocytosis
Andrew Botham;
Andrew Botham
1Division of Cardiovascular and Respiratory Studies, Postgraduate Medical Institute, Hull York Medical School, Castle Hill Hospital, Castle Road, Cottingham, East Yorkshire HU16 5JQ, U.K.
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Xiaodan Guo;
Xiaodan Guo
1Division of Cardiovascular and Respiratory Studies, Postgraduate Medical Institute, Hull York Medical School, Castle Hill Hospital, Castle Road, Cottingham, East Yorkshire HU16 5JQ, U.K.
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Yu Pei Xiao;
Yu Pei Xiao
1Division of Cardiovascular and Respiratory Studies, Postgraduate Medical Institute, Hull York Medical School, Castle Hill Hospital, Castle Road, Cottingham, East Yorkshire HU16 5JQ, U.K.
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Alyn H. Morice;
Alyn H. Morice
1Division of Cardiovascular and Respiratory Studies, Postgraduate Medical Institute, Hull York Medical School, Castle Hill Hospital, Castle Road, Cottingham, East Yorkshire HU16 5JQ, U.K.
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Steven J. Compton;
Steven J. Compton
1Division of Cardiovascular and Respiratory Studies, Postgraduate Medical Institute, Hull York Medical School, Castle Hill Hospital, Castle Road, Cottingham, East Yorkshire HU16 5JQ, U.K.
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Laura R. Sadofsky
Laura R. Sadofsky
1
1Division of Cardiovascular and Respiratory Studies, Postgraduate Medical Institute, Hull York Medical School, Castle Hill Hospital, Castle Road, Cottingham, East Yorkshire HU16 5JQ, U.K.
1To whom correspondence should be addressed (email L.R.Sadofsky@Hull.ac.uk).
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Publisher: Portland Press Ltd
Received:
November 25 2010
Revision Received:
May 24 2011
Accepted:
May 31 2011
Accepted Manuscript online:
May 31 2011
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2011 Biochemical Society
2011
Biochem J (2011) 438 (2): 359–367.
Article history
Received:
November 25 2010
Revision Received:
May 24 2011
Accepted:
May 31 2011
Accepted Manuscript online:
May 31 2011
Citation
Andrew Botham, Xiaodan Guo, Yu Pei Xiao, Alyn H. Morice, Steven J. Compton, Laura R. Sadofsky; Palmitoylation of human proteinase-activated receptor-2 differentially regulates receptor-triggered ERK1/2 activation, calcium signalling and endocytosis. Biochem J 1 September 2011; 438 (2): 359–367. doi: https://doi.org/10.1042/BJ20101958
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