Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.494854
Title: The role of intracellular and extracellular cysteines in regulating human proteinase-activated receptor-2 (hPAR2) function
Author: Botham, Andrew Michael
ISNI:       0000 0001 3471 5934
Awarding Body: University of Hull
Current Institution: University of Hull
Date of Award: 2007
Availability of Full Text:
Access through EThOS:
Access through Institution:
Abstract:
hPAR2, a member of the novel family of proteolytically-activated G-protein coupled receptors termed Proteinase-Activated Receptors (PAR), has recently been implicated in cardiovascular disease. Previous pharmacological studies have found that activation of hPAR2 by mast cell tryptase, (the major PAR2 activator outside the gastrointestinal tract) 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 three extracellular receptor cysteines (C22, C148, and C226) and one intracellular cysteine (C361).The putative disulphide bridging site (C148 in ECL1 and C226 in ECL2) of hPAR2 were mutagenically removed both individually and together. Mutagenic removal of C226 resulted in ablation of receptor cell surface expression and intracellular retention of receptor. Mutagenic removal of C148 resulted a receptor successfully expressed at the plasma membrane with only a small reduction in cell surface expression over wildtype. The hPAR2C148A mutant still retained the ability to signal through ERK MAP kinase and internalise post-activation with trypsin but was incapable of agonist mediated Ca2+ mobilisation. Removal of C22 resulted in a receptor with similar ERK signalling to wt-hPAR2 but altered agonist-mediated Ca2+ mobilisation. hPAR2C22A showed no change in sensitivity toward SLIGKV-NH2, but a slight decrease in sensitivity towards trypsin, increasing to similar to wt-hPAR2 on pre-treatment with thrombin.The role of the putative palmitoylation site (C361) in regulating hPAR2 function was explored. We demonstrated, using autoradiography, that C361 is the primary palmitoylation site of hPAR2. hPAR2C361A displayed greater cell surface expression compared to wt-hPAR2. The hPAR2C361A also showed a decreased sensitivity and efficacy (intracellular calcium signalling) towards both trypsin and SLIGKV-NH2. In stark contrast hPAR2C361A triggered greater and more prolonged ERK phosphorylation compared to that of wt-hPAR2. Inhibitor studies revealed that hPAR2C361A triggered the majority of the ERK signal through Gi, since pertussis toxin completely inhibited this receptors ability to activate ERK. Finally, flow cytometry was utilised to assess the rate, and extent of receptor internalisation following agonist challenge. hPAR2C361A displayed faster internalisation kinetics following trypsin activation, compared to wt-hPAR2, whilst SLIGKV-NH2 had negligible effect on internalisation for either receptor.This study has highlighted the importance of post-translational modifications in regulating PAR2 function. More specifically we have shown that palmitoylation of C361 on hPAR2 plays a pivotal role in regulating the ability of the receptor to signal to Gq and Gi. Thus, we have identified a potential target site within PAR2 that maybe useful in the design of novel therapeutic agents for cardiovascular disease.
Supervisor: Not available Sponsor: British Heart Foundation (Sponsor)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.494854  DOI: Not available
Keywords: Medicine
Share: