Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.787576
Title: Exploring the potential signalling interplay between WNK-SPAK/OSR1 kinases and the β2-adrenergic receptor
Author: Elzwawi, Abdulrahman
ISNI:       0000 0004 7972 6874
Awarding Body: University of Birmingham
Current Institution: University of Birmingham
Date of Award: 2019
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Abstract:
WNK, SPAK and OSR1 kinases are three serine/threonine protein kinases that regulate the function of cation chloride co-transporters (CCC). Such function has implicated these kinases in the regulation of electrolyte balance and hence blood pressure. This was confirmed by the discovery that genetic mutations in WNK kinases cause an inherited form of hypertension knowns as Gordon's syndrome. At the molecular level, WNK kinases become activated under osmotic stress leading to phosphorylation of SPAK and OSR1 kinases, which are their physiological substrates. Consequently, SPAK and OSR1 kinases phosphorylate CCCs and therefore switches on or off the transport of electrolytes inside and outside cells. To date, the only physiological substrates of SPAK and OSR1 reported are ion co-transporters. Aiming to identify novel substrates of SPAK and OSR1 kinases, we discovered that the human β2-adrenergic receptors (β2AR) contains an RFxV tetrapeptide, a unique peptide sequence of SPAK and OSR1 substrates. Thus, in this work, we focused on studying the possible interaction between the β2AR and SPAK /OSR1 kinases, and the role of such interaction on the function of this receptor. Using peptide pulldown assays, we showed that the β2AR-derived RFHV peptide binds endogenous SPAK and OSR1. Additionally, the binding of overexpressed SPAK and OSR1 kinases to overexpressed β2AR in HEK293 cells was competed out by titration of the RFQV peptide. Notably, SPAK and OSR1 binding to the β2AR was inhibited by mutating the β2AR-RFHV motif to AFHV or by single point mutations in SPAK and OSR1 C-terminal domains, the site that binds the RFxV tetrapeptides. Critically, the binding of the β2AR to SPAK and OSR1 kinases was confirmed using the pulldown assays of over-expressed proteins. Using in vitro kinase assays, OSR1 was able to phosphorylate the human β2AR and this phosphorylation was significantly reduced when the mutant β2AR-R239A was used or SPAK and OSR1 kinase inhibitors were used. Stimulation of the β2AR in cells by the agonist isoproterenol did not induce WNK-mediated SPAK and OSR1 activation. Overall, our results confirm a novel interaction between the human β2AR and SPAK and OSR1 kinases. Given, the central role the β2AR plays in many human diseases such as blood pressure, heart failure, asthma and COPD, our findings provide a link between the electrolyte balance regulation by SPAK and OSR1 kinases to these β2AR-involving diseases.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.787576  DOI: Not available
Keywords: Q Science (General) ; R Medicine (General)
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