dc.contributor.advisor |
Hay, D |
en |
dc.contributor.advisor |
Watkins, H |
en |
dc.contributor.author |
Abhayawardana, Rekhati |
en |
dc.date.accessioned |
2013-03-25T00:54:38Z |
en |
dc.date.issued |
2012 |
en |
dc.identifier.uri |
http://hdl.handle.net/2292/20335 |
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dc.description |
Full text is available to authenticated members of The University of Auckland only. |
en |
dc.description.abstract |
Adrenomedullin (AM) and Intermedin/Adrenomedullin 2 (AM2) mediate their effects through AM receptors, which are important targets in the development of new drugs for treating diseases. The AM1 and AM2 receptors are formed when the calcitonin receptor-like receptor (CLR) interacts with the receptor activity-modifying protein (RAMP) 2 and RAMP3 respectively. The elucidation of how these receptors are activated is an important first step towards designing new drugs for treating diseases. The aim of this study is to identify specific amino acid residues of the AM1 receptor that are directly involved in ligand binding and receptor activation. An alanine/leucine scan of extracellular loop 1 (ECL1) and the adjoining top of the transmembrane (TM) regions of CLR was carried out in HEK293S cells expressing wild-type (WT) CLR or mutants with RAMP2. cAMP assays were carried out using ALPHAScreen technology and concentration response curves were generated for AM and AM2. ELISA was also performed to study the cell surface expression. V198A, A199L, V205A, A206L and C212A decreased the potency of AM mediated cAMP responses while it was increased with L195A, Y221A and L222A. The potency of AM2 at L195A was decreased while at A197L, N200A, P209A, and L220A this was increased. For both AM and AM2 mediated responses, the potency of K213A was decreased while it was increased in Q216A. Interestingly more point mutants significantly reduced maximal cAMP response (Emax) in the AM2-mediated cAMP responses including A197L, N200A, N201A, A203L, P209A, S215A and I218A. In AM mediated responses A199L, and L222A decreased Emax whereas it was increased in A206L. In both AM and AM2 mediated responses, the Emax was reduced in L195A, V198A, V210A, C212A, K213A, L220A and Y221A while it was increased with Q216A. There were no significant changes in cell surface expression of the mutant receptors. The findings indicate that the predicted ECL1 of CLR and the adjoining TM regions are involved in both AM and AM2 mediated AM1 receptor activation. There are peptide specific and/or receptor specific effects produced by the individual amino acids and this provides clues about the unique mechanisms of AM1 receptor activation by AM and AM2. |
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dc.publisher |
ResearchSpace@Auckland |
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dc.relation.ispartof |
Masters Thesis - University of Auckland |
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dc.rights |
Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher. |
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dc.rights |
Restricted Item. Available to authenticated members of The University of Auckland. |
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dc.rights.uri |
https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm |
en |
dc.rights.uri |
http://creativecommons.org/licenses/by-nc-sa/3.0/nz/ |
en |
dc.title |
Investigation of the role of amino acid residues in the first extracellular loop of the Adrenomedullin 1 receptor |
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dc.type |
Thesis |
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thesis.degree.grantor |
The University of Auckland |
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thesis.degree.level |
Masters |
en |
dc.rights.holder |
Copyright: The Author |
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pubs.elements-id |
375356 |
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pubs.record-created-at-source-date |
2013-03-25 |
en |
dc.identifier.wikidata |
Q112888642 |
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