Characterisation of Melanocortin 4 Receptor Constitutive Activity: Signalling by Signature

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dc.contributor.advisor Mountjoy, K en
dc.contributor.advisor Montgomery, J en
dc.contributor.author Botha, Rikus en
dc.date.accessioned 2018-03-05T01:58:30Z en
dc.date.issued 2017 en
dc.identifier.uri http://hdl.handle.net/2292/36988 en
dc.description.abstract Cell signalling by the human melanocortin 4 receptor (hMC4R) suppresses food intake and increases energy expenditure, and defective mutation within the hMC4R represents the most common monogenetic cause of obesity. Perplexingly, six out of nine hMC4R constitutively active mutations (CAMs) associate with obesity, despite conferring a ligand-independent increase in cyclic adenosine monophosphate (cAMP) response element (CRE) reporter activity. Serendipitously discovered, hMC4R co-expression with human melanocortin 2 receptor accessory protein α (hMRAPα), but not related hMRAP2, robustly increased constitutive cAMP production by adenylyl cyclase (AC). Henceforth, a conundrum ensues – how can CAM hMC4R associate with obesity, and in light of this, would hMC4R co-expression with hMRAPα be beneficial? To address this conundrum, hypotheses were formulated that 1) at least two distinct CAM signalling conformations exist, one of which associates with obesity, and 2) hMC4R coexpression with hMRAPα may protect from obesity. To address these hypotheses, this study employed a hemagglutinin (HA) epitope tagged hMC4R wild-type (HA-hMC4R-WT) vector to generate nine CAM HA-hMC4Rs, as well as eight defective obesogenic HA-hMC4Rs. Mutant HA-hMC4R cell signalling was directly compared with HA-hMC4R co-expression with hMRAPα or hMRAP2, as well as HA-hMC4R-WT, within human embryonic kidney 293 (HEK293) cells. Only three CAM HA-hMC4Rs (obesogenic H76R and L250Q; non-obesogenic H158R) of 17 tested HA-hMC4Rs were confirmed to have constitutive CRE reporter activity, as did HA-hMC4R co-expression with hMRAPα (but not hMRAP2). Further molecular characterisation identified a common signalling signature between obesogenic CAM HAhMC4Rs and HA-hMC4R co-expression with hMRAPα. In addition to increased constitutive CRE reporter activity, the signalling signature consisted of increased constitutive AC activity, reduced cell surface and total cellular expression, normal α-melanocyte stimulating hormone (α- MSH) stimulated AC and extracellular regulated kinase (ERK) activity, but impaired α-MSH stimulated CRE reporter activity. At the molecular level, the signalling signature did not follow orthodox cAMP/protein kinase A/cAMP response element binding protein signal transduction, but was dependent on dynamin activity. This suggested constitutive activity was predominantly generated intracellularly. These findings confirm existence of two distinct constitutive signalling conformations, one obesogenic and one non-obesogenic, and if hMC4R co-expression with hMRAPα occurred in vivo a likely obesogenic signalling signature would ensue. en
dc.publisher ResearchSpace@Auckland en
dc.relation.ispartof PhD Thesis - University of Auckland en
dc.relation.isreferencedby UoA99265058112902091 en
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. en
dc.rights.uri https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm en
dc.rights.uri http://creativecommons.org/licenses/by-nc-nd/3.0/nz/ en
dc.title Characterisation of Melanocortin 4 Receptor Constitutive Activity: Signalling by Signature en
dc.type Thesis en
thesis.degree.discipline Biomedical Science en
thesis.degree.grantor The University of Auckland en
thesis.degree.level Doctoral en
thesis.degree.name PhD en
dc.rights.holder Copyright: The author en
dc.rights.accessrights http://purl.org/eprint/accessRights/OpenAccess en
pubs.elements-id 728670 en
pubs.record-created-at-source-date 2018-03-05 en


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