Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.441762
Title: Molecular heterogeneity of rodent and human histamine H₃ and H₄ receptors
Author: Shenton, Fiona C.
ISNI:       0000 0001 3402 9125
Awarding Body: Durham University
Current Institution: Durham University
Date of Award: 2007
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Abstract:
Histamine receptors H3 and H4 are classic G-protein coupled receptors (GPCRs) and are potential therapeutic targets for a number of CNS pathologies and inflammatory diseases, respectively.T hey are highly related both in terms of protein sequencea nd genes tructure, and are likely to share key molecular pharmacological features. Pharmacological heterogeneity between and within species has hindered efforts to bring H3R directed drugs to the clinic. Possible mechanisms underlying this variability are the subject of these investigations.C urrent opinion on GPCRs suggestst hat many function as obligate dimers or higher oligomers. In addition alternative splicing of H3 and H4R cDNA generatess plice variants which result in the potential expression of different receptor isoforms. Two common human H3R isoforms expressedin the basal ganglia are the focus of this thesis: the full length H3( 445i)s oform and the shorter H3( 329i)s oform. For the human H4 receptor only two isoforms have been identified to date. These are drastically truncated versions of the full length receptor. Many of the H3 isoforms and both the H4 isoforms appear to be non-functional with respect to ligand binding and signalling. To determine whether these attributes could be a source of receptor heterogeneity, five major hypotheses have been addressed: 1. Human H3 and H4 receptors are able to homo-oligomerise; 2. Human H3 and H4 receptorsa re able to hetero-oligomerisew ith their respectives plice variants; 3. Splice variants can act as dominant negative regulatory subunits; 4. N-glycosylation is a prerequisite for receptor dimerisation; 5. H3 and H4 histamine receptors are both expressed in brain and subserve distinct functions. A new panel of anti- H3R and H4R specific antibodies have been generated, and used as immunological probes to test the thesis hypotheses using a combination of biochemical,biophysical and pharmacological techniques. Cross-linking experiments suggested the presence of human and rodent H3R and human H4R homo-oligomers, further corroborated by FRET analysis for the hH3R, and by both nickel column purification and BRET in the case of the hH4R. FRET experiments also provided evidence for the existence of heterooligomers, consisting of the full length receptor with a respective shorter isoform. Neither of the truncated hH4 isoforms can bind or transduce a signal. The effect of the two isoforms on surface expression of the full length receptor was investigated using the surfaceb iotinylation technique.R esults confirmed that the shorter isoforms decreasedth e surface expressiono f the full length hH4 (390i)s oform in line with the effects of the two isoforms on the number of [3H] histamine binding sites. Both the human H3 and H4 receptors are modestly N-glycosylated, however N-glycosylation is not a prerequisite for receptor dimerisation. In the case of the H3 receptor preventing glycosylation has only a modest effect on ligand binding to the receptor. in conclusion,v ariability in the level of homo- and hetero-oligomerisationa nd propensity for N-glycosylation may contribute to H3 and H4 receptor heterogeneity. Furthermore shorter isoforms are able to act as dominant negative subunits which would have profound functional consequenceso n both H3 and H4 receptorsi n vivo. This study also provides the first evidence that both the H3 and H4 receptors are expressed in the human and rodent brain
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.441762  DOI: Not available
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