Henderson, James George ORCID: 0000-0002-0709-5124 (2021) Towards the Structures of the Parathyroid Hormone Receptors. PhD thesis, University of Leeds.
Abstract
G protein-coupled receptors (GPCRs) are a superfamily of membrane proteins that transduce extracellular signals to invoke physiological responses. Class B GPCRs are regulated by peptide hormones and are characterised by a seven transmembrane domain, a 120-150 residue extracellular domain (ECD), and an intracellular C-terminus. The parathyroid hormone 1/2 receptors (PTH1R/PTH2R) belong to this family and, despite being closely related, have different physiological roles: PTH1R is crucial in bone metabolism, whereas PTH2R function is significantly less well defined. The original aims of this research were to solve the structures of these two receptors. This involved creating various receptor constructs that would be suitable candidates for downstream structural trials. Insect cell expressed receptors were quantified through fluorescent intensity densitometry analysis and a potential candidate for further structural studies was found in the form of a mini Gs protein bound to an apocytochrome, b562RIL (BRIL) fusion protein following a FLAG purification. However, a constitutively active receptor, created by tethering PTH(1-14) to BRIL-PTH1R and verified through LANCE® cAMP assays, was unable to form this complex. PTH2R proved a major challenge, so attempts to solve the isolated ECD were made instead. This was pursued using Escherichia coli expressed protein in various oxidising environments, though due to low protein yields and poor stability it was impossible to obtain a candidate for further trials. Finally, the novel integral membrane protein stability selector (IMPROvER) program was used to create a thermostable PTH1R. Through fluorescent based thermostability assays, 40% of 20 mutations were stabilising with a maximum increase of 3.4 ± 1.1 °C. Combinations of these mutations resulted in a construct with an improved stability of 10 ± 0.76 °C. This research gives a strong foundation for future structural work regarding PTH1R and has highlighted that IMPROvER is a valid alternative to more laborious methods such as alanine scanning.
Metadata
Supervisors: | Goldman, Adrian and Donnelly, Dan |
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Related URLs: | |
Keywords: | Parathyroid hormone; PTH; PTH1R; PTH2R; IMPROvER; GPCR; Family B; Secretin-like; Structure |
Awarding institution: | University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) The University of Leeds > Faculty of Biological Sciences (Leeds) > Institute for Molecular and Cellular Biology (Leeds) The University of Leeds > Faculty of Biological Sciences (Leeds) > Institute of Membrane and Systems Biology (Leeds) |
Identification Number/EthosID: | uk.bl.ethos.837096 |
Depositing User: | Mr James Henderson |
Date Deposited: | 07 Sep 2021 09:38 |
Last Modified: | 11 Oct 2021 09:53 |
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