G-protein coupled receptors (GPCRs) represent the largest family of transmembrane proteins in the human genome. Their biological and medical significance has driven extensive research into their structure and function, yet a number of important aspects of their behaviour remain unresolved. Arguably the most contentious debate in the field concerns whether or not the receptors form stable homo- and hetero-oligomeric interactions, and there is currently no consensus on the extent or purpose of GPCR oligomerisation. In this thesis, the ‘typical’ stoichiometry of Rhodopsin family GPCRs is investigated via the examination of more than 60 receptors natively expressed by human HEK 293T cells using bioluminescence resonance energy transfer (BRET). Assaying receptors in the cells in which they are natively expressed maximises the likelihood of authentic assembly and trafficking while simultaneously providing an unbiased cross-section of the whole GPCR family. In order to make such an investigation possible, the sensitivity of existing BRET approaches for partial homodimers had to be, and was, confirmed, and a complementary competition-based BRET method suitable for a semi high-throughput analysis was developed. Application of these assays to the HEK 293T GPCR repertoire revealed that the Rhodopsin family is very predominantly monomeric but contains a small fraction of independently evolved dimers comprising small phylogenetic clusters of receptors. The mechanism of Rhodopsin family dimerisation was in some cases found to be reliant on interactions between transmembrane helices, in contrast to other families of GPCRs, which were observed or are known to be exclusively dimeric due to interactions between their N-terminal domains. The mechanism of dimerisation in Rhodopsin family GPCRs may preclude constitutive dimerisation but allow heterodimerisation of closely related receptors, as observed for a subset of receptors using a third type of assay designed to detect heterodimers. Taken together, these observations suggest a model of GPCR evolution in which dimers either have a selective disadvantage compared to monomers, or for which dimerisation offers no apparent selective advantage. These findings suggest that receptor stoichiometry is at least partly responsible for several of the remarkable features of GPCR family structure, including the very large size of the family as a whole, the great diversity of Rhodopsin family GPCRs, and the origins of sensory receptors.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:647545 |
| Date | January 2013 |
| Creators | Felce, James H. |
| Contributors | Davis, Simon J. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:ae078d13-fef7-4f1f-a8ea-c2e5412f59ff |
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