Nilsson Lock, Gun Antonia Evelina (2011) Mutational and computational characterization of transmembrane domains in the fungal G protein-coupled pheromone receptors STE2 and Mam2. PhD thesis, University of Warwick.

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Abstract

G protein-coupled receptors (GPCRs) comprise the largest family of cell-surface receptors involved in sensing a multitude of ligands and are consequently attractive pharmacological targets. Their study is complicated by cross-talk between signalling pathways and altered receptor pharmacology due to, for instance, receptor oligomerization. Difficulties in obtaining structural information of the receptors hinder the understanding of oligomerization and therefore it is desirable to develop alternative approaches in which to study this phenomenon. The fungal pheromone GPCRs, STE2 and Mam2, from Saccharomyces cerevisiae and Schizosaccharomyces pombe respectively are both known to oligomerize and a GxxxG motif in the first transmembrane (TM) domain of STE2 has previously been shown to mediate receptor oligomerization. Previous work on polytopic proteins suggest that individual TM helices may be treated as individually stable domains, and it may therefore be possible to study oligomerization via single TM peptides as opposed to full-length receptor. This thesis describes the use of STE2 and Mam2 to explore TM helix oligomerization and the effects of mutations on receptor trafficking, localization and cellular signalling. The development of a luminescent reporter assay for Sz. pombe, which proved more sensitive than previously used assays and is capable of generating high-throughput data, is also discussed. It was found that STE2 could couple to the Sz. pombe pheromone-response pathway and mutations in the GxxxG dimerization motif affected both signalling and trafficking. Expression of the first TM GxxxG containing domain of STE2 was insufficient for oligomerization, in line with previous reports suggesting that the presence of the second domain is required for receptor oligomerization. In Mam2, a motif was identified that appeared homologous to the STE2 dimerization motif and mutations of this motif also affected trafficking and signalling. This domain could oligomerize in isolation, and mutations of the motif abolished oligomerization. In contrast the study of more polar TM domains appeared more complicated. These findings suggest that relatively hydrophobic TM domains can be studied as individually stable units, whereas more polar domains may require the presence of other TM domains.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Cell receptors, Oligomers, Saccharomyces cerevisiae -- Physiology, Schizosaccharomyces pombe -- Physiology
Date:	September 2011
Institution:	University of Warwick
Theses Department:	Molecular Organisation and Assembly in Cells
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Ladds, Graham Robert ; Dixon, Ann, Ph.D.
Sponsors:	University of Warwick. Molecular Organisation and Assembly in Cells ; Engineering and Physical Sciences Research Council (EPSRC)
Extent:	xviii, 255 p. : ill., charts
Language:	eng
URI:	http://wrap.warwick.ac.uk/id/eprint/50223

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