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Mutational and computational characterization of transmembrane domains in the fungal G protein-coupled pheromone receptors STE2 and Mam2
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