McDonald lab Signalling and Structural Biology Laboratory
: Cell surface receptor activation and oncogenic deregulation
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To understand how extracellular signals are received at the cell membrane and trigger intracellular signalling pathway activation, we study the receptor tyrosine kinase (RET).
RET is crucial for embryonic and adult development and its mutation underlies three human diseases (Hirschsprung's disease, kidney agenesis and cancer).
We are interested in how RET becomes activated in response to binding a bipartite ligand that is comprised of a glial cell line derived neurotrophic factor (GDNF) family ligand and a GDNF family receptor a (GFRα) co-receptor.
To visualise this interaction, we have reconstituted several vertebrate RET ternary complexes containing both ligand and co-receptor, permitting a hybrid structural approach. The downstream consequence of RET ligand occupancy is the trans-phosphorylation of internal tyrosine sites within the tyrosine kinase catalytic domain.
Acareful analysis of the kinetics of tyrosine auto-phosphorylation for wild type and oncogenic RET mutants using label-free mass spectrometry has revealed surprising differences in the order of auto-phosphorylation.
We are continuing to characterise how regions flanking the core RET kinase domain contribute to ligand-dependent activation as well as how they are perturbed in an oncogenic context.
Our earlier structural work on RET identified a folding bottleneck that could be eliminated by removal of just two amino acids.
Removing these residues in a panel of RET Hirschsprung's (HSCR) disease mutants restored cell surface expression to most of these mutants instead of their being retained within the endoplasmic reticulum as immature forms.
Recently we carried out a small biased siRNA screen to identify components of the RET maturation pathway that could rescue HSCR mutants and influence cell surface levels of wild type RET.
Characterising factors that control RET maturation and export will help improve our understanding of how thresholds of wild type and pathological RET signalling are set.
