This proposal describes a structural biology research project aimed at determination of the three-dimensional structure of the extracellular domain of RET. RET is a unique member of the family of receptor tyrosine kinases. Its biological function is to med iate signalling by members of the GDNF family of neurotrophic factor across the plasma membrane. GDNF signalling through RET is crucial for survival of dopaminergic neurons in the substantia nigra. These neurons are progressively lost in Parkinson¿s diseas e. RET and GDNF are therefore important proteins in the search for treatments of Parkinson's disease. Signalling through RET is impaired in the congenital human disease Hirschsprung¿s disease, where point mutations in RET induce misfolding and prevents RET from reaching the membrane. Contrarily, mutations found in various parts of the ret gene lead to oncogenic transformation. Mutations found in the extracellular domain of RET cause a cancer known as multiple endocrine neoplasia 2A (MEN2A). To allow mechani stic analysis of the modus operandi of RET in both loss- and gain-of-function variants, we will determine the structure of extracellular domain of RET using x-ray crystallography. In addition to understanding the RET protein architecture, we will produce R ET mutant variants, which are misfolded in patients with Hirschsprung¿s disease, but which can be produced recombinantly in a functional state. A direct comparison of wild-type RET with a folding-rescued RET mutant involved in HSCR will the address the que stion whether major structural changes are induced in the RET structure by the mutations found in Hirschsprung¿s disease. Taken together, this proposal constitutes a natural extension of the research interest of the applicant and will provide cutting-edge information on the structural mechanisms of signal transduction, protein folding/misfolding and provides the applicant with advanced training in protein crystallography.
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