Mechanisms of transmembrane signalling by tetraspanins

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In this project the structures and interactions of the human tetraspanin proteins will be investigated by heteronuclear magnetic resonance spectroscopy. We have discovered that tetraspanin C-termini are bound by the tandem PDZ proteins of syntenin, and intend to elucidate the structural basis of these interactions in order to understand how they influence receptor signaling and endocytosis. The novel regulatory effects of PDZ domain phosphorylation and stabilizing terminal extensions will be investigated in order to build a model of the signaling mechanism. The extracellular domain has been expressed for NMR analysis of its lipid interaction sites and protein docking studies, providing a basis for defining its function as an cell surface receptor. Tetraspanins have been expressed as functionally intact full length proteins, and production will be scaled up to analyse their structural and binding properties in vitro. In particular, we seek to determine the oligomeric state and structural properties of the proteins in the free and ligand bound states using mixed micelles to solubilize the intact receptor. Together with collaborative in vivo cell biological studies this will provide a better understanding of the structural mechanism of tetraspanin signaling.