Aberrations in receptor-mediated signal transduction pathways that control cell-growth and differentiation are associated with several disorders, including cancers and immune-system malfunction. The early stages in these pathways involve an array of specific protein- protein interactions and membrane recruitment events, many of which are driven by the multiple modular domains within signaling proteins. These modules include Src homology (SH) SH2 and SH3 domains that mediate protein-protein interactions, and pleckstrin homology (PH) domains that appear to bind to the membrane surface, in some cases by interacting with phosphoinositides. The ultimate objective of the proposed research is to understand at a quantitative level how the multiple domains define specific inter-molecular interactions in cellular signaling. This understanding is essential for the appreciation of cross-talk between pathways, and for potential approaches to pharmacological intervention. In this proposal the focus is PH domain recognition of the membrane surface, and its importance for the function of PH domain-containing proteins. Binding to both phosphoinositides and their inositol phosphate head-groups will be studied for PH domains from four signaling molecules, selected on the basis of their phosphoinositide binding specificity.
The specific aims are: 1) To investigate the thermodynamic driving forces for membrane phosphoinositide binding and high affinity inositol phosphate recognition by the selected PH domains. 2) To determine the structures of two PH domains in complex with their inositol phosphate ligands, and to study the determinants of specificity in specific mutagenesis experiments. 3) To determine whether phosphoinositide binding by PH domains correlates with their physiological role, and to determine whether cooperation with other domains in the same protein is important. These studies will provide a fuller understanding of the way these small domains participate in recruitment of signaling molecules to the membrane surface following activation of cell-surface receptors.
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