Most eukaryotic proteins that receive and process signals are constructed from a combination of interaction and catalytic domains. Among the many interaction domains identified in the past decade, PDZ's are one of the most frequently encountered. They are often found in combination with other interaction modules and play a role in directing the specificity of receptor tyrosine kinase-mediated signaling, in establishing cell polarity, in directing protein trafficking, and in coordinating synaptic signaling. Their importance is underscored by the severe neuronal and developmental phenotypes observed in PDZ knockout mice and by their implication in human congenital diseases like Usher syndrome and Dejerine-Sottas neuropathy. The enormous diversity of PDZ function is manifest in their abundance; there are over 250 PDZ's encoded in the mouse genome. To understand their individual roles, it is necessary first to define their recognition properties in a comprehensive and relevant fashion. Previous efforts to define PDZ selectivity have focused on only a few domains and have relied on collections of randomized peptides, rather than on physiological ligands. In order to provide a genome-wide understanding of PDZ function, the selectivity of every PDZ encoded in the mouse genome will be investigated relative to a large collection of genomically-encoded ligands using protein microarray technology.
The aims of this proposal are: (1) to clone, express, and purify every mouse PDZ domain; (2) to screen every PDZ with every PDZ to reveal putative PDZ-PDZ interactions; (3) to screen every PDZ with 222 genomically-encoded peptide ligands to identify putative PDZ-protein interactions; (4) to investigate the physiological relevance of a subset of predicted interactions biochemically; and (5) to construct a relational database that integrates protein microarray data with published information on PDZ domains. Collectively, these efforts should provide the necessary foundation to understand PDZ function on a genome-wide level and aid future efforts to intervene appropriately when PDZ function goes awry.
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