The mechanism of GRP94 regulation remains an open topic. GRP94 is a homodimer containing two identical ligand-binding sites. These ligand-binding sites are targets for the natural anti-tumor compounds geldanamycin (GA) and radicicol (RD) and also function in ATP/ADP binding. Adenosine and its analogues are known to bind GRP94 at a stoichiometry of 1 mol ligand: GRP94 dimer and such binding is regulated through negative cooperativity. We hypothesize that the negative cooperativity of ATP/ADP binding functions to provide a highly sensitive response to cell stress conditions that elicit a decrease in cellular ATP/ADP levels. GA and RD compete nucleotide binding and are able to bind GRP94 at a stoichiometry of 2-mol ligand: GRP94 dimer. We hypothesize that inhibitor ligands that target the adenosine nucleotide binding pocket of GRP94 and override negative cooperativity will function as a novel class of cancer chemotherapeutics. To examine this hypothesis, a broad array of GRP94 chimera will be studied to identify the region(s) of the molecule responsible for the negative cooperativity. In addition to negative cooperativity, there are likely to be other regions of the molecule critical to GRP94 function. To define such additional roles, individual GRP94 domains will be over-expressed and screened for dominant negative inhibitor activity. Together, the proposed analysis will lead to a greater understanding of GRP94 as a novel anti-cancer target.
