MOLECULAR MECHANISM OF GRP94 FUNCTION

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The PI proposes to identify the functional, structural and regulatory properties of GRP94 which define it as a (poly) peptide binding protein. GRP94, the endoplasmic reticulum (ER) homolog of hsp90, is an abundant resident molecular chaperone of the ER lumen. In addition to its chaperone role, studies in animal carcinogenesis models have established that GRP94 can function as a tumor-specific vaccine. Vaccine activity is know to require the uptake of GRP94-peptide complexes by antigen-presenting cells, and the transfer of GRP94- bound peptides to nascent MHC class I molecules in the ER. The molecular signals governing the finding the release of the GRP94 associated peptides are currently unknown, but are certain to prove significant to the identification of the molecular mechanism of GRP94 function and to the development of GRP94 as an immunotherapy agent. To achieve this goal, the PI proposes 1) to define the kinetics and regulation of (poly) peptide binding to GRP94. To this end, the hypothesis that peptide binding by GRP94 is regulated by ribonucleotides will be tested and assays will be developed to identify potential regulatory contributions of resident ER integral and lumenal proteins to the GRP94 peptide binding and release reactions. 2) To perform structural analyses of GRP94 bound peptides and identify peptide binding motifs. Having developed procedures for the purification of native GRP94, a large scale, mass spectrometry based, bound peptide sequencing study will be initiated to test the hypothesis that GRP94 recognizes specific peptide structural motifs. 3) To identify the GRP94 peptide binding site(s) through techniques including chemical and photo-crosslinking, proteolytic domain structure studies, and in vitro binding studies with purified expression constructs and isolated structural domains. 4) To determine the role of GRP94 dimerization in the regulation of peptide binding activity and in vivo function. To test whether dimerization is necessary for function, mutations in the assembly domain that block dimerization will be assessed by in vitro studies of peptide binding and in vivo studies of protein assembly and secretion.