The heat shock protein gp96 forms non-covalent complexes with peptides. Gp96-peptide complexes, upon immunization, elicit peptide-specific MHC I-restricted CD8+ T lymphocyte responses. The high efficiency of this process (as little as I ng peptide complexed to 10 ug gp96 is sufficient) and the strigent requirement of professional antigen presenting cells (APCs) for the immunogenicity of gp96-peptide complexes led to the suggestion that APCs possess receptors for gp96 (gp96R). Gp96 bindings to the surface of APCs but not other cells in a saturable and competable manner and is internalized by APCs through receptor-mediated uptake by clathrin-coated pits. A 75kda gp96-binding molecule expressed on the surface of APCs but not other cells has been identified as a candidate gp96R. Uptake of gp96-bound peptides through gp96R leads to re-presentation of the peptides by MHC I of the APCs. The application aims to identify and characterize gp96R and its gene(s) and plans to carry out: 1. Identification and characterization of gp96-binding proteins on the surface of APCs. * Cross-linking of gp96 with gp96-binding proteins from APCs using reversible cross-linkers; * Chromatography of surface proteins of APCs over immobilized gp96 affinity columns; * Generation of monoclonal antibodies to gp96R using inhibition of re-presentation as assay; * Purification and characterization of gp96R. 2. Identification and characterization of transcripts specific for APCs which can re-present gp96-chaperoned peptides: * Subtraction of cDNA gp96R-cells from that of gp96R+cells; * Use of differential display to isolate gp96R transcripts; * Screening of expression library from gp96R+cells by gp96; 3. Determination if gp96,hsp90, hsp70 and Calreticulin share receptors on APCs: *Competition among the four HSPs for binding to APCs and re-presenting chaperoned peptides; 4. Characterization of the intracellular pathway through which gp96-peptide complexes traffic within the APC: *Ultrastructural analysis where gp96 and the peptide are tagged with different indicator moieties and the fate of each is followed through electron microscopy; *Analysis of epitope-flanking sequences which affect the processing and processing and presentation of gp96-chaperoned peptides in a manner that is different from that of un-chaperoned peptides.
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