Receptor mediated and fluid phase pathways for internalization of the ER Hsp90 chaperone GRP94 in murine macrophages

Immunization of mice with GRP94, the endoplasmic reticulum (ER) Hsp90, elic its cytotoxic T lymphocyte (CTL) responses to chaperone-bound, source cell- derived peptides, Elicitation of a CTL response requires that GRP94-associa ted peptides be transferred onto major histocompatability complex (MHC) cla ss I molecules, a process that is postulated to accompany GRP94 internaliza tion by antigen presenting cells, such as macrophages (M Phi) and dendritic cells (DC). In studies of GRP94 uptake in elicited M Phi, we report that M Phi display specific cell surface binding of GRP94, and that surface-bound GRP94 can be internalized via receptor mediated endocytosis. GRP94 interna lized by this pathway colocalized predominately with transferrin-positive e arly endosomes. At time periods of up to 20 minutes, little trafficking of GRP94 to the lysosomal compartment was observed. When GRP93 was present in the medium, and thus accessible to both receptor-mediated and fluid phase i nternalization pathways, internalization was modestly inhibited in the pres ence of yeast mannan, a competitive inhibitor of mannose/fucose receptor ac tivity, and substantially inhibited by dimethylamiloride, an inhibitor of m acropinocytosis. GRP94 internalized via macropinocytosis did not display pr ominent co-staining with the lysosomal marker LAMP-2, These data identify m ultiple pathways of GRP94 internalization and indicate that receptor-depend ent uptake of GRP94 is not dependent upon its high mannose oligosaccharide moiety, Most significantly, these data demonstrate the existence of cell su rface receptor(s), apparently unique to antigen presenting cells, that func tion in the binding and internalization of the ER chaperone GRP94.