Tyrosinase and gp100 function in melanin biosynthesis and are resident integral membrane proteins of a tissue-specific, lysosome-like organelle called the melanosome. Failure of melanocytes to properly sort tyrosinase or gp100 to the melanosome results in developmental and pigment defects such as oculocutaneous albinism. Melanosomal sorting may also affect immune responses to melanoma; tyrosinase and gp100 are among the few human tumor-associated antigens recognized by CD4+, major histocompatibility complex (MHC) class II-restricted T cells. The mechanisms by which these proteins are sorted to melanosomes and the relationship between melanosomal and MHC class II antigen sorting pathways are poorly understood. We hypothesize that these two pathways utilize similar intracellular sorting mechanisms and that proper sorting is necessary for MHC class II-dependent antigen presentation. In addition, tyrosinase is retained within the endoplasmic reticulum (ER) and degraded by the proteasome under certain developmental conditions. Proteasomal degradation may enhance antigen presentation by MHC class I molecules. We hypothesize that tissue-specific activities regulate the folding and/or assembly of tyrosinase and affect antigen processing in melanic and non-melanic cells.
The Specific Aims are: 1. To identify melanosomal sorting determinants within tyrosinase and gp100. Transfected cells will be analyzed morphologically for localization of full-length molecules with targeted mutations or of chimeric proteins containing isolated topologic domains derived from tyrosinase and gp100. Cellular proteins that interact with identified determinants will be characterized biochemically. 2. To characterize the determinants of tyrosinase retention within the endoplasmic reticulum of non-melanic cells. Cell type differences between melanic and non-melanic cells in the ER protein folding environment and in the assembly and stoichiometry of tyrosinase and chimeric proteins containing isolated tyrosinase topologic domains will be determined using biochemical and genetic criteria. 3. To determine whether sorting and folding affect tyrosinase (and gp100) recognition by CD4+ and CD8+ T lymphocytes, respectively. Melanosomal proteins will be colocalized with MHC molecules in melanoma and transfected non-melanic cells. The effect of altering the protein sorting and/or retention properties of tyrosinase and gp100 on recognition by specific T cell clones will be determined.
