Tyrosinase catalyzes the initial and rate limiting step in the biosynthesis of melanin. In addition, two other tyrosinase related proteins, gp75 (or TRP-1, encoded by the brown locus) and TRP-2 play important roles in pigmentation. Although these proteins are all related, they exhibit differences in the rates of intracellular synthesis and transport. A correlation of these differences with the structural differences among these proteins would provide fundamental insights into the cell biology of melanogenesis and the control of pigmentation. Moreover, how these proteins are differentially processed intracellularly impacts on the immune system responses in patients with vitiligo and melanoma.
The specific aims and proposed strategies are: 1) To investigate the role played by specific domains (luminal, membrane anchoring, and cytoplasmic) of tyrosinase, gp75 and TRP-2 on the rate of transport from the ER. Mutant and chimeric constructs of these proteins will be constructed and the kinetics of their biosynthesis and intracellular transport will be studied. 2) To investigate the role of molecular chaperones in the kinetics of intracellular transport of melanosomal proteins. Chemical cross-linking of newly synthesized wild type and chimeric proteins expressed in fibroblasts and in melanoma cells will be employed to study the kinetics of association of endoplasmic reticulum (ER) resident chaperones, calnexin and BiP, in relationship to the kinetics of transport from the ER. Similar cross-linking experiments will be employed to study protein-protein interactions. 3) To investigate the relationship of retention of the brown mutant gp75 in the ER to pigmentation by coexpression of a human brown mutant gp75 generated by in vitro mutagenesis.
