The long term goal of the proposed research is an understanding of the molecular basis of cutaneous pigmentation in normal and pathologic states. The overall goal during the four years for which support is requested is to delineate the basic mechanisms by which a melanocyte creates its specialized organelle, the melanosome, and to better understand the function of melanosomal proteins. Efforts will be directed at four specific aims: 1. Melanosomal-lysosomal relationship. The Stage I melanosome demonstrates characteristics of both the melanosomal and endosomal/lysosomal lineage of organelles. Cellular and biochemical techniques will be used to address the nature of this organelle which is the key to understanding """"""""melanolysosomal"""""""" disorders such as the Chediak- Higashi. Whether the melanosome is a """"""""specialized"""""""" lysosome or whether the Stage I melanosome is a point of divergence for the melanosomal and lysosomal lineage of organelles will be determined. 2. Delineation of the role of Tyrosinase-related protein-1. TRP-1 is a marker for the early melanosomal compartment. Mutations in this protein affect melanosomal structures and the quality and quantity of melanin made. Mouse coat color genetics, cell biologic and biochemical techniques will be employed to examine the consequences of mutations in TRP-1 on its subcellular distribution and its role in melanization. The hypothesis that racial differences in skin color are due to differences in the amount, distribution and function of TRP-1 will be tested. 3. Delineation of the role of Tyrosinase-related protein-2. The enzyme dopachrome tautomerase is identical to a second tyrosinase-related protein encoded at the slaty locus. DHICA, the product of the reaction catalyzed by this enzyme, is a major component of melanin. The synthesis and subcellular distribution of TRP-2 and the nature of the """"""""melanogenic complex"""""""" of which it is a part will be examined. The slaty mutation will be employed to gain insight into the first post-tyrosinase steps in melanogenesis. 4. Pharmacologic regulation of pigmentation. Both retinoic acid and hexamethylene bisacetamide inhibit pigmentation in cultured melanoma cells, but only retinoic acid causes a derangement of melanosomes. Melanocytes will be treated with these agents and their effects on the expression, subcellular distribution and function of the proteins of interest analyzed to understand how each drug works to affect pigmentation at the biochemical and cellular level.
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