Studies of oculocutaneous albinism (OCA) have identified two types of OCA, those with mutations in tyrosinase (Type I OCA), the enzyme responsible for the first step in melanin production, and the most common form of OCA, type II or OCA2. OCA2 results from defects in a chromosome 15q11-q13 gene, P, encoding a putative melanocyte-specific protein. Although, P is not directly involved in the enzymatic production of melanin, it appears from structural analysis that the P protein sequence likely has 12 membrane-spanning domains and may represent a novel class of transporter proteins. We suggest that P may be responsible for delivery of substrate or cofactors for melanin biosynthesis, such tyrosine or L-DOPA (3,4-dihydroxyphenylalanine). In this pilot proposal, we will examine the function of the P protein, its intracellular localization, and determine if differential expression of the P protein is associated with the variable degree of hypopigmentation seen in some patients. As subcellular fractionation of melanocytes is difficult and often results in cross-contamination of melanosomal fractions with other organelles (e.g. lysosomes), we will use melanocytes [normal (Melan-A), P-deficient (Melan-P), and tyrosinase- deficient (Melan-C)] in whole cell immunocytochemical analyses with a polyclonal P antibody. Double-labeling with melanosomal- and subcellular organelle-specific antibodies will be utilized to directly determine the intracellular localization of the P protein. In order to directly assay the function ascribed to the P protein, transport analyses with amino acid precursors and cofactors of melanin biosynthesis will be conducted on Melan-A, Melan-P, and Melan-C melanocytes stable-transfected with the human P cDNA. Although P mutations in OCA2 are recessive, single allele deletions of P in Prader- Willi and Angelman syndrome are associated with mild to moderate hypopigmentation in some but not all cases. We suggest that a promoter polymorphism results in differential expression of P and hence, the variable hypopigmentation seen in these patients. This will be tested by cloning of the complete P promoter and identification of polymorphisms in affected and normal individuals. The effects of these promoter polymorphisms will be modeled in expression constructs utilizing luciferase as a reporter gene. Our long term goal in the understanding of P function is the rational design of novel pharmacological agents for the treatment of patients with OCA2 and P- deletion patients with hypopigmentation in Prader-Willi and Angelman syndromes.
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