Defects in protein sorting and vesicular trafficking processes are known to result in human hypopigmentary disorders often associated with storage pool deficiency. Although cytosolic proteins and their interactions with organelle proteins appear to play a role in these processes, cytosolic proteins involved in the regulation of melanosome biogenesis have not been identified. Understanding the regulation of trafficking of melanosomal proteins has implications also for understanding autoimmune pigmentary disorders and immune responses in melanoma. In this proposal, we address the roles of melanosomal membrane protein TRP1/gp75, cytosolic PDZ protein GIPC and signaling protein APPL in melanosome biogenesis. Although gp75, an abundant melanosomal protein, is thought to influence biogenesis of melanosomes, specific role(s) it plays in human skin pigmentation is not known. We reasoned that interaction of cytosolic proteins with the unique C-terminal sequences of gp75 may be important for functions of gp75. We identified a PDZ protein GIPC that interacts with the C-terminus of newly synthesized gp75 and with an adaptor protein APPL. APPL, in turn, binds protein kinase B/Akt. Our working hypothesis is that interaction of GIPC with gp75 influences trafficking of gp75 and provides a link between gp75 sorting and signaling pathways that regulate melanosome biogenesis. The following experiments are designed: 1) We will test the hypothesis that interaction of GIPC with gp75 C-terminus plays a role in gp75 trafficking to premelanosomes. Sorting of newly synthesized mutant gp75 proteins lacking PDZ-binding motif and melanosomal targeting signal will be investigated. 2) We will test the hypothesis that GIPC influences trafficking by palmitoylation of gp75. Palmitoylation of gp75 by GIPC and the role of palmitoylation in gp75 trafficking will be assessed using mutant GIPC lacking acyl carrier domain and mutant gp75 protein lacking palmitoylation acceptor site. 3) We will test the hypothesis that interaction of GIPC with gp75 provides a link, via APPL, between gp75 sorting and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. We will utilize inhibitors of PI3 kinase and expression of mutant APPL proteins lacking Akt binding motif to evaluate the role of PI3 kinase/Akt signaling pathway in gp75 trafficking and melanosome biogenesis.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-GMA-1 (01))
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Moshell, Alan N
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University of Wisconsin Madison
Schools of Medicine
United States
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Kedlaya, Rajendra; Kandala, Gokul; Liu, Tie Fu et al. (2011) Interactions between GIPC-APPL and GIPC-TRP1 regulate melanosomal protein trafficking and melanogenesis in human melanocytes. Arch Biochem Biophys 508:227-33
Xu, Xinjie; Kedlaya, Rajendra; Higuchi, Hitoshi et al. (2010) Mutation in archain 1, a subunit of COPI coatomer complex, causes diluted coat color and Purkinje cell degeneration. PLoS Genet 6:e1000956
Kedlaya, Rajendra H; Bhat, Kumar M R; Mitchell, Julie et al. (2006) TRP1 interacting PDZ-domain protein GIPC forms oligomers and is localized to intracellular vesicles in human melanocytes. Arch Biochem Biophys 454:160-9