Jimbow K, Gomez P F, Toyofuku K, Chang D, Miura S, Tsujiya H, Park J S
University of Alberta, Division of Dermatology & Cutaneous Sciences, Edmonton.
Pigment Cell Res. 1997 Aug;10(4):206-13. doi: 10.1111/j.1600-0749.1997.tb00486.x.
Tyrosinase-related protein (TRP)-1 is one of the most abundant melanosomal glycoproteins involved in melanogenesis. This report summarizes our recent research efforts related to the biological role and biosynthesis of TRP-1 and its transport from TGN (trans-Golgi network) to the stage I melanosome. Our UV irradiation and tyrosinase and TRP-1 cDNA co-transfection studies indicated that human TRP-1 is involved in not only melanogenesis but also prevention of melanocyte death, which may occur during biosynthesis of melanin pigment in the presence of tyrosinase. Furthermore, a coordinated gene interaction was indicated between tyrosinase and TRP-1, resulting in upregulation of mRNA and protein expression of LAMP (lysosome-associated membrane protein)-1 that would directly prevent the tyrosinase-mediated programmed cell death of melanocytes. Similar to tyrosinase, however, TRP-1 appears to require a molecular chaperone, calnexin, which we have cloned recently. Our cDNA transfection study of tyrosinase with calnexin showed clearly the necessity of calnexin in order to have efficient, functional activity of melanosomal glycoprotein, especially tyrosinase. Once glycosylation is completed, TRP-1 will be transported from TGN to the stage I melanosome. At this stage, TRP-1 will have its own target signal, in particular, tyrosine-rich leucine residues in cytoplasmic tail. Our TRP-1 cDNA transfection and immunoelectron microscopy study shows that TRP-1 will be transported through small vesicles, probably non-clathrin-coated type, to large vacuoles, identical to the MPR (mannose-6-phosphate receptor)-positive, late endosomes. In this transport process a low molecular weight G-protein, rab-7, was isolated from the purified melanosomal protein on 2D-PAGE and identified by subsequent sequencing and PCR amplification. Confocal microscopy with double immunostaining and immunoelectron microscopy confirmed the co-localization of rab-7 and TRP-1 in the melanosomes with early stages of maturation (I-HI). Furthermore, this process will also be regulated by phosphatidylinositol 3-kinase (PI-3 kinase).
酪氨酸酶相关蛋白（TRP）-1是参与黑色素生成的最丰富的黑素小体糖蛋白之一。本报告总结了我们最近关于TRP-1的生物学作用、生物合成及其从反式高尔基体网络（TGN）运输到I期黑素小体的研究工作。我们的紫外线照射以及酪氨酸酶和TRP-1 cDNA共转染研究表明，人TRP-1不仅参与黑色素生成，还参与预防黑素细胞死亡，这种死亡可能在酪氨酸酶存在的情况下黑色素色素生物合成过程中发生。此外，酪氨酸酶和TRP-1之间存在协同基因相互作用，导致溶酶体相关膜蛋白（LAMP）-1的mRNA和蛋白表达上调，这将直接预防酪氨酸酶介导的黑素细胞程序性细胞死亡。然而，与酪氨酸酶类似，TRP-1似乎需要一种分子伴侣钙连蛋白，我们最近已克隆了该蛋白。我们用钙连蛋白进行的酪氨酸酶cDNA转染研究清楚地表明，钙连蛋白对于黑素小体糖蛋白尤其是酪氨酸酶具有高效、功能活性是必要的。一旦糖基化完成，TRP-1将从TGN运输到I期黑素小体。在此阶段，TRP-1将有其自身的靶向信号，特别是细胞质尾部富含酪氨酸的亮氨酸残基。我们的TRP-1 cDNA转染和免疫电子显微镜研究表明，TRP-1将通过小泡运输，可能是非网格蛋白包被型，运输到与甘露糖-6-磷酸受体（MPR）阳性的晚期内体相同的大液泡。在这个运输过程中，一种低分子量G蛋白rab-7从二维聚丙烯酰胺凝胶电泳（2D-PAGE）纯化的黑素小体蛋白中分离出来，并通过随后的测序和聚合酶链反应（PCR）扩增进行鉴定。共聚焦显微镜双免疫染色和免疫电子显微镜证实了rab-7和TRP-1在成熟早期（I-HI）的黑素小体中共定位。此外，这个过程也将受磷脂酰肌醇3激酶（PI-3激酶）调节。
