Search

Pmel17 is a melanocyte/melanoma-specific protein that is essential for the maturation of melanosomes to form mature, fibrillar, and pigmented organelles. Recently, we reported that the less glycosylated form of Pmel17 (termed iPmel17) is sorted via the plasma membrane in a manner distinct from mature Pmel17 (termed mPmel17), which is sorted directly to melanosomes. To clarify the mechanism(s) underlying the distinct processing and sorting of Pmel17, we generated a highly specific antibody (termed αPEP25h) against an epitope within the repeat domain of Pmel17 that is sensitive to changes in O-glycosylation, αPEP25h recognizes only iPmel17 and allows analysis of the processing and sorting of iPmel17 when compared with αPEP13h, an antibody that recognizes both iPmel17 and mPmel17. Our novel findings using otPEP25h demonstrate that iPmel17 differs from mPmel17 not only in its sensitivity to endoglycosidase H, but also in the content of core 10-glycans modified with sialic acid. This evidence reveals that iPmel17 is glycosylated differently in the Golgi and that it is sorted through the secretory pathway. Analysis of Pmel17 processing in glycosylation-deficient mutant cells reveals that Pmel17 lacking the correct addition of sialic acid and galactose loses the ability to form fibrils. Furthermore, we show that addition of sialic acid affects the stability and sorting of Pmel17 and reduces pigmentation. Alterations in sialyltransferase activity and substrates differ between normal and transformed melanocytes and may represent a critical change during malignant transformation. [ABSTRACT FROM AUTHOR]

ATP-binding cassette (ABC) transporters play a pivotal role in physiology and pathology. We identified and cloned two novel mRNA isoforms (ABCB 5α andABCB 5β) of the ABC transporterABCB 5 in human melanoma cells. The deducedABCB 5α protein appears to be an altered splice variant containing only a putative ABC, whereas theABCB 5β isoform shares approximately 70% similarity withABCB1 (MDR1) and has a deduced topological arrangement similar to that of the whole carboxyl terminal half of theABCB1 gene product, P-glycoprotein, including an intact ABC. Northern blot, real-time PCR, and conventional RT-PCR were used to verify the expression profiles ofABCB 5α/β. We found that the melanomas included among the NCI-60 panel of cell lines preferentially expressed bothABCB 5α andABCB 5β. However,ABCB 5α/β expression was undetectable in two amelanotic melanomas (M14 and LOX-IMVI). The expression profile ofABCB 5α/β in all of the other melanomas of the panel was confirmed both by RT-PCR and by sequencing. NeitherABCB 5α norABCB 5β expression was found in normal tissues such as liver, spleen, thymus, kidney, lung, colon, small intestines or placenta.ABCB 5α/β mRNAs were also expressed in normal melanocytes and in retinal pigment epithelial cells, suggesting thatABCB 5α/β expression is pigment cell-specific and might be involved in melanogenesis. Our findings indicate that expression ofABCB 5α/β might possibly provide two novel molecular markers for differential diagnosis of melanomas and constitute potential molecular targets for therapy of melanomas. [ABSTRACT FROM AUTHOR]

Pigmentation in mammals is stimulated by α-melanocyte-stimulating hormone (MSH), which binds to the melanocortin 1 receptor (Mc1r) and induces activation of melanogenic enzymes through stimulation of adenylate cyclase and protein kinase A. The antagonist agouti signal protein (ASP) interacts with the Mc1r and blocks its stimulation by MSH. We studied the structure and expression of Mclr transcripts in MSH-or ASP-treated murine melanocytes and to determine the influence of these transcripts on the translation process. [ABSTRACT FROM AUTHOR]

Adaptor proteins (AP) play important roles in the sorting of proteins from the trans-Golgi network, but how they function in the sorting of various melanosome-specific proteins such as Pmel17, an essential structural component of melanosomes, in melanocytes is unknown. We characterized the processing and trafficking of Pmel17 via adaptor protein complexes within melanocytic cells. Proteomics analysis detected Pmel17, AP1 and AP2, but not AP3 or AP4 in early melanosomes. Real-time PCR, immunolabeling and tissue in-situ hybridization confirmed the coexpression of AP1 isoforms µ1A and µ1B (expressed only in polarized cells) in melanocytes and keratinocytes, but expression of µ1B is missing in some melanoma cell lines. Transfection with AP1 isoforms (µ1A or µ1B) showed two distinct distribution patterns that involved Pmel17, and only µ1B was able to restore the sorting of Pmel17 to the plasma membrane in cells lacking µ1B expression. Finally, we established that expression of µ1B is regulated physiologically in melanocytes by UV radiation or DKK1. These results show that Pmel17 is sorted to melanosomes by various intracellular routes, directly or indirectly through the plasma membrane, and the presence of basolateral elements in melanocytes suggests their polarized nature. [ABSTRACT FROM AUTHOR]