New insights on the structure of the mouse silver locus and on the function of the silver protein.
The melanosomal proteins encoded by the silver locus play important roles in melanogenesis. The human locus yields two proteins, PMEL17 and GP100, by alternative mRNA splicing. The mouse si locus was reported to encode a Pmel17 protein, and later gp87, a GP100 homologue. When we re-examined the products of wild-type and silver-mutant mouse si loci, RT-PCR of wild-type RNA and genomic DNA sequence accounted for gp87 but excluded the occurrence of Pmel17. Analysis of cDNA from the silver (si/si) melanocyte line, melan-si, showed that the pathogenic mutation is a G to A substitution at nt 1808, which yields a premature stop codon and a predicted protein truncated in the C-terminus. This was confirmed by reaction of a specific anti-gp87 antiserum with si/si melanocyte extracts. To further explore gp87 function, we compared the DHICA oxidase activity of extracts from B16, melan-si (heterozygous for the brown mutation and homozygous for the silver mutation) and Cloudman S91 cells (homozygous for the brown mutation), since both TRP1 and gp87 are thought to be involved in DHICA oxidation/polymerization. Cloudman extracts do not oxidize significantly DHICA and its methyl ester, supporting the involvement of native mouse TRP1 in DHICA oxidation. Extracts from B16 and melan-si do not show significant differences for the oxidation of free acid and methylated dihydroxyindoles, indicating that the mechanism is not decarboxylative. Melan-si extracts are very efficient in catalyzing dihydroxyindole oxidation, in spite of being heterozygous for the TRP1 mutation, consistent with a stablin effect for the wild-type gp87 protein. On the other hand, aggregated and degraded forms of that mutant gp87 protein are found in the cytosolic fraction of melan-si, suggesting that misrouting and aberrant processing of the gp87 and tyrosinase may also be related to the high DHICA oxidase activity of these melanocytes.
Solano
F
F
Department of Biochemistry and Molecular Biology, School of Medicine, University of Murcia, Spain. psolano@fcu.um.es
Martínez-Esparza
M
M
Jiménez-Cervantes
C
C
Hill
S P
SP
Lozano
J A
JA
García-Borrón
J C
JC
D017398:Alternative Splicing; D000818:Animals; D002457:Cell Extracts; D003653:Decarboxylation; D006801:Humans; D008544:Melanocytes / Q000378:metabolism; D008562:Membrane Glycoproteins; D051379:Mice; D016296:Mutagenesis; D009363:Neoplasm Proteins / Q000235:genetics* / Q000502:physiology*; D010088:Oxidoreductases; D011506:Proteins / Q000378:metabolism; D013347:Subcellular Fractions; D058951:gp100 Melanoma Antigen
D002457:Cell Extracts; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; C548637:PMEL protein, human; C548642:Pmel protein, mouse; D011506:Proteins; D058951:gp100 Melanoma Antigen; D010088:Oxidoreductases; C489799:TYRP1 protein, human; C079621:Tyrp1 protein, mouse; C064056:tyrosinase-related protein-1