Human melanocytes and melanomas express novel mRNA isoforms of the tyrosinase-related protein-2/DOPAchrome tautomerase gene: Molecular and functionalcharacterization

We previously reported that a melanoma antigen, recognized by tumor-specifi c cytotoxic T lymphocytes, was encoded by intron sequences retained in a pa rtially spliced transcript of the tyrosinase-related protein-2/DOPAchrome t automerase gene. At difference with the mRNA encoding tyrosinase-related pr otein-2, this anomalous transcript was not expressed in melanocytes. This s tudy examined whether neoplastic and/or normal cells of the melanocytic lin eage could express additional forms of tyrosinase-related protein-2 mRNA. S creening of a melanoma-derived cDNA library with a tyrosinase-related prote in-2 probe allowed identification of two novel isoforms. The first, tyrosin ase-related protein-2-long tail, corresponds to the dominant transcript det ected on melanomas and melanocytes by northern blot analysis. Tyrosinase-re lated protein-2-long tail is identical to the tyrosinase-related protein-2- encoding published cDNA sequence except for an extended 3'-untranslated reg ion and is originated by alternative polyadenylation. This novel 3'-untrans lated region contains an alternatively spliced, tyrosinase-related protein- 2 last exon in the second isoform (tyrosinase-related protein-2-8b). The pr otein encoded by tyrosinase-related protein-2-8b is identical to tyrosinase -related protein-2 in its first 460 amino acids but possesses a different c arboxyl-terminus devoid of transmembrane domain. Tyrosinase-related protein -2-long tail exhibited DOPA-chrome tautomerase activity, when transiently t ransfected into COS-7 cells. On the contrary, no detectable activity was ex hibited by tyrosinase-related protein-2-8b. Reverse transcription-polymeras e chain reaction analysis indicated that tyrosinase-related protein-2-long tail and tyrosinase-related protein-2-8b are expressed by tyrosinase-relate d protein-2-positive melanomas and normal melanocytes. Moreover all cell li nes positive for tyrosinase-related protein-2 isoforms expressed tyrosinase and, all but one, tyrosinase-related protein-1. These data show that the h uman tyrosinase-related protein-2/DOPAchrome tautomerase gene can yield dif ferent isoforms by alternative poly(A) site usage or by alternative splicin g. The pattern of expression of these isoforms suggest that they might play a part in the normal pathway of melanin biosynthesis.