EXPRESSION OF TYROSINASE AND THE TYROSINASE-RELATED PROTEINS IN THE MITF(VIT) (VITILIGO) MOUSE EYE - IMPLICATIONS FOR THE FUNCTION OF THE MICROPHTHALMIA TRANSCRIPTION FACTOR (MITF)

Mitf (Microphthalmia transcription factor), a basic-helix-loop-helix z ipper protein, encoded at the microphthalmia (Mitf) locus, regulates t he transcription of the gene encoding tyrosinase, the rate-limiting en zyme in melanin biosynthesis, by binding the DNA sequence CATGTG, This binding site is present also in the genes encoding two tyrosinase rel ated proteins, TRP-1 and TRP-2. To gain insight into the function of M itf in vivo, we determined whether there was a difference in the Level s of these proteins in the RPE/choroid of the vitiligo (Mitf(vit)) mou se, in which there is a mutation of the Mitf gene. This mouse has alte ration of RPE pigmentation and function that presumably leads to slow progressive loss of photoreceptor cells. The RPE/choroid was dissected from eyes of vitiligo and C57BL/6 wild-type mice at postnatal ages 2, 4, 7, 10, 14, 21 and 42 days. Extracts of pooled tissues were subject ed to electrophoresis and immunoblotting. The levels of tyrosinase, TR P-1 and TRP-2 were determined densitometrically following immunodetect ion with rabbit antipeptide antisera. In addition, the tyrosine hydrox ylase activity of tyrosinase as assayed radiometrically. Levels of TRP -1 were 3-7 fold greater in control RPE/choroid compared with mutants. This marked difference in protein level was observed at the earliest age examined (P2) and persisted throughout the first two weeks. Tyrosi nase levels in mutants were similar to controls at P2 and P4, but were reduced at P10 and beyond. Tyrosinase activity was diminished also in mutants by P10, Levels of TRP-1 were similar between mutants and cont rols, although the typical decrease seen in controls after P14 was att enuated in the mutant mice. There is a significant reduction in the le vel of TRP-1 in the RPE/choroid of the Mitf(vit) mouse. The data sugge sts that transcription of the gene encoding TRP-1 is extremely depende nt upon functional Mitf. It provides in vivo evidence that Mitf regula tes the transcription of the gene encoding TRP-1 as well as tyrosinase . (C) 1998 Academic Press Limited.