MGRN1 depletion promotes intercellular adhesion in melanoma by upregulation of E-cadherin and inhibition of CDC42.

Mahogunin Ring Finger 1 is an E3-ubiquitin ligase encoded by the color gene MGRN1. Our previous in vitro and in vivo studies demonstrated that Mgrn1 deletion in mouse melanoma cells induced cell differentiation and adhesion, and decreased cell motility and invasion on collagen I, and lung colonization in an in vivo model. Here, we investigated the role of MGRN1 on human melanoma cell morphology, adhesion and expression of genes/proteins involved in an EMT-like transition. We demonstrated that wild-type BRAF human melanoma cells adopted a clustering-like morphology on collagen I, with permanent MGRN1 abrogation resulting in bigger cell clusters. Enhanced intercellular adhesion was mostly mediated by induction of E-cadherin and higher co-localization with ß-catenin. Transcriptional upregulation of E-cadherin likely occurred through downregulation of the ZEB1 repressor. Finally, pulldown assays showed reduced activation of CDC42 in the absence of MGRN1, which was reverted after E-cadherin silencing. Overall, these findings highlight a new MGRN1-dependent pathway regulating melanoma cell shape, motility, and invasion potential.

Variant amino acids in different domains of the human melanocortin 1 receptor impair cell surface expression.

The alpha melanocyte-stimulating hormone receptor (MC1R) is a heptahelical G protein-coupled receptor (GPCR) found in the plasma membrane of melanocytes. By mediating the melanogenic response to melanocortins, MC1R is a major determinant of mammalian pigmentation. The human MC1R gene is unusually polymorphic. Many loss-of-function alleles have been described, but the molecular basis for their functional impairment remains most often unknown. Here we report a study of two natural MC1R loss-of-function variants, Leu93Arg and Arg162Pro, and two artificial mutants, Cys35Ala and a deleted form missing the last five amino acids in the carboxyl tail. When expressed in HEK 293T cells, those mutants neither bound an iodinated hormone analogue nor elicited cAMP increases in response to saturating doses of a superpotent agonist. Cell surface expression of mutant receptors was dramatically decreased respect to the wild type form, in spite of smaller changes in total protein abundances and intracellular stability. Accordingly, aberrant processing with intracellular retention is the most likely cause of loss-of-function for those mutants. Therefore, mutations in virtually any region of the heptahelical protein, including its extracellular N terminus, a transmembrane fragment, intracellular loops or carboxyl terminal cytosolic extension, seem to compromise normal MC1R processing.

Antimelanoma effect of 4-S-cysteaminylcatechol, an activated form of 4-S-cysteaminylphenol.

Rational chemotherapy of malignant melanoma could be developed by taking advantage of the presence of melanogenic enzymes in melanoma cells. 4-S-Cysteaminylphenol (4-S-CAP) has been evaluated for melanocytotoxicity and antimelanoma effect. Although 4-S-CAP is selectively toxic to pigmented melanoma cells, it is not potent enough when applied as a single agent. To increase the efficacy of 4-S-CAP, we synthesized 4-S-cysteaminylcatechol (4-S-CAC), an activated form of 4-S-CAP, and compared its biochemical properties and antimelanoma effects with those of the isomers 3-S-cysteaminylcatechol (3-S-CAC) and 2-S-cysteaminyl-hydroquinone (2-S-CAH). 4-S-CAC was found to be a better substrate for melanoma tyrosinase than was L-3,4-dihydroxyphenylalanine, the natural catecholic substrate. 3-S-CAC was a poor substrate, whereas 2-S-CAH was not a substrate. 4-S-CAC was the most cytotoxic to three lines of melanoma cells in vitro, followed by 2-S-CAH and 3-S-CAC. When applied i.p. for 9 days at a dose of 100 mg/kg, 4-S-CAC.HCl, increased by 46-52% the life span of C57BL/6 mice inoculated i.p. with B16 melanoma; this effect was comparable to that of a 50 mg/kg dose of 5-(3,3-dimethyltriazenyl)-1H-imidazole-4-carboxamide. 3-S-CAC was marginally effective, whereas 2-S-CAH was toxic to the host. This systemic toxicity of 2-S-CAH reflected its susceptibility to autoxidation. Growth of B16 melanoma cells inoculated s.c. was significantly inhibited by i.p. administration of 4-S-CAC.HCl (200 mg/kg) for 5 days (P < 0.05). These results suggest that 4-S-CAC is a potent antimelanoma agent, the effect of which is mostly mediated through tyrosinase oxidation.

Activation by thyroid stimulating hormone of nerve growth factor-induced gene-B expression in thyrocytes in culture: relation with proliferation and specific gene expression.

Nerve growth factor-induced gene-B (NGFI-B) is an immediate early gene first found as a part of the PC12 cell response to NGF (Milbrandt, J., Science 238 (1987) 797-799). We have previously reported that NGFI-B mRNA is strongly upregulated by thyroid-stimulating hormone (TSH) in dog thyrocytes in culture (Pichon et al., Endocrinology 137 (1996) 4691-4698). In this study, we have analyzed the regulation of NGFI-B mRNA expression by a variety of agents acting on thyrocytes proliferation and/or differentiation. We show that: (1) the induction of NGFI-B mRNA is stronger after stimulation of the cAMP cascade, but it is not restricted to this signaling pathway; (2) the powerful mitogens for thyroid cells EGF and HGF have little or no effect on NGFI-B mRNA induction; (3) NGFI-B mRNA is induced by anisomycin at a subinhibitory concentration for protein synthesis, and is superinduced by the combination of TSH and anisomycin; this treatment decreases the TSH-induced proliferation levels, but does not inhibit the induction of some differentiation markers; and (4) both in dog and in pig thyrocytes, NGFI-B mRNA induction is observed after a variety of treatments stimulating differentiation, but without proliferative effects. Our results therefore suggest that NGFI-B mRNA induction might not be related to TSH-induced thyrocyte proliferation, but could participate in the differentiation program triggered by TSH.

Effect of detergents and endogenous lipids on the activity and properties of tyrosinase and its related proteins.

Within mammalian melanocytes, melanin biosynthesis is controlled by three enzymes structurally related: tyrosinase and two tyrosinase related proteins, TRP1 and TRP2. These melanosomal enzymes are integral membrane proteins with a carboxyl tail oriented to the cytoplasm, a single membrane-spanning helix and the bulk of the protein located inside the melanosome. Their solubilization is usually carried out by treatment of melanosomal preparations with non-ionic detergents, but, so far, no comparative study of the effect of the detergents employed on the properties of the solubilized proteins has been reported. We have compared the effect of the detergents Brij-35, Nonidet P-40, Tween-20, sodium deoxycholate and Triton X-114 on several properties of the melanogenic enzymes, including the solubilization yield, stability, electrophoretic behaviour and accessibility of epitopes located in the carboxyl tail to specific antibodies. Our data indicate that not only the total amount of enzymes solubilized, but also their relative proportions in the solubilized preparations depend on the detergent used. The non-ionic detergents apparently interact strongly with the melanogenic enzymes, affecting their mobility in SDS-PAGE, and might induce different conformations of the carboxyl tail. Complete replacement of lipids by the detergents results in a decreased stability that can be partially reversed by the addition of endogenous lipids. This treatment also produces a noticeable activation of the tyrosinase isoenzymes, which is higher for TRP1 than for tyrosinase. Taken together, these data show that the transmembrane and carboxyl fragments of the proteins of the tyrosinase family might modulate the stability and activity of the melanogenic enzymes.

Biochemical characterization of the melanogenic system in the eye of adult rodents.

The melanogenic activities in the eye of the adult gerbil (Meriones unguiculatus) have been investigated and compared to those found in the B16 mouse melanoma model. Eye extracts contain tyrosine hydroxylase, DOPA oxidase, DOPAchrome tautomerase and DHICA oxidase activities. The subcellular distribution of these activities was investigated by differential centrifugation and detergent solubilization of the particulate fractions. The distribution pattern closely resembled the one found for mouse melanoma, with a higher percentage of activity associated to the particulate fractions but a substantial proportion in the cytosolic fraction. The tyrosine hydroxylase activity was characterized by a KM of 62 microM for L-tyrosine and a stringent requirement for the co-factor L-DOPA (Ka 10.3 microM). The KM for L-DOPA was 0.41 mM. The sensitivity of the eye and mouse melanoma tyrosinase activity to a variety of substrate analogs and metal chelators was found to be identical. In keeping with these kinetic similarities, eye tyrosinase displayed some structural properties resembling those of the melanoma enzyme. The molecular weight of the enzyme, determined by SDS-PAGE and DOPA oxidase activity stain, was 75 kDa for the eye enzyme and 66.2 kDa for melanoma tyrosinase, and both enzymes were apparently dimeric in non ionic detergent solution. Immunoprecipitation with specific antibodies proved that at least 80% of the total tyrosinase activity could be immunoprecipitated with the specific anti-tyrosinase antibody alpha PEP7, while the anti-TRP-1 monoclonal antibody TMH-1 precipitated little, if any, tyrosinase activity. Taken together, these observations provide the first vis-à-vis comparison of an extracutaneous melanogenic system and the melanogenic system of melanoma. Our results prove that, at least in rodents, the melanogenic system in the eye is similar, but not identical, to the melanin biosynthesis machinery of epidermal melanocytes.

Melanin formation in the inner ear is catalyzed by a new tyrosine hydroxylase kinetically and structurally different from tyrosinase.

Detergent solubilized extracts of the cochleae of adult gerbils (Meriones unguiculatus) contain a tyrosine hydroxylase activity measurable by the radiometric method of Pomerantz. This activity is not related to Fenton-type reactions, since it is not inhibited by free radical scavengers and is heat and protease sensitive. It does not appear to be related to a peroxidase (EC 1.11.1.7) since it is neither dependent on H2O2, nor inhibited by catalase (EC 1.11.1.6). The involvement of a tyrosine hydroxylase (EC 1.14.16.2) related to catecholamine synthesis is also unlikely, since the activity is highly sensitive to 2-mercaptoethanol and is not increased by addition of tetrahydrobiopterin. The activity in crude inner ear extracts displayed an unusual maturation behaviour, with a slow activation upon aging at 4 degrees C. Fully active enzyme displayed Michaelis-Menten kinetics, with a Km for L-tyrosine of 47 microM. Cochlear tyrosine hydroxylase, but not melanoma tyrosinase (EC 1.14.18.1), was inhibited by o-phenanthroline, and was not dependent on L-DOPA as cofactor for full enzymatic activity. Crude extracts were also able to catalyze L-DOPA oxidation and melanin formation from either L-tyrosine or L-DOPA. The tyrosine hydroxylase, DOPA oxidase and melanin formation activities most probably resided in the same molecule, as suggested by inhibition studies. A tyrosine hydroxylase and melanin formation activity with identical properties was found in primary cultures of stria vascularis melanocytes. Immunochemical evidence confirmed the absence of either the tyrosinase encoded for by the albino locus, or the tyrosinase isoenzyme TRP1, encoded for by the brown locus. Conversely, an immunorreactive band of molecular weight 70 kDa was specifically recognized by a tyrosinase polyclonal antiserum in Western blot experiments. These results prove that melanogenesis in the cochlea, and likely in other extracutaneous locations such as the brain, is catalyzed by enzymatic systems different from, but related to tyrosinase.

The melanocortin-1 receptor carboxyl terminal pentapeptide is essential for MC1R function and expression on the cell surface.

The pigmentary actions of the melanocortins are mediated by the melanocortin-1 receptor (MC1R), a seven transmembrane domains receptor positively coupled to Gs and the cAMP cascade. In order to define the structure-function relationships of potentially relevant domains in MC1R, particularly its C-terminal cytosolic tail, we generated and analyzed several variants with C-terminal deletions, as well as point mutants in selected residues of the human MC1R. We show that the MC1R C-terminal pentapeptide is essential for proper receptor expression on the plasma membrane, and that residues Thr314, Cys315 and Trp317 are at least partially responsible for this effect.

Induction of nerve growth factor-induced gene-B (NGFI-B) as an early event in the cyclic adenosine monophosphate response of dog thyrocytes in primary culture.

We investigated the induction of nerve growth factor-induced gene-B (NGFI-B) in dog thyrocytes in primary culture stimulated by different agents. The dog NGFI-B complementary DNA (cDNA) was cloned from a cDNA library of dog thyrocytes and used to study, by Northern blotting, the level of NGFI-B messenger RNA (mRNA) in those cells. We have shown that TSH and forskolin, which both induce proliferation and differentiation of the thyroid cells by activation of the protein kinase A pathway, lead to a strong and transient expression of two NGFI-B mRNA species, which differ in the length of the poly(A) tail. In contrast, 12-O-tetradecanoyl-13-phorbol-acetate (TPA) and epidermal growth factor, which induce proliferation and dedifferentiation of those cells by activation of the protein kinase C and the protein tyrosine kinase cascade, respectively, lead to a weaker expression of NGFI-B mRNA. In parallel, we studied the transactivation capacity of NGFI-B in the same cell system by transient transfection of a chloramphenicol acetyl transferase reporter construction containing a NGFI-B-dependent synthetic promoter. The highest transactivation was observed after forskolin stimulation, whereas transactivation after TPA stimulation was weak and no significant transactivation was observed after epidermal growth factor stimulation. Taken together, these results show that NGFI-B is an immediate early gene product that is mainly induced by the cAMP-dependent pathway in dog thyrocytes. Moreover they suggest that NGFI-B expression could be one of the early transcriptional changes induced specifically by this cascade and leading to differentiation and/or proliferation of these cells.

Thr40 and Met122 are new partial loss-of-function natural mutations of the human melanocortin 1 receptor.

Activation by melanocortins of the melanocortin 1 receptor (MC1R), expressed in epidermal melanocytes, stimulates melanogenesis. Human MC1R gene loss-of-function mutations are associated with fair skin, poor tanning and increased skin cancer risk. We identified two natural alleles: Ile40Thr, probably associated with skin types I-II, and Val122Met. Val122Met bound [(125)I][Nle(4), D-Phe(7)]-alpha-melanocyte stimulating hormone with lower affinity than the wild-type. Dose-response curves of cAMP accumulation were right-shifted for both forms. The Val122Met form failed to achieve maximal cAMP responses comparable to the wild-type or Ile40Thr receptors. Thus, the Ile40Thr and Val122Met variants are partial loss-of-function natural mutations of MC1R.

Biosynthesis of hamster zona pellucida is restricted to the oocyte.

The zona pellucida (ZP) is an extracellular coat that surrounds the mammalian oocyte and the early embryo until implantation. This coat mediates several critical aspects of fertilization, including species-selective sperm recognition, the blocking of polyspermy and protection of the oocyte and the preimplantation embryo. Depending on the species, the ZP is composed of three to four different glycoproteins encoded by three or four genes. These genes have been cloned and sequenced for different species. However, controversy exists about the cell type specificity of the ZP glycoproteins, for which several models have been proposed. Different groups have reported that ZP is produced only by the oocytes, by the granulosa cells or by both cell types, depending on the species under study. We recently described the expression of four ZP proteins in the hamster ovary. By means of the complete set of the hamster ZP cDNAs, we undertook the study of the origin and expression pattern of the four ZP genes. In the present work, the expression of ZP1, ZP2, ZP3 and ZP4 is carefully analyzed by in situ hybridization (ISH) in hamster ovaries. Our data suggest that the four hamster ZP genes are expressed in a coordinate and oocyte-specific manner during folliculogenesis. Furthermore, this expression is maximal during the first stages of the oocyte development and declines in oocytes from later development stages, particularly within large antral follicles.

Hamster zona pellucida is formed by four glycoproteins: ZP1, ZP2, ZP3, and ZP4.

The zona pellucida (ZP) is an extracellular glycoprotein matrix that surrounds all mammalian oocytes. Recent data have shown the presence of four glycoproteins (ZP1, ZP2, ZP3, and ZP4) in the ZP of human and rat rather than the three glycoproteins proposed in the mouse model. In the hamster (Mesocricetus auratus), it was previously described that ZP was composed of three different glycoproteins, called ZP1, ZP2, and ZP3, even though only ZP2 and ZP3 have been cloned thus far. The aim of the study was to determine whether hamster might also express four, rather than three, ZP proteins. The full-length cDNAs encoding hamster ZP glycoproteins 1 and 4 were isolated using rapid amplification cDNA ends (RACE). The cDNA of ZP1 contains an open reading frame of 1851 nucleotides encoding a polypeptide of 616 amino acid residues. The amino acid sequence of ZP1 revealed a high homology with other mammalian species like human (66%), rat (80%), and mouse (80%). The cDNA of ZP4 contains an open reading frame of 1632 nucleotides encoding a polypeptide of 543 amino acid residues. The deduced amino acid sequence of ZP4 revealed high overall homology with rat (82%) and human (78%). Subsequent mass spectrometric analysis of the hamster ZP allowed identification of peptides from all four glycoproteins. The data presented in this study provide evidence, for the first time, that the hamster ZP matrix is composed of four glycoproteins.

Improved tyrosinase activity stains in polyacrylamide electrophoresis gels.

Mammalian tyrosinase exists in a variety of subcellular locations and maturation states that result from a complex post-translational processing with possible regulatory implications. So far, SDS-PAGE has proven to be the method of choice for the resolution of tyrosinase isoforms. However, the relatively poor sensitivity of the currently available specific activity stain based on incubation of the gels with L-dopa until the formation of melanin has severely limited the use of electrophoresis in regulation studies. Two alternative staining procedures are presented and discussed. The first one involves the fluorographic detection of radioactive melanin after incubation of the gels in the presence of L-[3-14C]-dopa. A similar method has already been used by others (Tsukamoto et al., 1992, Pigment Cell Res. [Suppl.] 2:84-89), but its performance has not yet been compared to the one of the dopa procedure. The sensitivity of this method can be varied by adjusting the isotopic dilution of the tracer and/or the time of exposure of the gel, but it is at least ten times higher than the one of the colorimetric stain. Moreover, the intensity of the bands is proportional to the initial tyrosinase activity over a wide range. Using this procedure, the activity present in the different subcellular fractions of melanocytes in culture can be easily detected. The second procedure involves the formation of a colored adduct between dopaquinone and MBTH.(ABSTRACT TRUNCATED AT 250 WORDS)

Tyrosinase isoenzymes in mammalian melanocytes. 2. Differential activation by alpha-melanocyte-stimulating hormone.

In mouse melanoma melanocytes, alpha-melanocyte-stimulating hormone (MSH) stimulates differentiation, melanin synthesis and tyrosinase activity. However, the molecular mechanisms underlying these events have not yet been characterized. We have studied the activation of tyrosinase by MSH. Treatment of B16 melanoma cells with either theophylline, MSH, or its superpotent analog [Ahx4, DPhe7]MSH promotes a larger induction of tyrosine hydroxylase than of dopa oxidase activity in whole cell extracts. This higher activation of tyrosine hydroxylation was found not only in the melanosomal but also in the microsomal fraction; it appears to be dependent on continued transcription and translation since it can be blocked by actinomycin and cycloheximide. The tyrosinase activity of control and theophylline-treated extracts displayed several kinetic differences, including different Km values for both substrates and requirements for the cofactor L-dopa. SDS/PAGE, followed by a sensitive specific activity stain, demonstrated that melanosomes of control cells contain one lower-electrophoretic-mobility form of tyrosinase, whereas melanosomes of cells treated with either theophylline or MSH display, in addition to the lower-mobility form, a faster-migrating activity band. These tyrosinase forms are not interconvertible by proteolysis or deglycosylation. Their nature is discussed as related to the properties of the previously described low- and high-electrophoretic-mobility tyrosinases (LEMT and HEMT), as well as of the proteins encoded by the c and b loci.

The melanogenic system of Xenopus laevis.

Melanin pigments in lower vertebrates are often found in locations other than the skin, thus forming an extracutaneous pigmentary system of unknown function. The cellular and biochemical structure of this system is still poorly characterized. This paper deals with the ultrastructural and biochemical features of the melanogenic system of Xenopus laevis. Melanin containing cells were identified in the dorsal and ventral skin, and in the lung, spleen, liver and connective tissue surrounding blood vessels. The pigment cells in the skin and the lungs appeared to be typical melanocytes. The spleen contained isolated melanocyte-like cells, but most of the pigment cells present in this organ were associated with melanomacrophage centers. Conversely, the liver appeared devoid of melanocytes and only displayed melanomacrophage centers. Tyrosinase activity was found in all pigment-containing organs except the liver. All organs containing tyrosinase activity also displayed melanin formation potential from L-tyrosine. Therefore, tyrosine hydroxylase and melanin formation activities could be detected only in those organs containing typical melanocytes but not in locations such as the liver, where only melanomacrophages centers were found.

The 5,6-dihydroxyindole-2-carboxylic acid (DHICA) oxidase activity of human tyrosinase.

Melanin synthesis in mammals is catalysed by at least three enzymic proteins, tyrosinase (monophenol dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) and tyrosinase-related proteins (tyrps) 1 and 2, whose genes map to the albino, brown and slaty loci in mice, respectively. Tyrosinase catalyses the rate-limiting generation of L-dopaquinone from L-tyrosine and is also able to oxidize L-dopa to L-dopaquinone. Conversely, mouse tyrp1, but not tyrosinase, catalyses the oxidation of the indolic intermediate 5,6-dihydroxyindole-2-carboxylic acid (DHICA) into the corresponding 5,6-indolequinone-2-carboxylic acid, thus promoting the incorporation of DHICA units into eumelanin. The catalytic activities of the human melanogenic enzymes are still debated. TYRP1 has been reported to lack DHICA oxidase activity, whereas tyrosinase appears to accelerate DHICA consumption, thus raising the question of DHICA metabolism in human melanocytes. Here we have used two different approaches, comparison of the catalytic activities of human melanocytic cell lines expressing the full set of melanogenic enzymes or deficient in TYRP1, and transient expression of TYR and tyr genes in COS7 cells, to demonstrate that human tyrosinase actually functions as a DHICA oxidase, as opposed to the mouse enzyme. Therefore, human tyrosinase displays a broader substrate specificity than its mouse counterpart, and might be at least partially responsible for the incorporation of DHICA units into human eumelanins.

Tyrosinase isoenzymes in mammalian melanocytes. 1. Biochemical characterization of two melanosomal tyrosinases from B16 mouse melanoma.

B-16 mouse melanoma melanosomes contain two forms of tyrosinase that can be resolved by SDS/PAGE. These forms interact to different extents with the ion-exchanger DEAE-Sephadex and with hydroxyapatite, and have different affinity for the melanosomal membrane and/or the intraorganular matrix. After partial purification and complete separation of the two tyrosinases, several kinetic parameters were analyzed. The form of lower electrophoretic mobility displayed a higher Km for 3,4-dihydroxy-L-phenylalanine (L-dopa) and L-tyrosine, an absolute requirement for the cofactor L-dopa in its tyrosine hydroxylase activity, and a lower ratio of tyrosine hydroxylation to Dopa oxidation. The form of higher electrophoretic mobility displayed lower values of Km for both substrates and was able to exhibit tyrosine hydroxylase activity after a lag period even in the absence of L-dopa. Both forms were stereospecific for the L isomers and sensitive to the specific tyrosinase inhibitor 2-phenylthiourea. These forms do not appear to result from different degrees of glycosylation, nor from limited proteolysis and are also present in the microsomal fraction of B16 mouse melanoma. They might correspond to different gene products, most likely derived from the b and c loci.

Tyrosinase isoenzymes: two melanosomal tyrosinases with different kinetic properties and susceptibility to inhibition by calcium.

Two forms of tyrosinase from B16 mouse melanoma were identified by nonreducing SDS-PAGE after solubilization of crude melanosomal preparations with the nonionic detergent Brij 35. These forms, named LEMT and HEMT (low and high electrophoretic mobility tyrosinase, respectively), were purified by a combination of differential detergent extraction and chromatographic techniques. They displayed tyrosine hydroxylase and dopa oxidase activity and were stereospecific and sensitive to phenylthiourea, providing that they are true tyrosinases. However, based on its kinetic parameters, HEMT is a much more efficient enzyme. Immunoprecipitation and Western blots performed with the specific antibody alpha PEP1, directed against the b protein carboxyl terminus, suggested that LEMT is identical to the b protein. Both forms of tyrosinase were noncompetitively inhibited by Ca2+ at physiologically relevant concentrations. However, the b protein was apparently more susceptible, since maximal inhibition was reached at lower Ca2+ concentrations for LEMT. Moreover, binding of Ca2+ to the tyrosinases resulted in a noticeable thermal destabilization of the enzymes, which was also more pronounced for LEMT.

The DHICA oxidase activity of the melanosomal tyrosinases LEMT and HEMT.

Although melanins can be formed in vitro by the unique action of tyrosinase on L-tyrosine, it is now well accepted that other enzymes termed tyrosinase-related proteins are involved in mammalian melanogenesis. However, some aspects of their roles in the regulation of the pathway are still unknown. The action of dopachrome tautomerase on L-dopachrome yields DHICA, a stable dihydroxyindole with a low rate of spontaneous oxidation. However, DHICA is efficiently incorporated to the pigment, as judged by the high content of carboxylated indole units in natural melanins. Therefore, the fate of this melanogenic intermediate and the mechanisms of its incorporation to the melanin polymer are major issues in the study of melanogenesis. We have recently shown that mouse melanosomes contain two electrophoretically distinguishable tyrosinase isoenzymes, LEMT and HEMT, that can be purified and completely resolved (Jiménez-Cervantes et al., 1993a). Herein, we have compared the ability of these tyrosinases to catalyze DHICA oxidation. Although highly purified LEMT shows a very low specific activity for dopa oxidation in comparison to HEMT, it is able to catalyze DHICA oxidation. However, the DHICA oxidase activity of HEMT was very low, if significant. The ability of purified LEMT to catalyze DHICA oxidation was abolished by heat, trypsin, or phenylthiourea treatments. LEMT acting on DHICA caused the formation of a brownish soluble color similar to DHICA-melanin. Immunoprecipitation of the DHICA oxidase activity of LEMT by specific antibodies suggests that this activity corresponds to TRP1. These results indicate that LEMT, most probably identical to the product of the b locus, is a tyrosinase having a specific DHICA oxidase activity.(ABSTRACT TRUNCATED AT 250 WORDS)