Pigment Cell Research Suppl. 2 67-78 (1992)

Recent Advances in the Molecular Biology of Pigmentation: Mouse Models RUTH HALABAN AND GISELA MOELLMANN Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut 06510

Pigmentationin mammals is determined by melanin and depends on pigment synthesis by melanocytes as well as transfer and redistribution of melanosomes to keratinocytes of epidermis and hair. Melanosomes contain melanocyte-specificenzymes and proteins whose functions are not yet known. The complexity of control of the pigmentary system is examplified by the gradations seen in the color of skin,hair, and eyes in humans and the multitude of coat color variations in animals. In mice, more than 50 genetic loci affecting pigmentation had been described by 1979 (1) and others have been identified since. These loci can be grouped into four major, not necessarily mutually exclusive, classes: those affecting the migration, proliferation, and survival of melanocytes; those controlling the amount of melanin produced, those that determine the kind of melanin synthesized (eumelanin and pheomelanin); and those reflected in the shape and ultrastructure of rnelanocytes. We focus in this review on the first two classes as represented by piebaldism, premam graying, and albinism. No molecular data are available for the other two classes.

ALBINISM Albinism is a generalized absence or drastic reduction of pigment in otherwise normally distributed pigment cells, including those of the visual tract. Strikingadvances have been made in the area of genes

affecting melauogenesis, rnspecifically, the genes for tyrosinase and the brown -locus protein,the l a m having been renamed catalaseB (2). Significant homologies in the amino acid sequence suggest that these two melanosomal oxidoreductases have originated from a common ancestor gene. As will be shown below, the conserved regions are critical for the normal function of both enzymes. Cloning of cDNAs and chromosomal mapping of tyrosinase and catalase B. For several years there was a controversy as to whether tyrosinase was encoded by a single gene or a family of genes, because two melanocyte-specific cDNA clones with high homology at strategic sites had been isolated and both ascribed to tyrosinase (3-6). The deduced amino acid sequences showed that the two clones coded for glycoproteins of similar size, with a presumptive signal peptide and membrane-spanning domain, and presumptive catalytic regions of high homology, with conserved positions of histidine and cysteine (3-7). Additional cDNA clones were isolated by hybridization of cDNA libraries with oligonucleotideprobes based on nucleotide sequences of Pmel-34 described by Kwon et al. (6, 8, 9). Tyrosinase was confirmed to be encoded by a single gene in both mouse and man (5, 6, 10, Il), as represented by the cDNA clones isolated by Yamamoto et al. (4) and Kwon et al. (5). The clone isolated by

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R. Halaban Shibahara et al. (3), was mapped to the brown locus on mouse chromosome 4 (12). Mouse tyrosinase maps to the c-locus on chromosome 7 (5, 6, ll), and human tyrosinase (TYR)to chromosome 11, region q14-->q21 (11). The mapping of the structural gene for mouse tyrosinase to the c-locus on chromosome 7 has once and for all overthrown the notion that this locus encodes a tyrosinase regulatory protein (13.14). An additional site of human tyrosinase-related sequences was, however, detected on the short arm of chromosome 1 1, near the centromere (region pll.Z-xen) (ll), that harbored a pseudogene. Characterization of the tyrosinase pseudogene showed that it contained only exons IV and V because only these two were recognizedand amplifed by the polymerase chain reaction (15). Functions of tyrosinase and the brownlocus protein. Tyrosinase is essential to melanogenesis. The enzyme catalyzes three reactions: hydroxylation of L-tyrosine to L-dihydroxyphenylalanine (L-dopa) (16) and the oxidations of Ldopa to Ldopa quinone (16) and 5.6-dihydmxyindole to indole-5,6 quinone (17). Tyrosinase is sufficient for melanin synthesis in vizro and may be sufficient in vivo, as shown by transfection of tyrosinase cDNA into fibroblasts, mammary carcinoma and amelanotic melanoma cells, and albino melanocytes, turning the nonmelanocytic and amelanotic cells into highly pigmented ones (9, 18-21). In addition, injection of tyrosinase minigenes into fertilized eggs from an albino mouse strain have yielded normally pigmented transgenic mice (22,23). The organelles, into which the melanin has been deposited, have not been identifed. In the case of albinos, these are assumed to be the albino melanosomes. In the cells of nonmelanocytic origin the melanin may be in dilated tranS-Golgi saccules or lysosomes, both of which have an acidic internal pH that would favor initiation of melanogenesis (24), or peroxisomes containing catalase. Both human and murine tyrosinases have the peroxisomal targeting sequence -SHL(25)at the carboxy terminus (26). In melanocytes, melanin production is modulated by mutations both inside and outside the tyrosinase locus, indicating the presence of factors or agents that

et al.

regulate tyrosinase activity and/or interfere with the synthesis of melanin. An agent belonging to the latter group is H202, suggesting a role for enzymes that catalyze the decomposition of H202 in normal melanin biosynthesis. H202 is probably formed during the catalytic conversion of tyrosine to melanin precursors and melanin (26, 27) and, unless removed, can destroy precursors and melanin. A highly abundant melanosomal protein, gp75 (28), was shown to be identical with the brown-locus protein (2,29), which in turn was shown to have catalase activity in vizro (catalase B) (2). Recurrent reports from VJ Hearing's laboratory (e.g., 30) to the contrary, this protein does not have tyrosinase activity and is not another tyrosinase (2,28, 31, 32). Tyrosinase copurifies with gp75 through several steps of the purification procedure (31) and is carried along as a contaminant of gp75. In addition, tyrosin= has weak CTOSS reactivity toward moAbTMH1, which has a high affinity for catalase B (2). The question of contaminating tyrosinase activity in preparations of the brown-locus protein has been resolved (2,31) and could be reconfimed easily by use of a murine albino melanocyte line, melan-c (32), which carries an inactive tyrosinase but wild-type catalase B. In addition, there are anti-gp75 antibodies that do not cross-react with tyrosinase, e.g., MEL-5 (Signet Laboratories, ref. 2, identical with TA99, refs. 28, 29, 31), 2G10 (33), and 3B5 (Natali, personal communication). The brown-locudgp75/catalase appears to be a highly antigenic glycoprotein. Monoclonal antibodies against it have been raised inadvertently in four independent laboratories by immunizing rodents with crude mixtures of melanosomal proteins in hopes of raising antibodies against tyrosinase (28, 33, 34, and personal communicationby Dr. K. Jimbow, Edmonton, Alberta, Canada). This antigenicity may be due to a heme- or porphyrin-binding epitope (2) known from other systems to evoke an intense immune response (35). Heme-containing oxidoreductases have been idenwied as autoantigensin autoimmune thyroiditis (36) and idiopathic autoimmune hepatitis (37) and

R. Halaban et a].

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BALB/c ALBINO

o \ \ u 0

0

I

I

GCys-

C at nucleotide 387 Ser at aminoacid 85

94 -

w

67 -

30 -

Figure 1 Murine BalWc albino (clc). This c-locus mutation causes complete failureof pigment formation (photograph) (1). Tyrosinase has no detectableactivity because of substitutionat residue 85, replacing Cys* with S a (10,39-41) in a region highly conserved between tyrosinase and catalaseB. 'pitated tyrosinasefrom cultured albino melanocy&s is low in abundance and appears mostly as dem-ucfs (38). Cys 86** in catalaseB is the site of the b-mutation causing substitution with Tyr (42).

HI MALAYAN

A-G

ot nucleotide 1342

His-Arg

ot ominooctd 402

402

Tyrosinose: Alo. Asn -Aloe Pro.Ile.Gly. His-Asn .Arg-Asp.Ser .Tyr

I I I I I I I I

-

m-locus protein: Gln * Asn * Alo. Pro- I l e Gly.His.Asn- Arq. Gln * Tyr -Asn

:otolose -8)

410

TEMPERATURE (TI

Figure 2

Murine bimalayan (&&. This c-locus mutation causeswhite coat color except at emme, cool body parts (photograph) (1). Himalayan tyminase is temperature sensitive, even in the physiologicalrange (graph) (38) because of a mutation at codon 402,substitutingHis with Arg (43)in a region conserved between tymsinase and catalase B (compareamino acid sequences). kft-haad gel shows tyrosinase pteins (arrows)from cultured wild type (W) and himalayan melanocytes (llight; : d dark himalayanmelanocytes). Radioactive cell extracts were subjected to i m r n e p i t a f i o n with antityrosinase antibodies, electrophoresis and fluomgraphy. The himalayantyrosinast appears mostly as the unprocessed species (lowertyrosinase band). The protein is deficient in carbohydrateresidues (right-hand gel) (38).

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catalase B(gp-75) is a melanorna-associated autoantigen (31). Mouse m u t a n t s of tyrosinase and catalase B. Recent molecular and biochemical analysis of melanocytes carrying mutations at the tyrosinase gene have greatly advanced our understanding of the basis for the albino phenotype. In inbred albino mice, tyrosinase activity is not detectable and the immunoprecipitated protein appears mostly as two low-molecular weight degradation products at extremely low levels, suggesting severe sensitivity to proteolytic degradation (38, and Fig. 1). Sequence analysis of the cDNA indicates a point mutation at exon 1: a G to C transition at nucleotide 387, which would substitute Ser at Cys 85 (10,39-41). The cysteine of tyrosinase and flanking amino acids are identical with those of the brown-locus protein, with the exception of one conservative substitution (Fig. 1). Cys 85 of tyrosinase is part of a sequence -CysGlyAsnCysthat may be a heme- or porphyrin-bindingdomain (2), a domain that is conserved in both tyrosinase and catalase B. In the murine brown-locus b mutation, Cys 86 is replaced by Tyr (42). The Balb/c albino mouse is homozygous for this mutation in addition to being homozygous for the albino mutation. figment cells from mice homozygous for the b mutation have little or no catalaseB activity (2). In homozygous blb animals that have wild type tyrosinase, the coat color is brown instead of black, probably because of a lower eumelanin/pheomelanin ratio. These examples show that point mutations in the highly conserved domains in the genes for tyrosinase and catalase B cause moderate to severereduction in enzyme activity. Another tyrosinase mutation analyzed at the nucleotide and protein levels is that in the himalayan mouse (chich)(Fig. 2). In himalayan melanocytes, tyrosinase activity is easily abolished by slight increases in temperature at physiological ranges or upon cell lysis in vim, regardless of the method used to break down the melanocytes (38, and Fig. 2). Analysis by immunoprecipitation showed that the post-transcriptional modification of the protein, i.e., the addition of carbohydrate residues, was deficient (38, and Fig. 2).

Sequencecomparison of wild-type and mutant tyrosinase cDNAs revealed a point mutation at exon 4 an A to G transition at nucleotide 1342, which would substitute Arg for His at amino acid 402 (43). The segment of His 402 and eight flanking amino acids is identical in tyrosinase and the brown-locus protein (Fig. 2). suggesting a domain important for maintaining the threedimensional structure of the protein andor the binding of copper (a metal critical for tyrosinase activity) or the iron in heme (a prosthetic group in many mammalian hydroperoxidases and oxidoreductases). It is thus possible that the loss of His 402 in himalayan tyrosinase leads to structural abnormalitiesthat reduce the efficiency of glycosylation. The reduction in carbohydrate residues, in tum, would confer instability on the protein, hence reduction and loss of activity. Another mutation at the murine b-locus, Bfr(Zighr), is discussed under the heading PIEBALDISM AND VITILIGO. Tyrosinase in human oculocutaneous albinism (OCA). Experience in sequencing tyrosinase and catalase B of murine strains has shown that substitutions of conserved amino acids, situated in regions of high homology, disrupt the stability of the respective enzyme. This disruption is expressed in part as an inability of the protein to become fully glycosylated. So far, the sequencing of human albino tyrosinase has shown the existence of diverse but not entirely random mutations in the tyrosinase gene. Two different nucleotide insertions leading to shifts in the reading frame and introducing early termination signals have been reported (44,45). The truncated protein was verified in one of the cases (45). This tyrosinase was present in the same abundance as normal tyrosinase in normal melanocytes, but mostly as an immature species of lower molecular size. Immunoprecipitation with antibodies directed against a 14-amino acid peptide comprising the carboxy terminal domain of human tyrosinase (30), confirmed the prediction that the protein suffered truncation in this domain. The melanocytes had extremely low tyrosinase activity.

R. Halaban et al. Three human albmo mutations have been idenfied in regions conserved between tyrosinase and catalaseB. They are: i) replacement of Pro 81 by Leu (46)(i.e., Pro 62 if the putative signal peptide of 18 amino acids is not counted and His is taken as the first amino acid at the amino tenninus of tyrosinase, see references 6-9,47); ii) a G to A transition, causing replacement of Gly 401 by Arg (48); and iii) a G to A transition at nuclmtide residue 312 causing an Arg to Gln substitution at position 59 (49). The first of these mutationsoccurred in 6 of30 unrelated American Caucasianpbands (46).the second was identified in an OCA family (48). The relevant Pro in the tyrosinase described in ref. 46,and the 5 amino acids preceding it, are conserved in both ryrosinase and catalase B (-AspAspArgGluAlaTrpPro-, amino acids 61-67of the brown-locus protein, the Ala being Ser in tyrosinase). The mutation identified in ref. 46 is located in a region which is part of one of the presumptive copper- (or in catalase B, heme-) binding sites. Arg 59 and the two flanking amino acids are conserved in tyrosinase and catalase B, suggesting that this segment is imponant for s m d integrity. It is also not clear whether a trans- or semidominant inheritance may be possible in albinism (see also c-kit and BIt below) since tyrosinase, like the blocus protein occurs in aggregates, and the defective protein, e n d e d by the mutant allele, may inactivate the normal protein, encoded by the wild-type allele. Mutually inactivating complexes of the c- and b-locus proteins have also not been ruled out.

PIEBALDISM AND VITILIGO Piebaldism, epitomized by the white forelock in humans and white spotting in animals, is characterized by the reduction in number, down to complete absence, of pigment cells in well delineated patches of skin. Vitiligo is patchy postnatal loss of pigment cells. Piebaldism is stable congenital white spotting, the patterns having been determined during embryogenesis. In contrast, vitiligo is acquired in childhood and adolescence, or even later in life, and may in rare cases progress a,total depigmentation. Several mouse models exist for the study of piebaldism and vitiligo (1, 50).

Mu~ationsthat result in piebaldism have been identified a! minimslly nine independent alleles (1). These m known as steel (st, chromosome 10). piebald spotting (s, chromosome 14), lethal spotting (Is,chromosome 2), belted (bt, chromosome 15), microphthalmia (mi, chromosome 6), and several that are clustered on chromosome 5: dominant spotting (W),patch (Ph), rump-white (Rw), and recessive spotting (rs). So far, only the W,Sl and Ph mutations have been studied at the molecular level. Among the possible animal models for vitiligo, molecular data are available for the murine Bit mutation (seebelow). None exist for human piebaldism and vitiligo. White spotting and c-Kit. Mutations at the W locus affect the proliferation of melanoblasts as well as hemopoietic stem and primordial germ cells during embryogenesis (51). Homozygous mice are ' by extensivewhite spotting and suffer fnrm Charactenzed anemia and sterility. The identification of c-kit as the gene at the W locus was an important step toward elucidating the mechanism by which W mutations exert their pleiotropic effects (52, 53). C-kit encodes a transmembrane tyrosine-kinase receptor and is the cellular homologue of v-kit, the oncogene of an acute transforming feline retrovirus that causes sarcomas in cats (54). The oncogene encodes a constitutively active tyrosine kinase as a result of truncation of the extracellulardomain. The c-kit k i n a can be re* as a prototype receptor tyrosine kinase whose proper control is critical for normal proliferation of the cells in which it is expressed. Down-regulation of activity causes extinction, or a reduction in number, of the affected cells; constitutive activity is associated with ~gnancy.

The ligand for the c-kit receptor tyrosine kinase is Mastcell Growth Factor (MGF), also referred to as the kit-ligand, stem-cell growth factor, or the steel ligand because it is encoded at the s&eZ locus (55-62). The c-kit kinase has homology to CSF- 1-R and PDGF-R (receptors for Colony Stimulating Factor 1 and PlateletDerived Growth Factor) (54, and Fig. 3). These are transmembrane receptor tyrosine kinases that have 5

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extracellular domain

transmembrane domain

plasma membrane

c-kit

cytoplasmic domain FGF-R

PDGF-R CSF-1-R

Melanocytes

Figure 3 Murine piebaldism (W'IWL).Top: Receptors with tyrosineh a s e activity, belonging to the immunoglobulin superfamily of proteins (adapted from references67and 68 ). Hatched areas in inmacellular regions of the receptors indicate kinase domains. Point mutations in the W locus (c-kir) so far identified are expressed in this region (51-54,6570). Left: Phosphorylationof p 1 4 P on tyrosine residues in melanwytes in response to MGF (Funasaka et al., manuscript in preparation). Photogmph: the W'IWLphenotype (1). Abbreviations: R, WeptoG PDGF, platelet-derived growth factor; CSF,colony-stimulatingfactm, FGF. fibroblast p w t h factar(s); IL, interleukin.

extracellular loops of immunoglobulin-like domains. Receptors for bFGF (basic Fibroblast Growth Factor), the all-important melanocyte growth factor in humans (63,64),are also receptor tyrosine kinases, albeit with 2 or 3 immunoglobulin-like loops at the extracellular domain (65-70). The phenotypic common denominator of mutations at the W locus is reduced c-kit receptor tyrosine kinase activity (52,53,71-73).The reduction may be due to a decreased abundance of a protein with apparently n o d kinase activity (the dominant alleles Wd,W,and W7),

or expression at normal levels of a defective enzyme (the dominant homozygous-lethal alleles W37 and W42,the moderately dominant homozygous alleles W41 and WJ, and the hetemzygous WIW). Reduced abundance may be due to rearrangements within the c-kit gene, in at least one case because of an insertion (52),affecting stability but not activity of the enzyme. Reduced activity may be due to missense mutations causing substitutions .in highly conservedresidues of the kinase domain (68-70). The surprisingly extensive white spotting in heterozygotes carrying a missense mutation in the

R. Halaban et al. defective allele is thought to be due to inhibition of the wild-type normal protein by the defective one (71-73). Mutations at the SZ locus cause defects in the same three cell lineages as do mutations in rhe W locus and cause similar phenotypes. However, mutations at the W locus originate within the affected cells, whereas mutations at the Sl locus affect the cells from without. Mclanocytes and mast cells daived from either wild type or steel fail to grow 011 steel fibroblasts or steel-derived exoIBceUularmatrix but thrive on wild-type fibroMasts or wild-type matrix (74,75). Studies of several steel alleles showed that X-irradiation caused complete deletion of mgf, or smaller deletions andlor genomic reanangements, whereas a chemical carcinogen caused point mutations (59). The ability of fibroblasts (which usually express MGF as a surface molecule) to bind genetically engineered soluble receptor, is missing in fibroblasts derived fmm steel mutants, confirming that MGF is absent or dysfunctional(60). Erom all the above it follows that the c-kit receptor kinase ligand, MGF, must stimulate melanocytes. That this is, indeed, the case (Fig. 3) is the subject of an upcoming publication by Funasaka et al., in which we show that c-kit kinase activity is important to normal human pigment cell proliferation and, possibly, maintenance of the differentiatedmelanocytic phenotype. The patch locus and PDGF-R. The gene at the patch locus in mice encodes the alpha subunit of the PDGF-R (PDGF-Ra), another receptor tyrosine kinase; the piebald P h mutation is a deletion (76). Because of the high sequence homology, structural and functional similarities, and the close topographical proximity on chromosome 5, it is possible that the c-kit receptor kinase and PDGF-Ra have evolved from tandem duplication of a single ancestor gene. The W'9* allele is a deletion on both the c-kiz and the PDGF-Ra genes (52, 76). The white spotting in the WJ9Hmutant mice is less pronounced than in the W and P h mutants, suggesting that an imbalance in the expression of these two receptors is mole deleterious to melanocyte development than a reduced but balanced expression (76). Whether the other two piebald genes, Rw and rs,tightly linked to

Wand Ph on chromosome 5, also share homology with receptor-tyrosine kinases, has not been explored. It is thus possible that homologous receptor kinases act in concert, not only to trigger signal transduction through phosphorylation of tyrosine residues on crucial substrates, but also through phosphorylation and inactivation of inhibitors, such as membrane bound protein-tyrosine phosphatase, and that a deficiency in one of these kinases reduces the efficiency of others. In support of this idea is the recurrent finding that normal human and murine melanocytes will not be sustained in vitro by addition of only one melanocyte peptide growth factor, unless the culture medium is supplementedwith cyclic AMP,"PA, or a second growth factor. It appears that the second growth factor cannot be chosen at random from the list of melanocyte-stimulating peptides but that there are distinct complementary pairs, such as bFGF plus HGF (HepatocyteGrowth Factor) (64). Whether inactivation of the PDGF-Ra can be as deleterious to human pigmentation as it is in mice is not yet clear. Neonatal human melanocytes do not express the PDGF-Ra in culture (77) and do not proliferate in response to PDGF (78) whereas murine melanocytes do (our unpublished results). Whether the PDGF-Ra is expressed in human melanoblasts during embryogenesis or prior to hair follicularrenewal, is a subject that has not been investigated. Premature graying and B*: Measured against this wealth of information, knowledge of molecular events in vitiligo is miniscule. The current prevalent hypothesis for the etiology of Vitiligo is a Combination of melanocytic autotoxicity and secondary immune response. The best advanced model for this view is the Smyth chicken (79) for which no molecular data are available at this writing. Neither are there such data for the CS7b3L-Ler.vitlvit mouse (50). There is, however, a murine model for early graying: the B1' (light) mutation at the b locus (80, Sl), which is being studied by us and Ian Jackson (Edinborough, Scotland). A single point mutation causes substitution of an original cysteine residue near the carboxy terminus of the putative signal peptide of catalase B (Ian Jackson, personal

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communication) and results in a protein with increased sensitivity to proteolytic degradation and undetectable catalase activity (2). Since, in contrast to the nondeleterious b mutation at this locus, the.presumptive heme epitope is intact in the BU mutant, it is possible that the melano-cytotoxicity inherent in this mutation is due to release of toxic amounts of iron from heme during breakdown of the enzyme. The phenotype produced by the Blt mutation is a hypopigmented coat color due to clumping, irregular distribution and reduced number of melanosomes, followed by premature death of follicular melanocytes (80, 81). The result is a lightly pigmented hair tip on an almost white hair shaft. Catalases in general exist as tetramers, and undenatured catalase B occurs as a homodimer or part of a larger aggregate (2). Therefore, the B1' mutation may create a condition similar to that described for the c-kit receptor tyrosine kinase: in heterozygotes the mutant protein may complex with and inactivate the normal wild-type enzyme, causing what is known as transdominant or semidominant inheritance of a theoretically recessive mutant trait. CONCLUSION We have shown in this review that mutations at diverse loci, or within the same locus at diverse sites, can produce almost indistinguishable phenotypes. As a consequence, identification of human carriers of such mutations for the purpose of genetic counseling will be more complicated than in some other inherited diseases for which single genetic markers are diagnostic.

The author gratefully acknowledges permission to reproduce in this article three figures (Figs. 1, 2, 3 ) from Silvers. The Coat Colors of Mice. Springer-Verlag, 1979.

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