IAN J. JACKSON COAT COLOUR

More to collour than meets the eye Two classical mouse coat colour genes have recently been cloned and found to encode the receptor for a-melanocyte

stimulating hormone and an antagonist of the hormone.

The study of mouse coat colour genetics is not as eso- teric as one might think. The pigment producing cells, melanocytes, are a model for numerous developmental processes, and the molecular nature of mutations that affect these processes often reveal important principles [I]. A growth factor and its receptor, a transcription fac- tor, a myosin-like protein, a membrane transport protein and several enzymes were all originally identified as muta- tions affecting mouse pigmentation. A number of recent papers, describing the cloning of, and mutations in, the mouse extension and ago&i genes add to the list, as the genes turn out to encode a G protein-coupled hormone receptor and an apparent antagonist of hormone bind- ing, respectively. The molecular findings nicely explain the considerable body of results from earlier genetic and developmental experiments.

Wild-type mouse hair contains two pigments: the tips and bases are coloured black with eumelanin, whereas in- between there is a transverse band of yellow phaeome- lanin. This pattern is formed by the melanocytes, which initially synthesize eumelanin, then switch mid- way through hair growth to making phaeomel.anin and finally switch back. Mutations at both the hgouti (A) or extension (E) loci affect this switch, so that hair can be entirely black or entirely yellow.

Dominant mutations of the A gene, such as Lethal yellow (Ay) and Viuble yellow (A@, result in a mouse that is completely, or almost completely, yellow; the melanocytes in the mutant mice make only phaeomelanin (although NY mice have some darker patches, see Fig. 1). In ad- dition, mice with either dominant mutation suffer from obesity and a disposition to develop various turnours. The recessive mutations, including non-agouti (a), result in mice with no phaeomelanin that are consequently al- most completely black (Fig. 1). The simplest explanation is that A encodes a protein involved in the switch from making eumelanin to making phaeomelanin, and that the switch is always on in the dominant mutants and never on in the recessives. Experiments done over 30 years ago, in which skin of one agouti genotype was grafted onto another, indicated that this switch is not carried out autonomously by the melanocytes, but is mediated by the microenvironment of the hair follicle. The switch, there- fore, involves a signal from the dermis to the melanocyte [21.

Similar experiments showed that the phenotype caused by dominant E muta@ons is, by contrast, determined autonomously by the genotype of the melanocytes. This might have indicated that E could encode the receptor for

Fig. 1. Mice showing the phenotypes caused by different agouti alleles. The mouse on the left carries the dominant allele A’+‘, as a result of which it is mostly yellow and also obese. On the right are an all black non-ago& mouse (top), a wild-type Agouti mouse (middle) and a tan-bellied black-and-tan mouse (bottom). (Photograph courtesy T Cerniglio and E Michaud).

the signal to switch, but the phenotype of the mutations suggested not, as the dominance series of E alleles runs opposite to that of the A alleles. Thus, the dominant, gain- of-function E mutations Sombre (Eso) and Tobacco dark- enin& (Etob) result in black hairs, whereas the recessive extension (e) phenotype: is completely yellow.

Melanogenesis is regulated, to some extent, by CL- melanocyte stimulating hormone (CX-MSH). Melanocytes or melanoma cells in vitro respond to a-MSH by an increase in mRNA and/or activity (possibly due to pro- tein phosphotylation) of the melanogenic enzyme tyrosi- nase, and a consequent increase in melanin synthesis. If a-MSH is injected into the skin of yellow, &-‘, mice, the melanocytes respond locally by making black eumelanin. In contrast, if the same injection is made into recessive yellow, e mice, there is no eumelanin synthesis. The over- expressed agouti switch in N animals can be overcome by a-MSH, but the presumed lack of gene product in e animals cannot be rescued [ 31. A model that accounts for all these observations, and that the recent molec- ular data fully support, is that the E locus encodes the c~-MSH receptor (MSH-R), and the A gene product antag- onizes the ligand-receptor complex (Fig. 2). Antagonists of the binding of the related hormone ACTH to its re- ceptor have been isolated. An alternative model has been proposed [4], in which the A protein has a separate, as yet undiscovered receptor and its binding to this receptor

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independently switches the cells to phaeomelanin synthe- sis. ,This would require that excess a-MSH at the MSH-R could override the second-site binding of A to its receptor. Furthermore, the data with dominant E alleles (see below) strongly support the antagonist hypothesis.

Inhibition A

‘, !‘,% “,;,f, ” ;_ .,_ : MELANIN SYNTHESIS

Fig. 2. A model for the interaction of a-MSH, the MSH receptor (MSH-R) and the A antagonist in the control of phaeomelanin and eumelanin synthesis. A and a-MSH are suggested to compete for binding to MSH-R, A thus preventing activation of MSH-R by CZ- MSH. MSH-R signalling leads to increased tyrosinase activity and supply of the tyrosinase substrate (by activating transcription of a gene encoding a putative tyrosine transport molecule). A muta- tions alter production of the A protein, extending or eliminating its antagonistic effect; E mutations either remove the MSH-R, and thus eliminate signalling, or abolish its dependence on a-MSH, so that there is continuous signalling, or enhance its signalling response to a-MSH.

The A locus is one of those used in large mutagenesis experiments, and there are thus many newly-produced alleles available. In one of these experiments, a muta- tion occurred that simultaneously affected both A and another gene on chromosome 2, limb-deformity (Id), and that turned out,to be a chromosomal inversion with a breakpoint in both genes. As the Id gene had already been cloned, the DNAsurrounding the second breakpoint and containing the A gene was easily isolated [ 561. The A gene turns out to encode a 131 amino-acid protein with

a sequence suggesting that is secreted from cells; char acteristics of the expression and mutations of the gene are consistent with its proposed role in pigmentation. The gene is expressed in skin, and not in melanocytes. During hair growth there is a pulse of A mRNA synthesis, corresponding to the time when the yellow band is made. Dominant yellow A alleles express the A mRNA constantly, and the recessive alleles express defective or no A mRNA

The molecular nature of some of the A alleles is interest- ing. The classical non-ago& (a) mutation produces no detectable mRNA in this allele there is a large insertion in the DNA upstream of the gene, which presumably inacti- vates transcription [ 51. Another mutation, known as black and tan (al), repeatedly found as a partial reversion of a, produces black pigment dorsally but is yellow on the ven- u-urn, where A mRNA is expressed (Fig. 1). The DNA in sertion is still present 5’ of the gene in at but it is reduced in size compared with the insertion in a. Presumably the smaller insertion still prevents normal gene expression, but somehow now results in deregulated expression in the ventrum [4].

Both dominant mutations result in continuous A mRNA expression, not only in the skin but in all tissues [5-71. AY has no obvious DNA change to explain the deregula- tion. However, -ill, which is also an embryonic lethal muta- tion when homozygous, has undergone a rearrangement upstream of the gene. The result of this rearrangment is that another gene, several hundred kilobases away, no longer produces a functional mRNA but instead splices its first, non-coding exon onto the coding exons of the A gene. This upstream gene, now known as Raly, encodes a previously undescribed hnRNP protein, The ubiquitous expression of Raly means that in Ay animals an abnor- mal mRNA encoding normal A protein, is expressed ev- erywhere. Furthermore, if Raly is an essential gene, this may explain the recessive lethality of AY [5-i’].

The MSH-R was known to be a member of the large fam ily of receptors that are coupled to G proteins and thus activate adenylyl cyclase. Members of this family charac- teristically have seven transmembrane domains and are sufficiently related in sequence that primers for the poly- merase chain reaction can be designed that will amplify DNA sequences encoding many or all of them. Apply- ing this approach to cDNAs made from melanoma mRNA yielded one sequence representing a melanocyte-specific mRNA [ 81. Expression of this mRNA in heterologous cells demonstrated that it would activate adenylyl cyclase in response to a-MSH treatment. As a-MSH binding sites are found in many tissues other than melanocytes, there are presumably different receptor molecules expressed in these other tissues.

The MSH-R gene was mapped in the mouse genome at or near the E locus, and use of the cloned MSH-R cDNA has now confirmed that E is the MSH-R gene 191. Four mutant E alleles have been sequenced, and all turn out to be .point mutations in the MSH-R coding region (Fig. 3). The properties of the mutant receptors were examined by expressing in transfected cells. The recessive e mutation is

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a single base deletion, resulting in a frameshift and prema- ture termination of the receptor protein after the fourth transmembrane domain; the truncated protein is not f&c- t,fona.l. All three dominant alleles have m&sense mutations: the two Son&-e mutations, Es0 and Es@3J, occur very close together-in the second transmembrane domain, while Etob has a substitution in the intracellular loop that precedes this domain.

One might predict that the dominant mutations would generate receptors that signal constitutively, This turns out to be so for Es@34 the product of which has a basal sig- nalling level (measured as adenylyl cyclase activity) about four-fold higher than the unstimulated, and half that of the stimulated, wild-type receptor, but which is unresponsive to a-MSH. The Es@3J mutation adds further weight to the antagonist model, as the constitutive signalling from the mutant protein is rather less than the wild-type stimulated receptor but nevertheless can switch the melanin pathway from phaeomelanin to eumelanin even in the presence of A protein. The Etob mutation, on the other hand, gener- ates a receptor that has a basal activity only slightly higher than the wild-type receptor, but that responds to a-MSH by activating adenylyl cyclase to a level more than three- times higher than the maximum achieved by the wild-type receptor.

The elegant analyses of the A and E genes put on a molec- ular level what genetic and developmental studies have al ready hinted at. They also, of course, raise plenty of ques- tions. The eumelanin to phaeomelanin switch is probably mediated by A protein-antagonism of o!-MSH binding to its receptor, implying that the default state of pigment synthesis .is phaeomelanogenesis. This is surprising, as melanocytes and melanoma cells, in culture are often eumelanic. Perhaps the microenvironment in vivo sup- plies other factors which maintain a phaeomelanic state. Alternatively, it may be that conditions required to keep the cells in culture, whether growth media or mutations

that generated the melanoma, bypass the a-MSH require- ment for eumelanin synthesis, It also appears that the melanocytes of the pigmented retinal epithelium of the eye do not require the same hormone-receptor interac- tion, as they produce eumelanin even in AY or e mutant mice. Perhaps these melanocytes do respond to a-MSH, but the response is mediated through a different recep- tor - at least two other, neural-specific, MSH-R types have been identified [9]. This would fit with the differ- ent embryological origin of the pigmented retinal epi- thelium, which arises from neurectoderm in situ, unlike hair melanocytes which come from migratory neural crest cells.

The question of precisely how the phaeomelanin to eume- lanin switch occurs is still open: that is, what does MSH-R regulate? Unfortunately, the biochemistry of the pathways by which the two pigments are synthesized is not fully un- derstood: synthesis of both begins with the enzyme tyrosi- nase, but what determines the metabolism beyond that is unclear. It has been proposed that simply increasing the activity of tyrosinase would alter me synthetic pathway from phaeomelanin to eumelanin by changing the sub- strate concentrations downstream in the pathway [lo]. The putative tyrosine transporter protein, P, is only ex- pressed in eumelanic skin [ 111, and tyrosinase enzyme activity and mRNA level are both increased in response to a-MSH in cultured cells. Hormonal up-regulation of tyrosinase and the supply of its substrate, leading to an increase in concentration of intermediate products, might be enough to effect a switch from phaeomelanin to eumelanin synthesis.

How the A protein antagonizes cc-MSH binding to its receptor is an interesting question: does it act on the hormone, the receptor or the bound complex? The antagonism can be overridden in two ways. Injection of {excess a-MSH overcomes the excess A protein in the dominant mutants,,whereas the Etob mutant receptor

Fig. 3. Pseudostructural diagram of the mouse MSH-R protein, with transmem- brane domains predicted by hydropa- thy analysis and comparison with other members of the same receptor fam- ily. Green residues are conserved be- tween mouse and human MSH-R and the human adrenocorticotropin recep- tor. The location and nature of the four E locus mutations are shown. (Adapted from L91).

causes darkening in the presence of normal amounts of the A protein, apparently because it has a much. greater response to a-MSH. However, this hyperactive rlesponse requires normal levels of hormone - the mutant recep- tor is not hypersensitive. The first result shows that o?-MSH and A cornpete, so they either interact directly or lbind the MSH-R at the same site. The second observation indicates that Etob mice must have some receptors left free from an- tagonism that can bind hormone, and high enough levels of free a-MSH available even in the presence of A. protein to generate a response. I suggest, therefore, that the in- teraction occurs by competition between A protein and a-MSH at the hormone-binding site of the MSH-II.

The pleiotropic effects of the dominant A mutation, which include obesity and a propensity to develop tumours, .caused by ectopic A expression, suggest that the A pro- tein may antagonize other hormone-receptor interactions elsewhere in the body. The pleiotropic effects cannot be due to the same interaction as in the skin, as they are not seen in e mutant mice, which lack a functional MSH-R. Given the sign&cant medical interest in both obesity and cancer, and the possibility that identification of the recep- tors or ligands affected would open up areas of potential pharmaceutical intervention, this is likely to be a major area of study and provide a justification other than simple curiosity - if one were needed - for continued study of apparently esoteric fields.

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References

1. JACK.SON IJ: Mouse coat colour mutations: a molecular

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genetic resource which spans the centuries. Bioesays 1991, 13:43‘+446. SILVERS WK: The coat colors of mice: a model for gene action and interaction. New York: Springer; 1979.

TAMATE HB, TAKEUCHI T: Action of the e locus of mice in the response of phaeomelanic hair follicles to a-melanocyte stimulating hormone in vitro. Science 1984, 224:1241-1242. CONKLIN BR, BOURNE HR: Mouse coat colour reconsidered. Na- ture 1993, 364:llO. BULTMAN SJ, MICHAUD EJ, WOYCHLK RP: Molecular characteriza- tion of the mouse ago&i locus. Cell 1992, 71:1195-1204.

MILLER MW, DLIHL DMJ, VRIELING H, CORDES SP, OLLMANN MM, WINKES BM, BARTH GS: Cloning of the mouse ago&i gene pre- dicts a secreted protein ubiquitously expressed in mice car- rying the Lethal yellow mutation. Genes Dev 1993, 7:454-467. MICHAUD EJ, BULTMAN SJ, S~VSBS LJ, WOYCHM RP: The embry- onic lethality of homozygous lethal yellow mice is associated with the disruption of a novel RNA-Finding protein. Genes Dev, in press. MOUNTJOY KG, ROBBINS IS, MORTRLID MT, CONE RD: The cloning of a family of genes that encode the melanocortin receptors. Science 1992, 257324%1251. ROBBWS IS, NADEAU JH, JOHNSON KR, KELLY m ROSELU-REHFUSS L, BAACK E, MOUNTJOY KG, Coh?z RD: Pigmentation phenotypes of variant extension locus alleles result from point mutations that alter MSH receptor function. Cell 1993, 72:827-834. IT0 S: High-performance liquid chromatography analysis of eu- and pheomelanin in melanogenesis control. J Invest Demzatol 1993, 2S166S171S. RINCHIK EM, BULTMAN SJ, HORSTHEMKE B, LEE S-T, STRUNK KM, SPRITZ Q AVIDANO KM, JONG MTC, NICHOLLS RD: A gene for the mouse pink-eyed dilution locus and for human type II oculocutaneous albllsm. Nature 1993, 361~72-76.

Ian J. Jackson, MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, UK.