Functional variation of MC1R alleles from red-haired individuals

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1 2001 Oxford University Press Human Molecular Genetics, 2001, Vol. 10, No Functional variation of MC1R alleles from red-haired individuals Eugene Healy, Siobhán A. Jordan, Peter S. Budd, Ruth Suffolk, Jonathan L. Rees 1 and Ian J. Jackson* MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK and 1 Department of Dermatology, University of Edinburgh, Lauriston Building, The Royal Infirmary, Edinburgh EH3 9YW, UK Received June 8, 2001; Revised and Accepted July 30, 2001 Red hair in humans is associated with variant alleles of the αmsh receptor gene, MC1R. Loss of MC1R function in other mammals results in red or yellow hair pigmentation. We show that a mouse bacterial artificial chromosome (BAC) which contains Mc1r will efficiently rescue loss of Mc1r in transgenic mice, and that overexpression of the receptor suppresses the effect of the endogenous antagonist, agouti protein. We engineered the BAC to replace the mouse coding region with the human MC1R sequence and used this in the transgenic assay. The human receptor also efficiently rescued Mc1r deficiency, and in addition, appeared to be completely resistant to the effects of agouti, suggesting agouti protein may not play a role in human pigmentary variation. Three human variant alleles account for 60% of all cases of red hair. We engineered each of these in turn into the BAC and find that they have reduced, but not completely absent, function in transgenic mice. Comparison of the phenotypes of αmsh-deficient mice and humans in conjunction with this data suggests that red hair may not be the null phenotype of MC1R. INTRODUCTION The wide range of hair and skin colour in mammals, including humans, is partly due to different contributions of two melanin types, black/brown eumelanin and red/yellow phaeomelanin (1 3). Eumelanin synthesis is stimulated in melanocytes by activation of the α melanocyte stimulating hormone (αmsh) receptor, MC1R (4). Mutations of MC1R produce altered pigmentation in mice and other mammals (5 11). Loss of function of MC1R results in a red coat in guinea pigs, horses and pigs, a yellow coat in mice and either red or yellow hair (depending on the breed) in dogs. Point mutations within MC1R that confer constitutive or ligand-independent activity result in a dominant black phenotype in mice, cattle, sheep, foxes and possibly in dogs. An endogenous antagonist of MC1R, agouti protein, binds to the receptor and results in phaeomelanin synthesis in the melanocyte (12 14). In mice, agouti is expressed in a pulse during hair growth, resulting in a yellow subapical band in the otherwise black hair. Overexpression of agouti in mutant mice produces a dominant yellow phenotype, whereas loss of agouti results in hairs that are uniformly black (11). Surprisingly, mice that lack proopiomelanocortin (POMC), the precursor of αmsh, are not yellow but are quite dark (15), suggesting that there is substantial ligand-independent signalling from MC1R. Furthermore, these mice show an agouti pattern on their hairs, indicating that agouti protein modulates this signalling. Human MC1R sequence variants are associated with red hair and fair skin in the Caucasian population (16 19). These variant alleles are extremely common; in northern European populations <50% of the MC1R genes encode the wild-type or consensus protein. Three alleles in particular, Arg151Cys, Arg160Trp and Asp294His together make up 22% of the MC1R genes and account for 60% of all cases of red hair. In most families MC1R alleles behave as normal recessive mutations, although there are a few individuals with red hair in whom no MC1R variant can be found. Rare individuals lack POMC and therefore lack αmsh, and consequently have red hair, adrenal insufficiency and obesity because of lack of stimulation of the whole family of melanocortin receptors (20). However, this does not seem to be the explanation for the vast majority of red-haired individuals without MC1R variants, and other, yet to be identified genes are probably playing a role. When introduced into cells in culture, the common red-hair associated variants produce a very severely impaired camp response to αmsh (21,22). However, these experiments provide only a crude measure of receptor function, relying on a single assay (camp level) of activation in non-melanogenic cells. Therefore, we have developed a sensitive in vivo assay using transgenic rescue of Mc1r mutant mice. We find that Mc1r-containing bacterial artificial chromosome (BAC) transgenes efficiently rescue or over-rescue the yellow phenotype of Mc1r-deficient mice in a copy number dependent manner. Human MC1R also rescues efficiently, and the human receptor is much less sensitive to the antagonistic action of mouse agouti protein, suggesting that agouti may not *To whom correspondence should be addressed. Tel: ; Fax: ; ian.jackson@hgu.mrc.ac.uk The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors Present address: Eugene Healy, Dermatopharmacology, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, UK

2 2398 Human Molecular Genetics, 2001, Vol. 10, No. 21 Table 1. Pigmentation and transgene copy number in transgenic recessive yellow lines rescued by Mc1r Line Coat colour Transgene copy number A134 Agouti 1 A139 Agouti 1 A139.3 Agouti 1 A135.2 Agouti 2 3 A139.1 Dark agouti 3 4 A138.1 Very dark agouti 3 6 A139.2 Dark agouti ND Coat colour was determined on the first hair cycle. In general, the altered pigmentation was maintained throughout subsequent hair cycles, with some loss of darker (eumelanin) pigmentation after 9 months. play a significant role in human hair colour. BAC transgenes containing any of three common human MC1R variant alleles result in partial rescue of coat colour. The common alleles that result in red hair in humans alter melanin synthesis in vivo but they are not complete loss-of-function mutations. RESULTS The BAC, CITB-484H6, contains the mouse Mc1r gene and 65 and 50 kb flanking the gene on the 5 and 3 sides, respectively. We injected the BAC into mouse embryos homozygous for the recessive yellow (Mc1r e ) mutation. Nine positive transgenic founders gave rise to seven lines, all of which showed rescue of the coat colour from yellow to agouti or darker (Table 1). In addition to darkening of hair, the skin of the ear and tail was also rescued from pale to darkly pigmented (Fig. 1A) consistent with a role for MC1R in interfollicular as well as follicular pigmentation. Comparison of BAC copy number with the pigmentation in the rescued lines indicated a dosage effect (Table 1). The agouti pattern of dorsal hair pigmentation is characterized by a stripe of yellow phaeomelanin below the tip of a black, eumelanic, hair. This is produced by a pulse of synthesis of agouti protein near the start of the hair growth cycle. Transgenic Mc1r e mice carrying one or two copies of the BAC transgene were agouti in colour, indicating that the transgenic MC1R responds normally to agouti protein. Animals carrying three or four copies had coats darker than wild-type, in which the sub-apical yellow band was reduced in length, whereas in mice with up to six BAC transgene copies the transgenic hairs are almost entirely black. It appears that the antagonist effect of agouti at MC1R can be overcome by increased copy number (and presumably overexpression) of the Mc1r gene. This could be due to summation of a low level of constitutive signalling by MC1R even in the presence of agouti, or to saturation of a limited pool of agouti protein by increased receptor number. We modified the BAC by recombination in Escherichia coli, and replaced the mouse Mc1r coding region with that encoding human consensus MC1R, upstream of an internal ribosome entry site (IRES) and a lacz reporter (23). Transgenic lines were produced containing this modified BAC in recessive Figure 1. Coat colour of recessive yellow (homozygous Mc1r e ) mice rescued with Mc1r transgenic BAC. (A) Top, non-transgenic, agouti mouse; bottom, transgenic recessive yellow mouse rescued with BAC encoding mouse Mc1r (line A138.1, three to six copies). (B) Left, non-transgenic recessive yellow mouse; right, transgenic mouse rescued with BAC carrying human wild-type MC1R (line A167, five to seven copies). (C) Left, transgenic recessive yellow mouse rescued with BAC encoding human wild-type MC1R (line A168, three to five copies); right, non-agouti mouse. Note the non-agouti mouse has yellow fur behind the ears, which is black in the transgeneic animal. (D) Top, non-transgenic recessive yellow mouse; bottom, transgenic mouse rescued with BAC encoding Arg151Cys MC1R variant. (E) Bottom, non-transgenic recessive yellow mouse; top, transgenic mouse rescued with BAC encoding Arg160Trp MC1R variant. (F) Right, non-transgenic recessive yellow mouse; left, transgenic mouse rescued with BAC encoding Asp294His MC1R variant. yellow mice. Copy number analysis indicated that these animals contained approximately four and six copies of the transgene, respectively (Table 2). We assayed expression of the human transgene by comparison with the endogenous Mc1r (Table 2) and found the levels of the human MC1R mrna equivalent to an output per gene of 50% of the endogenous gene (i.e. four transgene copies express mrna equivalent to two endogenous copies), possibly because the IRES:lacZ destabilizes the mrna. Expression of human MC1R-encoding mrna not only rescues the Mc1r e mutant phenotype but in both lines the hair colour is rescued from yellow to entirely black with no sign of any phaeomelanin synthesis (Fig. 1B and C, and Fig. 2). In addition, the ear and tail skin shows over-rescue to a black colour. This degree of over-rescue is much greater than that produced by up to four or more copies of the BAC encoding mouse Mc1r. Human MC1R is reported to be super-sensitive to melanocortin peptides; assayed as a camp response in transfected cells, it is eight times more sensitive to αmsh and about 200 times more

3 Human Molecular Genetics, 2001, Vol. 10, No Table 2. Pigmentation of human wild-type and variant MC1R transgenic recessive yellow lines Line MC1R construct Coat colour a Transgene copy number b Total transgene expression c (N) A167 Wild-type Black ± 0.37 (5) A168 Wild-type Black ± 0.80 (8) A184 Arg160Trp Grey ± 0.22 (4) A185.2 Arg151Cys Grey ± 0.25 (6) A186 Asp294His Dark yellow ± 0.14 (6) A207 Asp294His Dark yellow 1 ND A207 homozygote Asp294His Dark yellow 2 ND Coat colour was determined on the first hair cycle and was maintained throughout subsequent hair cycles. Line A207 was bred to homozygosity to allow comparison of pigmentation between variant MC1R animals containing identical transgene copy numbers. a Figure 1. b Whole number range as determined by Southern blot analysis. c Assayed on neonatal skin RNA by primer extension, mean ± SD. sensitive to ACTH than the mouse receptor (24). The human MC1R appears to be relatively insensitive to antagonism by mouse agouti protein, with the consequence that MC1R signalling is maintained throughout the hair growth cycle, resulting in an overall black phenotype. This insensitivity to agouti may be direct (i.e. the human receptor binds the antagonist less well) but is more likely the result of a higher level of signalling from the more sensitive human MC1R such that signalling occurs even in the presence of agouti. Data from the variant receptors (below) also support the latter. It is worth noting that in these experiments we are expressing the human receptor from the mouse promoter and so may be generating a nonphysiological level of MC1R, and consequently a stronger signalling response. Previous work has shown that mouse agouti protein is 10 times more effective than the human agouti protein in inhibition of the melanogenic response from MC1R on human melanocytes (14). Given the lack of effect of the more potent mouse agouti protein on human MC1R in our transgenic assay, it is possible that agouti may not play a significant role in determination of human hair colour; however, the consequence of a much lower expression in human melanocytes remains to be determined. We used a photometric assay as an objective measure of melanin content in dorsal hair from age-matched transgenic mice (3). Phaeomelanin is more soluble than eumelanin at high ph and so the fraction of alkaline soluble melanin quantifies the phaeomelanin:eumelanin ratio. Yellow mice lacking Mc1r have a ratio approximately 0.6, black, non-agouti mice have a ratio <0.08, whilst in agouti mice it is approximately 0.1. Mice expressing the human MC1R transgene have an average ratio of 0.08, equivalent to that in black non-agouti animals (Fig. 2). Three human MC1R variant alleles, Arg151Cys, Arg160Trp and Asp294His, together account for >60% of all cases of red hair and we therefore engineered each of these changes separately into the human coding sequence of the BAC (followed by the IRES:lacZ sequence). Transgenic lines were generated, containing each of the variants in recessive yellow mice. The expression of each of the transgenes is shown in Table 2 and the effect on the coat in Figure 1D F. All MC1R variant transgenic lines showed some degree of rescue of eumelanin synthesis and all had a coat darker than recessive yellow, but paler than wild-type agouti, although none showed visible rescue of tail and ear skin. The hair melanin type ratio in the transgenic mice is intermediate between yellow and agouti (Fig. 2). Arg151Cys and Arg160Trp result in very similar ratios, 50% that of the Asp294His lines. Interestingly, all lines showed the agouti hair pattern, unlike those rescued by the consensus MC1R, indicating that the variant receptors were antagonized by agouti protein. The phenotype also appears to be less dosage sensitive. Line A207, carrying one transgene copy of the Asp294His variant, was bred to make the transgene homozygous to double the copy number and to compare the phenotype with the lines carrying equivalent copies of Arg151Cys and Arg160Trp variants. Homozygote A207 mice were no different in phenotype from hemizygotes, still much paler than mice carrying either of the other two variants. As the variant transgenic MC1R responds to agouti protein, we asked whether the paler colour was due simply to an increased sensitivity of the receptor to agouti antagonism or if they also produced less eumelanin. We crossed the Arg151Cys and Arg160Trp transgenic lines onto a homozygous nonagouti background. Mice with wild-type Mc1r on a non-agouti background are black. The variant transgenic mice were dark grey in colour, indicating that the variant receptors are able to elicit a eumelanogenic response, although of a lower intensity than normal. The variant receptors respond normally to agouti antagonism. The reduced melanogenic response from the Arg151Cys and Arg160Trp variant MC1Rs is probably not due to less efficient binding by the hormone but to less effective signalling. Schioth et al. (21) demonstrated that hormone binding to these variants in transfected cells is indistinguishable from wild-type. The Asp294His variant receptor, in contrast, binds hormone slightly less well, and the paler coat of these transgenic mice might in part be accounted for by this. To try and overcome the poorer binding we administered αmsh to neonatal transgenic pups by subcutaneous injection four times over a 7 day period, a protocol which normally darkens coat colour (25) but which had no effect on the colour of the two Asp294His lines (data not shown).

4 2400 Human Molecular Genetics, 2001, Vol. 10, No. 21 Figure 2. Melanin content of hair samples of transgenic and control mice. Bar chart showing ratio of alkali-soluble melanin to total melanin (ASM/TM) in hair samples from transgenic and control mice. Three mice were sampled from each line and each is shown as a separate bar. Samples of hair from each line are shown below the baseline. DISCUSSION We have demonstrated that Mc1r-containing BAC transgenes are able to very efficiently rescue the yellow, loss-of-function phenotype of Mc1r e homozygous mice. Furthermore, the transgenic mice show a strong dosage-sensitive phenotype. Addition of only one or two copies in excess of the normal diploid complement darkens the coat by reduction of the yellow, agouti-induced band on the hair. A total Mc1r complement of approximately six copies almost completely suppresses the effect of agouti. We do not know whether the suppression of agouti is due to the increase in MC1R on the melanocyte surface saturating out a limited amount of agouti protein or whether there is normally a low level of signalling from the receptors even in the presence of agouti, which has an additive effect with increased receptor number and overcomes the antagonism. Transgenic mice expressing human MC1R show a much greater suppression of agouti. Indeed, mice expressing only the normal diploid complement of human MC1R have no detectable phaemelanin and are completely black. This is not due to a lack of activity of mouse agouti protein on the human receptor, as others have demonstrated that in cells in culture the human MC1R is 10-fold more sensitive to mouse agouti than to the human protein (14). There is evidence to suggest that human MC1R is more sensitive to stimulation by hormone than the mouse receptor and this may be sufficient to overcome the antagonsim by agouti. It also appears that the density of MC1R on human melanocytes is considerably lower than the receptor density on mouse melanocytes (24). The transgenic system we describe here, in which the human receptor is expressed from the mouse promoter, may result in MC1R being present at non-physiological, high levels. The variant human MC1R receptors in transgenic mice do respond to agouti protein, as shown by the phaeomelanic patterning on their hairs. These receptors have attenuated signalling (demonstrated by the pale eumelanin synthesized on a non-agouti background) which appears to allow agouti to act. Our data indicate that although the variant MC1R proteins are clearly defective, in accordance with population and family data associating them with red hair, none has complete loss of function when assayed in transgenic mice. Rare patients mutated in the POMC gene and thus lacking αmsh, have red hair that is apparently the same as the MC1R-associated red hair (20). However, POMC-deficient mice do not have the same phenotype as loss of Mc1r (15). Our finding that red-hair associated variant human receptors still allow significant eumelanin synthesis, may indicate that red hair is not the null phenotype. Although a few MC1R frameshift alleles that clearly do not produce functional protein have been described (17,26,27), they are rare and no homozygotes have been found. Whether MC1R-null individuals have different or additional phenotypes which means they have not been included in case control or population studies is unclear at this stage. MC1R has been suggested to have a role in melanocyte proliferation and in various non-melanocytic functions such as inflammation. There is no evidence that MC1R-associated red-haired individuals have any additional phenotype. However, identification and study of individuals homozygous for MC1R loss of function might reveal further roles for MC1R in humans. MATERIALS AND METHODS Generation of transgenic mice We isolated a mouse genomic BAC clone (Research Genetics, CITB-484H6, insert size 120 kb) which contained the Mc1r coding region approximately central in the cloned segment. Sequencing of the BAC Mc1r coding region confirmed that it was identical to the GenBank database sequence X65635 apart from a 2 bp inversion resulting in alanine rather than valine at

5 Human Molecular Genetics, 2001, Vol. 10, No codon 216. We generated transgenic recessive yellow (Mc1r e / Mc1r e ) mice by pronuclear injection of the circularized modified or unmodified BAC directly into homozygous embryos. Genotyping was performed by PCR using primers directed at the BAC vector sequences adjacent to both ends of the insert (5 -TAACTATGCGGCATCAGAGC-3 and 5 -GTCGTATT- ACAATTCACTGGC-3, product 268 bp; 5 -GGCGTAATCA- TGGTCATAGC-3 and 5 -TCATTAATGCAGCTGGCACG-3, product 190 bp). BAC modification To substitute the murine Mc1r coding region (bases in X65635) with human MC1R wild-type and variant coding sequences (bases in X65634), homologous recombination based modification of the BAC in recombination-deficient E.coli, was employed as described by Yang et al. (23), using human MC1R sequences which had been amplified from human volunteers by PCR and cloned (21). The insert in the homologously recombined BACs also contained an IRES appended to a lacz marker (23). The sequence of the encoded wild-type human protein was confirmed by sequencing to be identical to the Swissprot entry and the three variants contained only the single variant amino acid stated. Transgene copy number Copy number of the Mc1r-encoding BAC was determined by assay of the ratio of PCR amplified Mc1r (endogenous plus transgenic) to endogenous Mc5r. PCR was performed on genomic DNA, extracted from tail tips of 6 8-week-old transgenic animals, using primers at conserved sequences (5 -AACCTGCACTCRCCCATGTA-3 and 32 P-radiolabelled 5 -GCATAGAAGATGGWGATGTA-3 ), which amplified Mc1r and Mc5r simultaneously, and the product digested with AluI prior to 6.5% non-denaturing PAGE. The transgene copy number was calculated from the area ratios of the Mc1r (101 bp, endogenous and transgenic) and Mc5r (209 bp, endogenous) AluI-digested products on a Storm phosphoimager system (Molecular Dynamics). Copy number of transgenic BACs encoding human MC1R was assayed by Southern blot hybridization on tail tip DNA digested with BamHI or with NcoI using mouse Mc1r 3 -untranslated region as probe. Transgene copy number was calculated from the peak area ratio of MC1R to Mc1r fragments using a Storm phosphoimager system. Copy number was also calculated by single nucleotide primer extension (SnuPE) using the SnaPshot ddntp kit (PE Biosystems) as described below. Single nucleotide primer extension The PCR product from amplification of genomic DNA or random primed cdna was analysed by primer extension to determine the content of transgenic versus endogenous Mc1r/ MC1R. Total RNA was isolated from dorsal trunk skin of 4-day-old animals using LiCl/urea (28) and reverse transcription carried out with random primers. Mouse transgenic and endogenous Mc1r genomic DNA was amplified using primers 5 -AGCATCGTCTCCAGCACC-3 and 5 -CATGTGGGCATACAGAATCG-3, and products of the transgene (wild-type) and endogenous (Mc1r e ) distinguished by extension of the primer, 5 -GGCAGAGCAGAACG- GCTGT-3. The template from the transgenic transcript added ddgtp, whereas that from endogenous added ddttp. Transcript levels from the mutant, Mc1r e, allele compared to the wild-type Mc1r were also assessed in heterozygous animals to control for differential transcription and/or stability. Human transgene content was assayed following amplification with primers 5 -GGCCCCTTCTTCCTGCATC-3 and 5 - TGCGGAGCTCCTGGCTG-3, which amplify both the mouse and human genes, followed by extension of the primer 5 -GTTGAAGTTCTTGAAGATGCAGC-3, which primes from both mouse and human sequences, by adding ddttp (mouse) or ddctp (human). Extension products were separated by capillary electrophoresis on an ABI 310 and peak areas measured using GeneScan 2.1. Melanin assays Total and alkali-soluble melanin was determined by modification of a previous report (3). Briefly, 0.9 ml of Soluene-350 was added to 1 mg of mouse hair in 0.1 ml of sterile water and heated for 45 min in a boiling water bath. The resulting solutions were analysed in duplicate for absorbances at 500 nm (A 500 ) and these values are referred to as total melanin. To determine alkali-soluble melanin, 800 µl of 8 M urea/1 M NaOH solution was added to 2 mg of mouse hair in 200 µl of water and incubated with shaking at 37 C for 30 min. The samples were vortexed and centrifuged at g for 10 min. The supernatants were analysed for absorbances at 400 nm (A 400 ), the values being referred to as alkali-soluble melanin. Determination of melanin and alkali-soluble melanin was performed in duplicate for each hair sample. ACKNOWLEDGEMENTS We thank Brendan Doe and the staff of the Transgenic Unit for their assistance, N.Heintz for providing the temperature-sensitive shuttle vector for homologous recombination, and S.Philips for providing expression vector pcr3.1 (Invitrogen) containing human wild-type and variant MC1R sequences. E.H. is a Medical Research Council Clinician Scientist Fellow. REFERENCES 1. Prota, G. (1992) Melanins and Melanogenesis. Academic Press, New York. 2. Prota, G., Lamoreux, M.L., Muller, J., Kobayashi, T., Napolitano, A., Vincensi, M.R., Sakai, C. and Hearing, V.J. (1995) Comparative analysis of melanins and melanosomes produced by various coat color mutants. Pigment Cell Res., 8, Ozeki, H., Ito, S., Wakamatsu, K. and Hirobe T. (1995) Chemical characterisation of hair melanins in various coat-colour mutants of mice. J. Invest. Dermatol., 105, Tamate, H.B. and Takeuchi, T. 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