PubMed · 8385273
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I J Jackson. 1993-04-15. Molecular genetics. Colour-coded switches.. https://doi.org/10.1038/362587a0
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The classical mouse fancy Agouti gene is responsible for the wild-type coat color where hairs are banded black and yellow. The Agouti gene encodes a 131-amino-acid secreted protein product that regulates phaeomelanin synthesis by melanocytes in mice. Mice with a dominant mutation at this locus, Ay, develop a yellow coat color, obesity, and diabetes, as the result of a deletion that results in ectopic overexpression of the Agouti gene mRNA in all tissues examined. Obesity and diabetes in Ay mutant mice could be caused by circulation of the protein, or localized action in specific tissues as a paracrine factor acting in cell-cell communication. To test these two possibilities, the Agouti cDNA was overexpressed in the skin of transgenic mice using either the Tyrosinase-Related Protein-1 or the keratin-14 (K14) promoter, the latter with and without an intron. The K14 promoter directed high constitutive levels of expression of Agouti mRNA in the skin, and several lines of transgenic mice exhibited coat colors resembling dominant Agouti allele phenotypes. Two highly expressing K14-Agouti transgenic lines, with light-yellow pelage, were analyzed for obesity and hyperglycemia. The transgenic mice were not significantly different from the controls (P > 0.05), indicating that the Agouti product does not act as an endocrine factor. RNase protection assays revealed a correlation between the levels of dorsal and ventral skin expression with pigmentation/phaeomelanin phenotypes. Co-injection experiments with the Agouti transgenes and other transgenes demonstrated co-integration of the two constructs at the same chromosomal site in approximately 95% of F1 progeny, allowing transgene inheritance to be visibly detected.
Agouti protein is known to antagonize cAMP formation, tyrosinase activation and melanogenesis in mouse B16-F1 melanoma cells induced by alpha-melanocyte-stimulating hormone (alpha-MSH). We now demonstrate that although agouti binds to the melanocortin receptor MC1-R with an almost identical affinity to that of alpha-MSH, it does not antagonize the inhibitory action of alpha-MSH on the growth of B16-F1 cells. Instead it has a similar antiproliferative action with a half-maximal effective concentration of 13 nM. In G4F cells lacking MC1-R, agouti is without effect. Agouti was also found to induce MC1-R down-regulation with identical kinetics and magnitude as alpha-MSH. Thus, the different effects of agouti on B16-F1 cells proceed via interaction with MC1-R but are not exclusively antagonistic.
The pleiotropic effects of the viable yellow mutation (Avy), an allele of the mouse agouti coat-color locus, include increased susceptibility to spontaneous and chemically induced tumors that affect a wide variety of tissues. As a first step toward understanding the molecular basis of this phenomenon, we established permanent fibroblast-like cell lines from newborn Avy/a and control congenic a/a mice and compared their growth characteristics in vitro. From the VY/WffC3Hf/Nctr and YS/WffCH3f/Nctr-Avy inbre strains, each of which carries the Avy allele on a congenic background, 38 clonal Avy/a and 16 clonal a/a lines were established. Regardless of inbred strain, all Avy/a cell lines exhibited a significant degree of spontaneous transformation, as assessed by focus formation in monolayer culture, whereas none of the a/a cell lines formed foci in prolonged cultures. To test whether changes in dosage of the Avy- or a-bearing chromosomes were related to these events, we analyzed each cell line with a closely linked molecular probe from the Emv-15 locus, which in the VY strain detects a restriction fragment length variant (RFLV) informative for the Avy- and a-bearing chromosomes. Most of the transformed foci maintained heterozygosity for RFLVs detected by the probe, but two of the transformants lost the a-associated RFLV, and at least one of the transformants exhibited amplification of the Avy-associated RFLV. When the transformants were analyzed with 5' sequences derived from the recently cloned agouti gene, three of eight transformants lost the a-associated RFLV, and two of the transformants showed amplification of the Avy-associated RFLV. Reverse transcriptase-polymerase chain reaction assays indicated that agouti RNA was detected in Avy/a, not a/a cell lines. Surprisingly, some of the Avy/a transformants lacked agouti RNA. These results suggest that deregulated expression of the Avy allele is required for the initiation but not for the maintenance of transformation of the Avy/a cell cultures. These cell lines may provide an in vitro culture system for studying the effect of the agouti gene on tumorigenicity as well as to potentially study other pleiotropic phenotypes.