A Dominant Mutation in Gαs-Protein Increases Hair Pigmentation

Abstract

We have identified a chemically induced mouse mutation which increases the eumelanic hair pigmentation. We identify a coding mutation, A3533G, resulting in an amino acid substitution Y1133C, in the Gnas gene encoding the G_αs subunit of the tripartite G-protein, consistent with an activation of signalling via MC1R. In addition heterozygous mutant females are significantly lighter than wild type littermates. In cultured melanocytes, derived from mutant mice crossed to C57BL6 mice carrying Cdkn2a^tm1Rdp, basal pigmentation is higher than wild type melanocytes derived from litter mates. However, the addition of exogenous NDP-MSH does not increase pigmentation in mutant melanocytes in contrast to the pigmentation response of non-mutant melanocytes. The mutant and wild type cells respond in the same way to agouti signalling protein (ASP), consistent with ASP signalling mediated through a pathway other than G_αs-protein.

Keywords: ASP; G‐alpha S; MC1R; McCune Albright syndrome; NDP‐MSH; melanocytes; pigmentation.

FIGURE 1

(A) Wild‐type (right) and Goth mutant (left) mice. (B) Growth curves of male Goth mutant and wild type mice. Weights of 6 wild type and 7 Goth measured at intervals between 20 and 100 days of age. Data is expressed as mean weight in grams ± SEM. (C) Growth curves of female Goth mutant and wild type mice. Weights of 3 wild type and 7 mutants, expressed as above. Data is in Table S1.

FIGURE 2

(A) Representative paired phase‐contrast and bright field microscopy images of wild‐type control (melan‐a9) and Gnas‐mutant (melan‐Gnas1) melanocytes at steady‐state or treated with 100 pM NDP‐MSH for 7 days. Scale bar = 100 μm. Boxed regions are enlarged 5x next to the corresponding image for both phase‐contrast and bright field images. Scale bar of enlarged images = 25 μm. (B) Comparison of the basal melanin content of wild‐type (melan‐a9, −a10 and ‐a11) and Gnas‐mutant (melan‐Gnas1, ‐Gnas2 and ‐Gnas3). Melanin content was quantified after 7 days in culture, normalised to cell number and expressed as mean pg melanin/cell ± SEM. Data are the mean of 3 technical replicates for each cell line. The average pg melanin/cell were combined for each genotype and compared by two‐tailed t‐test. **** = p < 0.0001. Dose–response relationship between melanin content and NDP‐MSH concentration of wild‐type (C) and Gnas‐mutant melanocytes (D). Cells were treated with NDP‐MSH for 7 days, melanin content was quantified, normalised to cell number and expressed as mean pg. melanin/cell ± SEM. Data are the means of 3 technical replicates for each cell line. A four‐parameter dose–response curve is shown for each cell line. Dotted line is the average pg melanin/cell of all three untreated wild type melanocytes lines from (B). (E) Comparison of the basal cAMP concentration of wild‐type (melan‐a9, ‐a10 and ‐a11) and Gnas‐mutant (melan‐Gnas1, ‐Gnas2 and ‐Gnas3) melanocytes. Absolute cAMP concentration quantified by luminescence‐based assay and expressed as nM cAMP ± SEM. Data are the mean of 3 technical replicates for each cell line. Mean basal cAMP concentration of the three wild‐type lines (8.16 ± 2.36 nM) were not significantly different to the three Gnas‐mutant lines (11.26 ± 2.29 nM), (p = 0.42 by unpaired two‐tailed t‐test). The dose‐dependent effects of NDP‐MSH on intracellular cAMP concentration of wild‐type (F) and Gnas‐mutant (G) melanocytes. Cells were treated with NDP‐MSH for 20 min, with absolute cAMP concentration quantified by luminescence‐based assay and expressed as nM cAMP ± SEM. Data are the means of 3 technical replicates for each cell line. A four‐parameter dose–response curve is shown for each cell line. The average basal cAMP concentration for each genotype is represented by the dotted line.

FIGURE 3

(A) Representative paired phase‐contrast and bright field microscopy images of wild‐type control (melan‐a9) and Gnas‐mutant (melan‐Gnas1) melanocytes at steady‐state or treated with 10 nM ASP. Scale bar = 100 μm. The effects of 10 nM ASP on the melanin content of wild‐type (B) and Gnas‐mutant (C) melanocytes compared to untreated controls. Melanin content was quantified after 7 days in culture, normalised to cell number and expressed as mean pg melanin/cell ± SEM. Data are the mean of 3 technical replicates for each cell line. The average pg melanin/cell were combined for each genotype and compared by two‐tailed t‐test. **** = p < 0.0001. The effects of ASP on intracellular cAMP concentration of wild‐type (D) and Gnas‐mutant (E) melanocytes. Cells were treated with 10 nM ASP for 20 min, with absolute cAMP concentration quantified by luminescence‐based assay and expressed as nM cAMP ± SEM. Data are the means of 3 technical replicates for each cell line. A four‐parameter dose–response curve is shown for each cell line. The average basal cAMP concentration for each genotype were combined and compared by two‐tailed t‐test. ns, not significant.