A Val85Met mutation in melanocortin-1 receptor is associated with reductions in eumelanic pigmentation and cell surface expression in domestic rock pigeons (Columba livia)

Abstract

Variation in the melanocortin-1 receptor (Mc1r) is associated with pigmentation diversity in wild and domesticated populations of vertebrates, including several species of birds. Among domestic bird species, pigmentation variation in the rock pigeon (Columbalivia) is particularly diverse. To determine the potential contribution of Mc1r variants to pigment diversity in pigeons, we sequenced Mc1r in a wide range of pigeon breeds and identified several single nucleotide polymorphisms, including a variant that codes for an amino acid substitution (Val85Met). In contrast to the association between Val85Met and eumelanism in other avian species, this change was associated with pheomelanism in pigeons. In vitro cAMP accumulation and protein expression assays revealed that Val85Met leads to decreased receptor function and reduced cell surface expression of the mutant protein. The reduced in vitro function is consistent with the observed association with reduced eumelanic pigmentation. Comparative genetic and cellular studies provide important insights about the range of mechanisms underlying diversity among vertebrates, including different phenotypic associations with similar mutations in different species.

Figure 1. Examples of plumage pigment variation in domestic rock pigeons.

All birds are show homer breed unless otherwise noted. A–C. Eumelanic phenotypes: A, black check; B, blue bar (same color as A, but different pattern); C, brown (Mookee). D–G. Pheomelanic phenotypes: D, ash-red check; E, ash-red bar (same color as D, but different pattern); F, yellow check (dilute form of phenotype in D); G, recessive red (Chinese owl). H. White (white carneau). I. Fantail, a breed examined in subset of association tests. Panels H and I modified after [27,48], respectively; photos courtesy of Eric Domyan (A–F) and Sydney Stringham (H).

Figure 2. Amino acid sequence variation among avian orthologs of Mc1r.

Several variants have been implicated in pigment variation within avian species. The Val85Met mutation found in domestic pigeons is associated with eumelanism in the lesser snow goose and red-footed booby. Functional studies of Mc1r protein variants have been conducted for chicken [14] and pigeon (this study). Additional mutations in chicken have been identified on the E92K background [13] but are not shown here. Figure based on previous review of avian Mc1r diversity [2].

Figure 3. Met85 alleles of Mc1r are associated with pheomelanism in the domestic rock pigeon.

A. Genotype frequencies across all sampled breeds, with birds categorized as eumelanic or pheomelanic. Met85 genotypes are significantly enriched in pheomelanic birds (p < 0.03, Chi-square test). B. Met85 genotypes are not significantly enriched in pheomelanic recessive red birds (p = 0.21, Chi-square test). C. Met85 genotypes are significantly enriched in pheomelanic standard and Indian fantails (p < 0.04, Chi-square test).

Figure 4. Functional differences between Val85 and Met85 alleles of Mc1r.

A. cAMP production in response to αMSH stimulation is reduced in cells transfected with the Met85 allele relative to the Val85 allele (p < 0.02, paired t-test). Error bars, ± SEM. B. Total cellular protein expression of HA/FLAG-tagged Mc1r is equivalent in COS-7 cells transfected with pigeon Val85 and Met85 alleles. The non-specific OD_{492/620 nm} value (GFP) was 0.008 ± 0.001 (0% set point) and the OD_{492/620 nm} value of the Val85 allele was 0.415 ± 0.070 (100% set point). C. Surface protein expression of the Met85 allele is reduced relative to Val85 allele (p < 0.007, one-sample t-test). Non-specific OD_{492/620 nm} value (GFP) was 0.053 ± 0.050 (0%) and the OD_{492/620 nm} value of the Val85 allele was 0.501 ± 0.049 (100%).