MBE Advance Access originally published online on July 3, 2007
Molecular Biology and Evolution 2007 24(9):2001-2008; doi:10.1093/molbev/msm134
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Research Articles |
High Diversity in Functional Properties of Melanocortin 1 Receptor (MC1R) in Divergent Primate Species Is More Strongly Associated with Phylogeny than Coat Color
* Department of Neuroscience, Division of Pharmacology, Biomedical Center, Uppsala University, Uppsala, Sweden
Department of Biology, Anglia Ruskin University, Cambridge, United Kingdom
Department of Zoology, University of Cambridge, Cambridge, United Kingdom
E-mail: helgis{at}bmc.uu.se.
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Abstract |
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We have characterized the biochemical function of the melanocortin 1 receptor (MC1R), a critical regulator of melanin synthesis, from 9 phylogenetically diverse primate species with varying coat colors. There is substantial diversity in melanocyte-stimulating hormone (MSH) binding affinity and basal levels of activity in the cloned MC1Rs. MSH binding was lost independently in lemur and New World monkey lineages, whereas high basal levels of MC1R activity occur in lemurs and some New World monkeys and Old World monkeys. Highest levels of basal activity were found in the MC1R of ruffed lemurs, which have the E94K mutation that leads to constitutive activation in other species. In 3 species (2 lemurs and the howler monkey), we report the novel finding that binding and inhibition of MC1R by agouti signaling protein (ASIP) can occur when MSH binding has been lost, thus enabling continuing regulation of the melanin type via ASIP expression. Together, these findings can explain the previous paradox of a predominantly pheomelanic coat in the red ruffed lemur (Varecia rubra). The presence of a functional, MSH-responsive MC1R in orangutan demonstrates that the mechanism of red hair generation in this ape is different from the prevalent mechanism in European human populations. Overall, we have found unexpected diversity in MC1R function among primates and show that the evolution of the regulatory control of MC1R activity occurs by independent variation of 3 distinct mechanisms: basal MC1R activity, MSH binding and activation, and ASIP binding and inhibition. This diversity of function is broadly associated with primate phylogeny and does not have a simple relation to coat color phenotype within primate clades.
Key Words: melanocortin • MSH • primate • coat color • G protein-coupled receptor • MC1
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The melanocortin 1 receptor (MC1R) is a G protein–coupled receptor expressed on the cell surface of melanocytes and is a key regulator of melanin pigment formation during hair development (Robbins et al. 1993
; Schioth 2001). High MC1R activity leads to a high cyclic adenosine monophosphate (cAMP) concentration and predominantly black/brown eumelanin synthesis, whereas low MC1R activity leads to a switch to red/yellow pheomelanin synthesis (or absence of melanin synthesis in some cases, e.g., Ritland et al. 2001). High MC1R activity is generally achieved by stimulation of MC1R by melanocyte-stimulating hormone (MSH) but can also be caused by high intrinsic basal (constitutive) activity. Low MC1R activity occurs in the absence of MSH or the presence of the inhibitor/inverse agonist agouti signaling protein (ASIP) (Siracusa 1994).
The role of MC1R mutations in coat pigmentation has been extensively studied in domestic and laboratory mammals and birds. It has been shown that gain-of-function mutations in MC1R result in increased production of eumelanin in coats, whereas loss-of-function mutations result in increased pheomelanin production (mice: Robbins et al. 1993; cattle: Klungland et al. 1995; horses: Marklund et al. 1996; pigs: Kijas et al. 1998; sheep: Vage et al. 1999; dogs: Newton et al. 2000; chicken: Takeuchi et al. 1996; Ling et al. 2003). MC1R variation is also associated with color variation in a wide range of wild vertebrates, ranging from mammals—black bears (Ritland et al. 2001), rodents (Nachman et al. 2003; Hoekstra et al. 2006), cats (Eizirik et al. 2003)—to birds (Theron et al. 2001; Mundy et al. 2003) and lizards (Rosenblum et al. 2004). Functional characterization of MC1R variants has been carried out in several domestic species (e.g., Robbins et al. 1993; Ling et al. 2003) and, more recently, in beach mice and woolly mammoth (Hoekstra et al. 2006; Rompler et al. 2006). However, all these studies have concentrated on within-species variation and little is known about the evolution of MC1R function within vertebrate groups over timescales of millions of years.
MC1R variation is well characterized in human populations. Several loss-of-function MC1R variants segregate in European populations, and these are associated with red hair and pale skin (Valverde et al. 1995; Schioth et al. 1999). In nonhuman primates, the functional diversity of MC1R is of interest because of their substantial variation in coat color. A recent study examined MC1R sequence variation in a broad range of primate species (Mundy and Kelly 2003). Convergent evolution of an 8 amino acid deletion in MC1R with jaguarundi cats suggested that MC1R variation was involved in the melanic coat of golden-headed lion tamarins (Eizirik et al. 2003; Majerus and Mundy 2003). There were also several cases where a role for MC1R in coat color variation could be excluded among closely related taxa. In some other taxa, including the completely reddish (pheomelanic) orangutans and the black (eumelanic) Goeldi's monkey, fixed MC1R differences were present but the absence of close relatives made the potential role of MC1R in coat color phenotype uncertain (Mundy and Kelly 2003). The orangutan MC1R has 13 fixed amino acid differences from human MC1R, and therefore, it remains an open question whether the pheomelanic coat of orangutans represents an MC1R loss-of-function phenotype as in humans. Finally, some mutations in important functional sites in MC1R were identified in which the expected phenotypic effects were not present. Most interestingly, black-and-white ruffed lemur and red ruffed lemur have MC1R with an E94K mutation that causes constitutive activation and dark coat coloration in other species, even though the red ruffed lemur (Varecia rubra) has predominantly pheomelanin hairs (Mundy and Kelly 2003).
As MC1R represents a critical control point in melanin-type switching, a general point of interest concerns molecular evolution of 3 potential mechanisms by which MC1R activity could be affected: basal activity, activation by MSH, and inhibition by ASIP. There is little information available on this from previous studies, although one relevant observation is that constitutive activation has tended to be associated with loss of MSH binding (Robbins et al. 1993; Lu et al. 1998).
In the present study, we expressed and pharmacologically characterized MC1Rs from a phylogenetically diverse range of primate species (apes, Old World monkeys, New World monkeys, and lemurs) in order to answer the following questions: Is there variation in MC1R biochemical function among primates, and, if so, does this variation relate to primate phylogeny and/or coat color? Is the mechanism of red hair generation the same in orangutans and humans? Is there evidence for evolution of the relative importance of different control mechanisms on MC1R activity? Pharmacological characterization involved 2 types of assay: melanocortin ([Nle4, D-Phe7]
-MSH and -MSH) -binding assays and second messenger (cAMP) assays in response to agonist (-MSH) and inhibitor/inverse agonist (ASIP).
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Materials and Methods |
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Receptor Clones
MC1R clones from orangutan (Pongo pygmaeus) Ppy4; Old World monkeys: black-and-white colobus (Colobus guereza) Cgu2, ebony (Javan) langur (Trachypithecus auratus) Tau1, and pig-tailed macaque (Macaca nemestrina) Mne2; New World monkeys: common marmoset (Callithrix jacchus) Cja13, Goeldi's monkey (Callimico goeldii) Cgo20, and mantled howler (Alouatta palliata) Apa1; and lemurs: bamboo lemur (Hapalemur griseus) Hgr1 and red ruffed lemur (V. rubra) Vvr5 were obtained from a previous study (Mundy and Kelly 2003
). The full-length MC1R genes were reamplified by polymerase chain reaction and transferred into a modified pCEP4-Turbo expression vector (Marklund et al. 2002). Human MC1R clone in pCEP4 vector was obtained from previous study (Ringholm et al. 2004). All constructs were sequenced to confirm their identity to original clones.
Expression of Receptors
Human embryonic kidney (HEK)-293 cells with Epstein–Barr nuclear antigen (EBNA) grown to 60–70% confluence were transfected with 10 µg of the construct using FuGENE 6 transfection reagent (Roche, Bromma, Sweden) according to the manufacturers' instruction. The cells were grown in Dulbecco's modified eagle media and F-12 nutrient mixture (D-MEM/F-12; 1:1) with GlutaMAX I, 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin B, and 250 µg/ml geneticin G-418 (Invitrogen, Lidingö, Sweden) in humidified atmosphere with 5% CO2 at 37 °C. Semistable cell lines, expressing target receptors, were obtained by selecting for growth in the presence of 100 µg/ml hygromycin B (Invitrogen), first added 24 h after transfection.
Radioligand-Binding Assay
HEK-293-EBNA cells expressing MC1Rs were harvested from plate and resuspended in binding buffer composed of 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2.5 mM CaCl2, 1 mM MgCl2, and 0.2% bacitracin, pH adjusted to 7.4. To obtain the membranes, cells were mechanically homogenized with Ultra Turrax. Cell suspensions were centrifuged for 3 min at 190 x g, and membranes were collected from supernatant by centrifugation for 15 min at 20,000 x g. The pellet was resuspended in binding buffer. The binding was performed in a final volume of 100 µl at room temperature for 2 h. Saturation experiments were carried out with serial dilutions of [125I]NDP-MSH ([Nle4, D-Phe7]-MSH), labeled by the Chloramine-T method. Nonspecific binding was determined in the presence of 1 µM unlabeled NDP-MSH. Competition experiments were performed with constant 0.4 nM concentration of [125I]NDP-MSH and serial dilutions of competing unlabeled human ligands: NDP-MSH and -MSH (Neosystem, Strasbourg, France). The membranes were collected by filtration on glass fiber filters, Filtermat A (PerkinElmer), using a Tomtec Mach III cell harvester (Orange, CT). The filters were washed with 5 ml per well of 5 mM Tris–HCl (pH 7.4) and dried at 50 °C. MeltiLex A scintillator sheets (PerkinElmer) were melted on dried filters, and radioactivity was counted with an automatic Microbeta counter 1450 (PerkinElmer, Upplands Väsby, Sweden). Binding assays were performed in duplicate with at least 3 independent experiments. Nontransfected cells did not show any specific binding with [125I]NDP-MSH. The results were analyzed with Prism 3.0 software package (GraphPad, San Diego, CA). Ki values were calculated with human MC1R Kd = 0.0851 nmol/l.
cAMP Detection Assay
cAMP production was determined on semistable HEK-293-EBNA cells expressing the target MC1Rs. A confluent layer of cells was incubated for 3 h with 2.5 µCi/ml of [3H] adenosine triphosphate (ATP; GE Healthcare, Uppsala, Sweden). Cells were collected, washed, and resuspended in buffer containing 137 mM NaCl, 5 mM KCl, 0.44 mM KH2PO4, 4.2 mM NaHCO3, 1.2 mM MgCl2 x 6H2O, 20 mM HEPES, 1 mM CaCl2, 10 mM glucose, and 0.5 mM isobutylmethylxanthine (Sigma-Aldrich, Stockholm, Sweden), pH adjusted to 7.4. Resuspended cells were incubated at 37 °C for 10 min. Stimulation reaction was performed at 37 °C for 20 min in a final volume of 150 µl containing approximately 2 x 105 cells and various concentrations of -MSH, ASIP-conditioned medium (obtained from previous study, Cerda-Reverter et al. 2005), and 15 µM forskolin, alone or with ASIP. After incubation, cells were centrifuged and 200 µl of 0.33 M perchloric acid was added to pellets to lyse the cells. The cells were frozen, thawed, and centrifuged. In total, 200 µl of lysate was added to Dowex 50W-X4 resin columns (Bio-Rad), previously washed with 2 x 10 ml H2O. As an internal standard, 750 µl of 0.33 M perchloric acid containing 0.5 nCi/ml [14C]cAMP (GE Healthcare) was added to each column. Columns were washed with 2 ml H2O to remove ATP, which was collected in scintillation vials to estimate the amount of unconverted [3H]ATP. Four microliters of Ready Safe scintillation cocktail (PerkinElmer) was added to the vials before counting. Dowex columns were then placed over alumina (Sigma-Aldrich) columns (prewashed with 8 ml of 0.1 M imidazole), and the cAMP was transferred into the alumina column using 10 ml H2O. cAMP was eluted from alumina column with 4 ml of 0.1 M imidazole and collected into scintillation vials to which 7 ml of scintillation fluid was added. 3H and 14C were counted on Tri-carb liquid scintillation beta counter. The amount of obtained [14C]cAMP was expressed as a fraction of total [14C]cAMP ([14C]cAMP/total [14C]cAMP) and was used to estimate column efficiency in order to standardize [3H]cAMP. Results were calculated as the percent of total [3H]ATP (obtained as a sum of [3H]ATP from first column and [3H]cAMP from second column) to [3H]cAMP and used to determine EC50 values by nonlinear regression using Prism 3.0 software. All experiments were performed in duplicate and repeated 3 times.
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Results |
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An alignment of nonhuman primate MC1Rs together with human MC1R is presented in figure 1. In order to pharmacologically characterize the wild-type MC1Rs from different primate species, the genes were transferred into expression vector, transfected into eukaryotic cells, and tested in radioligand-binding and second messenger assays.
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Radioligand-Binding Assay
Clones from orangutan, colobus, langur, macaque, and marmoset were able to bind NDP-MSH and
-MSH. Clones from bamboo and ruffed lemurs and howler and Goeldi's monkey did not display any binding of tested melanocortin peptides. The Ki values obtained from the competition experiments are presented in table 1. The curves for NDP-MSH and -MSH are displayed in figures 2 and 3, respectively. The affinity for NDP-MSH and -MSH was highest for human MC1R. Among tested primate MC1Rs, langur had the highest affinity for NDP-MSH, which was 5.3-fold lower than affinity of human MC1R. Orangutan had 5.8-fold lower, colobus had 7.9-fold lower, marmoset had 8.8-fold lower, and macaque had 9.4-fold lower affinity for NDP-MSH than human MC1R. Colobus displayed 1.9-fold lower potency for -MSH compared with human MC1R. Langur had 4-fold, orangutan had 8.2-fold, and macaque had 27-fold lower affinity for -MSH compared with human MC1R.
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cAMP Detection Assay
The production of intracellular cAMP in response to different concentrations of
-MSH is presented in figure 4. Stimulation of orangutan and the 3 Old World monkey (colobus, langur, and macaque) MC1Rs with different concentrations of -MSH resulted in production of intracellular cAMP in a dose-dependent manner. The calculated EC50 values are presented in table 1. EC50 values for -MSH were similar between nonhuman primate and human MC1Rs. The MC1R clones from lemurs (ruffed and bamboo), Goeldi's monkey, and howler did not induce production of cAMP in response to -MSH. On the other hand, all tested nonhuman primate MC1R clones displayed basal cAMP production. Macaque, ruffed lemur, langur, and bamboo lemur exhibited highest levels of basal cAMP production. The MC1R clones from Goeldi's monkey, howler, bamboo, and ruffed lemurs, which did not bind melanocortin peptides, were treated with goldfish ASIP, which has been reported to inhibit cAMP production of MC1R (Cerda-Reverter et al. 2005). Howler, bamboo, and ruffed lemur MC1R clones displayed lower basal cAMP production and lower forskolin-induced cAMP production in the presence of ASIP (see fig. 5).
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Discussion |
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Here, we present extensive pharmacological characterization of MC1Rs representing 4 primate clades: lemurs, New World monkeys, Old World monkeys, and hominoids (apes and humans; fig. 6). The lemurs are the most phylogenetically distant from human and have the most divergent MC1R sequence. It seems that the binding site for MSH peptides is nonfunctional as there was no binding detected for MC1R of either bamboo or ruffed lemur. An aspartate at position 117 in human MC1R has been identified as important for ligand binding and constitutive activation (Lu et al. 1998
). The derived substitution of this residue to a nonpolar amino acid, D117G, in both species is likely to destroy the MSH binding site, and the additional D121N substitution in ruffed lemurs also occurs at a residue previously shown to be crucial for binding (Lu et al. 1998). Furthermore, both lemurs share a C315F substitution, removing an intracellular residue that is normally palmitoylated (Rouzaud et al. 2006). Interestingly, however, these mutations have not rendered these MC1Rs nonfunctional as we detected high levels of basal cAMP production by them. A mutation from glutamate to lysine at position 94 or its equivalent, as found in ruffed lemurs, has been shown to cause constitutive activation of MC1R (i.e., a continually active MC1R in the absence of the MSH ligand) and a black coat in mice (Robbins et al. 1993), and the same mutation is associated with black plumage in domestic chickens (Takeuchi et al. 1996), quail (Nadeau et al. 2006), and a passerine bird, bananaquit (Theron et al. 2001). It is therefore interesting that the constitutive activation in the ruffed lemur clone was the highest among the nonbinding MC1Rs in this study, which is likely contributed to by the E94K substitution. Because the E94 position is unchanged in Hgr1, it is interesting to speculate which positions are responsible for the constitutive activation of this receptor. A likely candidate is D117G because previous mutagenesis studies indicate that substitutions in D117 lead to constitutive activation (Lu et al. 1998). From previous studies, constitutive activation of the MC1R is expected to be associated with dark, eumelanic, coat color. It is therefore surprising that both ruffed and bamboo lemurs have both eumelanin and pheomelanin in their coats, and pheomelanin is normally produced during inhibition of MC1R. It was therefore of interest to test whether ASIP, the inhibitor/inverse agonist of MC1R that can inhibit MC1R activity in the absence of MSH (Siegrist et al. 1997), had activity on the lemur receptors. Surprisingly, in both lemur species, we saw the suppression of constitutive activity of MC1Rs by ASIP, and an effect was confirmed by inhibition of forskolin-stimulated cAMP production by ASIP. This is the first demonstration that loss of MSH binding in MC1R is not necessarily coupled to loss of ASIP binding and functional inhibition by ASIP. In addition, it provides a plausible mechanism for the combination of a constitutively active MC1R and presence of pheomelanin bands in the hairs of bamboo and red ruffed lemurs via inhibition by temporally regulated ASIP expression. The black-and-white ruffed lemur (Varecia variegata) is a sister species to the red ruffed lemur that has no pheomelanin and an identical MC1R sequence (Mundy and Kelly 2003). Regulation of ASIP expression in the red ruffed lemur could therefore be the major mechanism for the difference in coloration between these sister species.
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" represents increase in basal level of activity of MC1R. The ancestral condition is reconstructed to be similar to the mouse outgroup, that is, presence of MSH binding and a moderate level of basal MC1R activity.Our results show that within the group of New World monkeys, there is binding diversity in response of MC1Rs to ligands. The mantled howler has a MC1R with a similar profile to that of the 2 lemurs, with lack of MSH binding, high basal activity, and ASIP responsiveness. The howler has a distinctive D117Q substitution that could explain this pharmacological profile. The 2 other New World monkeys investigated, marmoset and Goeldi's monkey, are closely related to Callitrichids, whose MC1R sequences differ by 11 amino acid residues. They displayed strikingly dissimilar binding profiles. The MC1R from the common marmoset, which has an agouti coat, appears to have a normally functional MC1R, which is able to bind MSH peptides, whereas that from Goeldi's monkey with solid black coat did not bind MSH peptides and displayed constitutive activity. In this case, the constitutive activity may underlie the extremely black phenotype of this primate. The amino acid residues responsible for the functional difference from marmoset are unclear; the L87V and A107V substitutions are in the region of the protein important for MSH binding and constitutive activation, although A107V is shared with humans. The MC1Rs of both marmoset and Goeldi's monkey possess an extended carboxy terminal, and so our results demonstrate that this does not affect MSH binding or cAMP activation. This is in line with the alternatively spliced long carboxy terminal of the human MC1R that also does not affect the binding or activation (Tan et al. 1999
). Although both MC1Rs have a substitution in the functionally important negatively charged aspartate at position 117, this is replaced by a positively charged histidine, and it has been shown previously that substitution from negatively to positively charged residues at this position (such as D117K) does not have an effect on MSH binding (Lu et al. 1998).
The orangutan MC1R has 13 fixed amino acid differences from human, 3 of which are reconstructed to have occurred along the lineage to orangutans after the split with the other hominoids (Mundy and Kelly 2003). MC1R therefore remained a plausible candidate gene for the solid pheomelanic coat color of this species. However, the pharmacological characterization in the orangutan revealed a functional MC1R in binding and intracellular response, with the only difference from human MC1R being lower intracellular response to -MSH and slightly elevated basal cAMP level. This demonstrates that the convergent evolution of red hair in orangutans and European human populations has a different genetic basis, as loss-of-function MC1R mutations account for the phenotype in humans (Valverde et al. 1995; Schioth et al. 1999). A separate genetic basis for coloration differences has been demonstrated in closely related populations of rodents (Nachman et al. 2003; Hoekstra et al. 2006), but this is one of the first demonstrations of this at deeper evolutionary timescales. Another possible cause of constant pheomelanin production in orangutans is overexpression of ASIP, as occurs in several mouse mutants (Duhl et al. 1994). The coding sequence of ASIP in orangutans was recently reported to be very similar to humans (Mundy and Kelly 2006), but the promoter regions, where gain-of-function mutations have been found in mice (Jackson 1997), have yet to be studied.
MC1Rs from Old World monkeys displayed similar pharmacological profiles to those of orangutan, showing dose-dependent MSH binding and activation, with some variation in levels of basal activity. Overall therefore, the catarrhine primates (Old World monkeys and hominoids) display strong conservation of MC1R function in contrast to that found in lemurs and New World monkeys.
A reconstruction of MC1R function during primate evolution is shown in figure 6. This is based on parsimony, using laboratory mouse (Mus musculus) MC1R as an outgroup. The mouse MC1R displays MSH and ASIP binding and activation/inhibition, and moderate levels of basal activity, which is apparent from the retention of eumelanin in mouse knockout mutations of POMC, the gene that encodes MSH (Slominski et al. 2005). The only equivocal part of the reconstruction is the loss of MSH binding in New World monkeys, which we consider to most likely have occurred twice independently in the howler and Goeldi's monkey lineages (rather than a single loss in the basal lineage followed by regain in the marmoset). The reconstruction highlights the amount of evolutionary change in MC1R function in lemurs and New World monkeys and the relative conservation of function in catarrhine primates. It also shows that changes in MSH binding and level of basal activity do not always evolve together.
In general, we find that substitutions at previously identified important functional sites (e.g., 94, 117) have a predictable effect on MC1R function. This demonstrates that functional effects of MC1R mutations are to a large extent independent of variation at other sites, that is, there is low intragenic epistasis and increases the confidence in predicting MC1R biochemical function from primary structure. However, the link between MC1R activity and coat color phenotype is not so strong. In particular, whereas in some cases (e.g., Goeldi's monkey), the presence of high constitutive activity is associated with a strongly eumelanic coat, as expected from previous studies, there are other cases where pheomelanin can still occur, as seen in the ruffed and bamboo lemurs. Unsurprisingly therefore, there is no one-to-one relationship between variation in DNA sequence and function at a single locus and coat color across a whole vertebrate order.
Overall, the results show that the relative importance of the 3 different mechanisms regulating MC1R activity has changed several times during primate evolution. The basal activity, which varies from low to high, sets the basic activity of MC1R, which can then be modified by spatiotemporal regulation of MSH and/or ASIP concentrations. Low or high basal activity can be associated with low or high MC1R responsiveness to MSH, showing that these can evolve independently. In the case where MC1R binding is lost, regulation of activity can still be maintained via ASIP reverse agonism, as long as there is moderate to high basal activity. Although we have not documented differences in ASIP effects on MC1R here, it is interesting to note that this mechanism of regulation has been lost in gibbons, which have a genomic deletion of the ASIP locus (Nakayama and Ishida 2006). An interesting question is whether evolutionary shifts in the relative importance of these mechanisms have an influence on the evolution of coat color diversity and, in particular, whether loss of one mechanism (e.g., MSH binding) leads to a reduction in color diversity. Although much more data on MC1R function, and rigorous comparative analyses, would be necessary to address this question robustly, no obvious genetic constraints emerge from our findings. For example, the lemurid lemurs studied here have evolved substantial variation in coat color diversity following the loss of MSH binding that probably occurred with the D117G substitution in their common ancestor.
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Acknowledgements |
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The studies were supported by the Swedish Research Council (VR, medicin), the Swedish Society for Medical Research (SSMF), Åke Wibergs Stiftelse, Svenska Läkaresällskapet, Sweden (H.B. Schiöth), and the Leverhulme Trust and Biotechnology and Biological Sciences Research Council (N.I.M. and J.K.).
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Footnotes |
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Naruya Saitou, Associate Editor
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