The results indicate that the structural requirements that allow the receptor to adapt an active conformation without binding to a ligand, as a con-sequence of this E/K mutation, are not
Trang 1Association of feather colour with constitutively active
melanocortin 1 receptors in chicken
Maria K Ling1, Malin C Lagerstro¨m1, Robert Fredriksson1, Ronald Okimoto2, Nicholas I Mundy3,
Sakae Takeuchi4and Helgi B Schio¨th1
1
Department of Neuroscience, Uppsala University, Uppsala, Sweden;2Department of Poultry Science, University of Arkansas, Fayetteville, Arkansas, USA;3Department of Biological Anthropology, University of Oxford, Oxford, UK;
4
Department of Biology, Faculty of Science, Okayama University, Okayama, Japan
Seven alleles of the chicken melanocortin (MC) 1 receptor
were cloned into expression vectors, expressed in
mam-malian cells and pharmacologicallycharacterized Four of
the clones e+R, e+B&D, ewh/ey, ERfayoumigave receptors to
which melanocortin stimulating hormone (a-MSH) and
NDP-MSH bound with similar IC50values and responded
to a-MSH byincreasing intracellular cAMP levels in a
dose-dependent manner Three of the cMC1 receptors; eb, Eand
ER, did not show anyspecific binding to the radioligand, but
were found to be constitutivelyactive in the cAMP assay
The E and ERalleles are associated with black feather
col-our in chicken while the eb allele gives rise to brownish
pigmentation The three constitutivelyactive receptors share
a mutation of Glu to Lys in position 92 This mutation was
previouslyfound in darklypigmented sombre mice, but
constitutivelyactive MC receptors have not previouslybeen
shown in anynonmammalian species We also inserted the
Glu to Lys mutation in the human MC1 and MC4
recep-tors In contrast with the chicken clones, the hMC1-E94K receptor bound to the ligand, but was still constitutively active independentlyof ligand concentration The hMC4-E100K receptor did not bind to the MSH ligand and was not constitutivelyactive The results indicate that the structural requirements that allow the receptor to adapt an active conformation without binding to a ligand, as a con-sequence of this E/K mutation, are not conserved within the
MC receptors The results are discussed in relationship to feather colour in chicken, molecular receptor structures and evolution We suggest that properties for the E92K switch mechanism mayhave evolved in an ancestor common to chicken and mammals and were maintained over long time periods through evolutionarypressure, probablyon closely linked structural features
Keywords: G-protein coupled; MSH; melanocortin receptor; polymorphism
Spontaneous or constitutive G-protein coupled receptor
(GPCR) activitywas first convincinglydescribed for the
d-opioid receptor [1], and was further established by
the demonstration of constitutive activityin chimeras of
the a1Band a2receptors (summarized in [2]) Later it was
shown that mutations in the human rhodopsin gene can
constitutivelyactivate transducin in the absence of retinal
and light [3] It is now known that naturallyoccurring
constitutivelyactive GPCRs are found to be responsible
for a diverse arrayof inherited as well as somatic genetic
disorders [4,5]
The melanocortins (a-MSH/ACTH), secreted from a
frog pituitary, were in 1912 found to cause pigmentation
In higher vertebrates including aves and mammals, these peptides are expressed throughout the body, and are involved in a varietyof physiological regulatoryfunctions [6–8] In the skin, the melanocortins are synthesized locally (for birds [9], for mammals [10]), and act through a GPCR named melanocortin (MC) 1 receptor to regulate melano-genesis Keratinocytes probably serve as the main physio-logical source of melanocortins acting on melanocytes in the epidermis and hair follicle The MC1 receptor couples through G proteins to adenylate cyclase to stimulate tyrosinase, the rate-limiting enzyme in the synthesis of both classes of melanin pigments, eumelanin and phaeomelanin
A low level of tyrosinase expression leads to increased phaeomelanin synthesis, while elevated levels of tyrosinase, that can result from a-MSH stimulation of melanocytes, divert the intermediates primarilyalong the eumelanin synthetic pathway (for reviews see [8,10–11])
The extension (E) locus, together with agouti (A) locus, regulates the relative amount of black pigment (eumelanin) and red/yellow pigment (phaeomelanin) in mammals [12] The E locus encodes the MC1 receptor and the A locus encodes the agouti peptide, an antagonist of the MC1 receptor Dominant alleles at extension result in dark brown
or black coat colour, while animals homozygous for recessive alleles have yellow or red coats The opposite is true for the agouti allele A dominant mutation at the A
Correspondence to H B Schio¨th, Department of Neuroscience,
Biomedical Center, Box 593, 75 124 Uppsala, Sweden.
Fax: + 46 18 51 15 40,
E-mail: helgis@bmc.uu.se
Abbreviations: GPCR, G-protein coupled receptor; IC 50 , 50%
inhibitoryconcentration; EC 50 , 50% effective concentration;
IL, intracellular loops; MC, melanocortin; MSH, melanocortin
stimulating hormone; NDP-MSH, [Nle4, D -Phe7]a-MSH;
TM2, transmembrane region 2.
(Received 3 December 2002, revised 26 January2003,
accepted 6 February2003)
Trang 2locus (Ay/A) in mice causes uniform yellow coat and obesity.
A similar phenotype, but with normal body weight, is found
in recessive yellow mice (e/e), where a loss of function
mutation in the MC1 receptor results in animals producing
phaeomelanin [13] The dominant extension locus alleles,
sombre(ESOand ESO)3J) and tobacco (Etob), which all have
dominant melanizing effects, result from point mutations
that produce constitutivelyactive receptors The sombre
alleles produce a fairlyuniform black coat, while tobacco
darkening onlyinvolves the dorsal portion of the animal
The tobacco alleles produce a receptor that remains
hormone responsive but produces a greater activation of
adenylyl cyclase than does the wild-type allele Both sombre
alleles were found to produce constitutivelyactive receptors,
defined as being able to significantly elevate adenylyl cyclase
activityin the absence of ligand, and therebyenhance the
eumelanin production resulting in a dark pigmentation in
mice [13] Null-mutations of POMC, the precursor for the
melanocortins, causes yellow coat, obesity and adrenal
insufficiency These mice are somewhat darker, dirtyblond
suggesting that the basal MC1 receptor activityin the
absence of ligands maybe higher than a nonfunctioning
receptor [14]
Mutations in MC1 receptors, related to hair or skin
colour, have been found in several other mammalian
species, although pharmacological characterization of these
changes has usuallynot been carried out Two species,
whose receptors have been pharmacologicallyinvestigated,
are fox and sheep Va˚ge cloned and characterized the fox
MC1 receptor and found a mutation that caused
constitu-tive activityin the dark coated Alaska Silver fox [15] This
dominant mutation was however, also found in foxes with
significant red coat colouration, and it was suggested that
the fox agouti protein counteracted the signalling activityof
a constitutivelyactive fox MC1 receptor In sheep, two
mutations in the MC1 receptor showed complete
cosegre-gation with dominant black coat colour in a familylineage
These mutations were transferred into the corresponding
mouse receptor in which theyproduced constitutive activity
[16] Loss of function mutations are common in the human
MC1 receptor and these are over-represented in Northern
European redheads and in individuals with pale skin [17,18]
These variants are a risk factor, possiblyindependent of skin
type, for melanoma [19]
Variants of the MC1 receptor gene were found to be
associated with the extension (E) locus in chickens and it
was proposed that theymight be linked to feather
pigmentation [20] A dominant mutation, identical to
one of the mutations in the sombre mouse, was found in
chicken with black feathers [20] The verysame mutation,
Glu to Lys in transmembrane region 2 (TM2), has also
been shown to be present in melanic individuals of the
bananaquit, a neotropical passerine bird, and absent in all
yellow individuals [21] The molecular pharmacology of
these mutations has however, not yet been investigated
In this study, we performed the first expression studies on
avian MC receptor genes We expressed and
pharmacologi-callycharacterized seven polymorphic variants of the
chicken MC1 receptors derived from different E locus
alleles We also introduced the Glu to Lys mutation in to the
human MC1 and MC4 receptors to investigate the
pharma-cological impact of these mutations
Experimental
Receptor clones Oligonucleotide primers were designed to amplifythe entire coding region of each cMC1 receptor variant To facilitate cloning and establishment of orientation of the PCR-amplified DNA fragments, recognition sites for HindIII and BamHI were introduced into 5¢ and 3¢ primers, respectively, byaltering original nucleotide sequences The primer sequences were 5¢-GGAAGCTTTGTAGGTGCTGCA GTT-3¢ for the 5¢ primer and 5¢-CATGGATCCTCCTC CTGTCTGTGCCGC-3¢ for the 3¢ primer, corresponding
to positions)55 to )78 and 1049–1072, respectively, of the cMC1 receptor gene, where the A of the translation initiation codon ATG was defined as +1 The amplified DNA fragments were cloned into pGEM3Zf(+), sequenced, and subsequentlysubcloned into pCEP4 Turbo expression vector [22] and resequenced The human MC1 [23] and human MC4 receptors [24] were used as templates for the mutagenesis
Site-directed mutagenesis The E94K mutation in hMC1 and E100K mutation in hMC4, were introduced into the receptor coding sequence byPCR Two complementaryoligonucleotides were designed to contain the required mutation The hMC1-E94K primers were (5¢ primer) 5¢-CAGGAGGATGA CGGCCGTCTTCAGCACGTTGCTCCC-3¢ and (3¢ pri-mer) 5¢-GGGAGCAACGTGCTGAAGACGGCCGTCA TCCTCCTG-3¢ The hMC4-E100K primers were (5¢ primer) 5¢-TAGGGTGATGATAATGGTTTTTGATCC ATTTGAAAC-3¢ and (3¢ primer) 5¢-GTTTCAAATG GATCAAAAACCATTATCATCACCCTA-3¢ The pri-mers were hybridized to opposite strands of the receptor gene and the complete MC receptor coding sequence was amplified The end-primer was complementaryto 3¢ or 5¢ depending on which mutagenesis primer was used (forward
or reverse) The two products were used as templates and linked together in a second PCR, in which onlythe end-primers were used
Expression DNA for transfection was prepared using Qiagen Plasmid Maxi Kit (Merck) HEK 293 EBNA cells 50–70% confluent
on 10-cm plates, were transfected with 15 lg of the construct using FuGENETMTransfection Reagent (Boeh-ringer Mannheim), diluted in Optimem medium (Gibco BRL) After transfection, cells were grown in Dulbecco’s MEM/Nut Mix F-12 (Gibco BRL) containing 10% foetal bovine serum (Biotech Line), 2.4 mM L-glutamine, 2.5 mgÆmL)1 G418, 2.5 lgÆmL)1 amphotericin, and
100 lgÆmL)1 kanamycin solution (all from Gibco BRL) until harvesting, after 48 h
Binding assays Intact transfected cells were re-suspended in 25 mMHepes buffer (pH 7.4) containing 2.5 mM CaCl2, 1 mM MgCl2 and 2 gÆL)1 bacitracin Competition experiments were
1442 M K Ling et al (Eur J Biochem 270) FEBS 2003
Trang 3performed in a final volume of 100 lL The cells were
incubated in 96-well plates for 3 h at 37C with constant
concentration of [125I]NDP-MSH and appropriate
concen-trations of competing unlabelled ligands, [Nle4, D-Phe7]
a-MSH (NDP-MSH) or a-MSH The incubations were
terminated byfiltration through Glass Fibre Filters,
Filter-mat A (Wallac Oy, Turku, Finland), which had been
presoaked in 0.3% poly(ethylenimine), using a TOMTEC
Mach III cell harvester (Orange, CT, USA) The filters were
washed with 5.0 mL 50 mMTris pH 7.4 at 4C and dried
at 60C The dried filters were then treated with MeltiLex A
(Perkin Elmer) melt-on scintillator sheets and counted in a
Wallac 1450 (Wizard automatic Microbeta counter) The
results were analysed with a software package suitable for
radioligand binding data analysis (PRISM 3.0, Graphpad,
San Diego, CA, USA) The binding assays were performed
in duplicate and repeated three times Nontransfected
HEK293-EBNA cells did not show anyspecific binding to
[125I]NDP-MSH NDP-MSH was radio-iodinated bythe
chloramine T method and purified byHPLC NDP-MSH
and a-MSH were purchased from Neosystem (France)
cAMP assay
Semi-stable cells, expressing the receptors of interest, were
harvested in growth media containing
3-isobutyl-1-methyl-xanthine from Sigma Twohundred microlitres of the cells
were added to tubes containing appropriate
concentra-tions of a-MSH and incubated for 30 min at 37C After
stimulation the cells were lysed and cAMP extracted using
4.4M perchloric acid The cAMP extract was then
neutralized with 5MKOH and centrifuged The
intracel-lular cAMP produced was measured in 50 lL of the
supernatant after addition of [3H]cAMP and bovine
adrenal binding protein and incubation for 2 h at 4C
Standards containing nonlabeled cAMP were assayed in
the same manner The incubates were then harvested by
adding activated carbon After centrifugation, the
super-natant was removed into scintillation tubes and counted
in a Tri-carb Liquid scintillation analyser after addition of
3 mL scint solution (ReadySafeTM; Beckman Coulter)
The cAMP assaywas performed in duplicate and
repeated three times The results were analysed using the
PRISM 3.0 software package (Graphpad, San Diego, CA,
USA) The protein concentrations were measured using
Bio-Rad Protein Assay(Bio-Rad, Solna, Sweden) with
BSA as standard
Results
The amino acid sequences for the seven polymorphic cMC1 receptors derived from different E locus alleles are shown in Table 1 Four clones; e+R, e+B&D, ewh/ey, ERfayoumiresulted
in competition curves for a-MSH and NDP-MSH using iodinated [125I]NDP-MSH as radioligand The binding curves are shown in Fig 1 The IC50for the MSH ligands are shown in Table 2 Three clones, eb, E and ER, did not induce anyspecific binding on the transfected cells We also measured the intracellular cAMP in response to varying concentrations of a-MSH The cells transfected with the four cMC1 receptors, that showed competition curves in the binding assays, also responded to a-MSH byaccumulation
of intracellular cAMP in a dose-dependent manner The cAMP curves are shown in Fig 2, and the EC50values are shown in Table 2
The three cMC1 receptors; eb, Eand ER, did not show anyspecific binding to the radioligand or respond to a-MSH byaccumulation of intracellular cAMP The results are shown in Fig 3 The basal levels were, however, significantlyincreased for the cells transfected with the three clones in all experimental points, as compared with nontransfected HEK-293 EBNA cells, that were used as a control The cells transfected with the receptors (e+R,
e+B&D, ewh/ey, ERfayoumi) also served as controls, as at the initial level and at the lowest concentrations of a-MSH, the cAMP levels were lower than that observed for the cells transfected with eb, E and ER All experiments were performed with a similar number of cells; the amount of protein per well was determined and the cAMP values were normalized accordingly The cAMP levels for these three clones were significantlyhigher for all experimental points, also when corrected for the cell number The experiments were repeated three times and qualitativelythe results were the same each time The activation level did, however, not reach the maximum level of the other clones eb, Eand ER cMC1 receptors were partiallyactivated to 20–60% of the maximal activation of a normally functioning cMC1 receptor (ewh/ey) The cAMP levels did, however, vary between the repeats and the internal order between the three clones was not always the same Therefore, we do not draw the conclusion that the cAMP levels for the eb, Eand ER cMC1 receptors transfected cells differed from each other The Glu92 residue in TM2 (see Table 1) is conserved within the familyof MC receptor subtypes The eb, Eand
ERcMC1 receptors have Lys in this position We inserted a
Table 1 Amino acid positions in the seven different cMC1 receptors Dominance: E >ER> ewh> e+> eb> ey.
– Indicates that the amino acid in this position is the same as the one as described in the allele at the top Note that ewhand eyare identical in amino acid sequence.
Trang 4Lys in the corresponding position in the human MC1 and
MC4 receptors bysite-directed mutagenesis and generated
clones termed hMC1-E94K and hMC4-E100K The clones
were expressed and assayed pharmacologically in the same
manner as the clones mentioned above The binding results
are shown in Fig 4 The cells transfected with hMC1-E94K,
in contrast with the chicken receptors with Lys in position
92, did bind NPD-MSH The IC50value was about 18-fold
lower than that of the wild-type human MC1 receptor The
cells transfected with hMC4-E100K did not, however, show
anyspecific binding The cAMP results are shown in Fig 5
The cells transfected with hMC1-E94K showed significantly
higher cAMP values at all experimental points, independent
of concentration of a-MSH, as compared with the
non-transfected cells The cells non-transfected with hMC4-E100K,
in contrast with the chicken receptors with Lys in position
92, showed the same low levels as nontransfected cells
Discussion
Like the extension locus in mammals, the extended black (E) locus of the chicken controls the relative amount of eumelanin and phaeomelanin in melanocytes The locus was originallylocalized on chromosome 1, but recent genetic and FISH analyses revealed that it is located on a microchromosome [25,26] Several alleles exhibiting differ-ent pigmdiffer-entation have been described and there is an intricate hierarchyamong them But in general, alleles that make more eumelanin are dominant over those that make less eumelanin Unlike the mammalian extension locus, the phenotype of each allele is expressed mainly in chicks and adult females In fact, the phenotypes of adult males with different nonmelanic alleles, including e+, ewh, ey, and eb, are similar, having black-breasted red feather pattern The only phenotypic difference observed among them is the under-colour, the fluff of the feathers next to the skin; it is white or cream for the ewhand eymales and grey for the e+and eb In melanic alleles (E, ER, and ERfayoumi), adult males are black
in all areas and the flight feathers are also black for E, while the other melanic alleles (ER and ERfayoumi) give half-red half-black feathers Fig 6 shows the adult E locus colour patterns on a wild-type background for all other feather colour genes
Our results indicate that the polymorphic eb, E and ER chicken MC1 receptors are constitutivelyactive These three receptors all share a mutation of Glu to Lys in position 92 (see Table 1) Previouslyit has been shown that constitutive activation of the MC1 receptor in darklypigmented sombre mice results from the verysame mutation in position 92 [13] L98P mutation in the MC1 receptor in mouse, D119N in
Fig 1 Competition curves of [Nle4, D -Phe7]a-MSH (u) and a-MSH (m) obtained with transfected HEK-293 (EBNA) cells using a fixed concen-tration of 0.2 n M [ 125 I][Nle 4 , D -Phe 7 ]a-MSH for four cMC1 receptors, e+R, e+B&D, ewh/ey, ERfayoumi Data points represent means of duplicates and error bars indicate standard error of the mean (SEM).
Table 2 Pharmacological characterization of chicken MC1 receptors
after expression in HEK cells The IC 50 values were obtained from
competition using [ 125 I] [Nle4, D-Phe7] a-MSH as radioligand and
a-MSH and [Nle4, D-Phe7] a-MSH as competitors The EC 50 values
are obtained in intracellular cAMP assayusing a-MSH as stimulator.
Receptor
a-MSH (IC 50 )
(nmolÆL)1)
NDP-MSH (IC 50 ) (nmolÆL)1)
a-MSH (EC 50 ) (nmolÆL)1)
e +(B & D) 601 ± 117 6.00 ± 0.40 21.9 ± 5.3
ERfayoumi 1080 ± 137 4.79 ± 0.20 36.2 ± 0.5
1444 M K Ling et al (Eur J Biochem 270) FEBS 2003
Trang 5sheep and C125R in Alaska silver fox that cause dark
pigmentation have also been shown to be constitutively
active [15,27] The pharmacologyof constitutivelyactive
MC receptors has not been previouslyshown in any
nonmammalian species, and these results show that the
function of this mutation seems to be similar in chicken and
mice The results add further support to the hypothesis that the same point mutation (E92K) in the bananaquit, Coereba flaveolaassociated with the melanic plumage morph [21] is constitutivelyactive
The clear association between the mainlyblack feather colour of chickens possessing E and ERand the presence
of constitutivelyactive MC1 receptor is in line with the previous observation of the E92K mutation in other species Males possessing the eballele are described above Among the females, the allele gives rise to brownish pigmentation, although according to the results from the cAMP assayfor this receptor, perhaps a darker pheno-type would have been expected, as for the other two constitutivelyactive cMC1 receptors Likewise, ERfayoumi and ewh/ey alleles exhibiting phenotypes similar to ER allele and yellow-red pigmentation, respectively, were found to encode normallyfunctioning cMC1 receptors, which bind agonist and couple to G-protein in a ‘normal’ manner Thus, our pharmacological results cannot com-pletelyexplain the association of cMC1 variants with the corresponding phenotypes It is possible that the expres-sion levels of cMC1 receptor varywith different alleles, which results in phenotypic difference in alleles encoding cMC1 receptors with similar pharmacological character-istics Alternatively, specific amino acid substitutions observed in each allele alter the interaction of cMC1 receptor with unidentified factors expressed specificallyin melanocytes Further analyses are thus required to clarify molecular mechanisms for all the different feather pig-mentation patterns in chicken
Considering the other polymorphic residues in the chicken clones, the results indicate that the changes of
Fig 2 Generation of cAMP in HEK-293 (EBNA) cells transfected with four cMC1 receptors: e +R , e +B&D , e wh /e y , E Rfayoumi in response to a-MSH The cAMP levels were normalized to the amount of protein Data points represent means of duplicates and error bars indicate standard error of the mean (SEM).
Fig 3 Cells transfected with the three cMC1 receptors eb(n), E (s),
E R (m) show elevated cAMP levels and independence of the concentration
of a-MSH in comparison to basal cAMP levels in HEK-293 (EBNA)
cells (j) The cAMP levels were normalized to the amount of protein.
e wh /e y (d) (from Fig 2) is shown for comparison Data points
repre-sent means of duplicates and error bars indicate standard error of the
mean (SEM).
Trang 6Met71 to Thr, Leu133 to Gln, Thr143 to Ala, Arg213 to
Cys, or His215 to Pro do not influence the pharmacological
function of the receptors Met71, Leu133 and His215 are
conserved through all the MC receptors cloned so far These
residues are believed to be in the first, second and third
intracellular loops (IL), respectively Thr143 alternates
between Thr and Ser, and is found in IL2, while Arg213
alternates between Arg and Cys, and is found in IL3, within
the entire MC receptor family It is interesting that mutation
of Cys215 to Gly in the human MC1 receptor
(correspond-ing to Arg213) resulted in failure to generate cAMP signal in
response to the agonists a-MSH [28] It seems therefore that
the function of the MC receptor requires a polar residue in
this position, while a hydrophobic nonpolar residue causes
disconnection of the signal transduction As the keyresidues
within the MC receptor familyare highlyconserved, our
new results provide additional information for generating
molecular models of the binding and activation process of
the MC receptors that is an important part of rational drug
discovery[8,29]
In order to shed further light on the structural
require-ments needed for generating a constitutivelyactive MC
receptor, we introduced the corresponding E92K mutation
into the human MC1 and MC4 receptors It was surprising
that the mutant human MC1 receptor showed differences in
pharmacologyas compared with the mutant chicken MC1
receptor It is intriguing that in contrast with the chicken
receptor with Lys, hMC1-E94K bound the ligand but was
still constitutivelyactive, independentlyof the ligand This
pharmacologyis remarkable, as this receptor seems to have
the abilityto bind the agonist peptide ligand with high
(albeit slightlylower) affinity, and undergo the
conforma-tional changes that this binding interaction is proposed to
have, without influencing the interaction with the G-protein
Even though inactivating mutations associated with red hair
are common in the hMC1 receptors, no constitutive
activating MC1 receptor has yet been found in humans It
seems clear that the structural properties of the hMC1
receptor are different from those of chicken, mouse and fox, where the constitutive E/K mutation leads to loss of binding It is possible that the evolutionarypressure on the hMC1 receptor is altered due to changes in the physiological importance of bodyhair colour and therefore the structural pharmacologyis evolving differentlyas compared to MC1 receptors of species in which colour has a more defined function It would be interesting to see if this propertyis unique to humans or if it has evolved earlier in primates
In order to find out if these pharmacological properties are shared beyond the MC1 receptors, we also introduced the E92K mutation into the hMC4 receptor This receptor is mainlyexpressed in the central regions of the brain, including the hypothalamus where it is an important regulator of food intake The MC4 receptor shares 60%
of the amino acids with the MC1 receptor, but has a completelydifferent physiological role with no known overlap in function The MC1 and MC4 receptors do, however, share the unique propertythat theyboth have natural antagonists, the agouti and the agouti-related peptide, respectively, in addition to their natural agonist, a-MSH The hMC4-E100K, in contrast with the hMC1 receptor mutation, did not bind the MSH ligand and was not constitutivelyactive No naturallyoccurring activation mutants have been found for MC receptors other than MC1, but several inactivation mutants of the MC4 recep-tors are related to obesity[30,31] The results indicate that the structural abilityto form constitutivelyactive receptors with a single amino acid mutation of the conserved Glu in TM2 has either not been maintained, or was never present
in the MC4 receptors Unlike constitutivelyactivating mutations in manyGPCRs that give increased agonist efficacyor affinity, these MC1 receptor mutations have the opposite effect The molecular mechanism of constitutive activation of the MC1 receptor has been studied byinserting the mutations into the mouse MC1 receptor [16] These authors proposed a ligand-mimetic model which explains
Fig 4 Competition curve of [Nle4, D -Phe7]a-MSH obtained with
transfected HEK-293 (EBNA) cells using a fixed concentration of 0.2 n M
[ 125 I][Nle 4 , D -Phe 7 ]a-MSH for the human wild-type MC1 (u), human
MC1 (E94K) (m), and wild-type MC4 (j), human MC4 (E100K) (r)
receptors Data points represent means of duplicates and error bars
indicate standard error of the mean (SEM).
Fig 5 cAMP levels of normal cAMP in HEK-293 (EBNA) cells (j) and cells transfected with human MC1 (E94K) (d), human MC4 (E100K) (u) receptors, showing cAMP levels independent of the con-centration a-MSH The cAMP levels were adjusted to the amount of protein ewh/ey(.) (from Fig 2) is shown for comparison Data points represent means of duplications and error bars indicate standard deviations.
1446 M K Ling et al (Eur J Biochem 270) FEBS 2003
Trang 7the lower (read nonexistent) ligand affinityand efficacy It
was proposed that the mutations transformed the receptor
into its active form, not bydisrupting the internal constraint
as proposed bythe earlyrhodopsin studies and the ternary
allosteric model, but byindirectlymimicking
ligand-bind-ing The amino acid residues that were mutated in the
mouse MC1 receptor are all conserved within the human
MC1 and further studies are thus needed to explain the
pharmacological differences between the mouse and human
MC1 receptors
The reptilian ancestors of chickens diverged about 300–
350 million years ago from the lineage leading to mammals
[32] It is remarkable that the same single amino acid
mutation forming a constitutive active receptor is found in
so evolutionarydistant species Recent studies show
remarkable evolutionaryconservation within the primary
sequence and pharmacologyof MC receptors (including the
E92) for at least 450 million years [33] Our mutations of the
hMC1 and hMC4 receptors indicate that the structural
requirements that allows the receptors to fall into a
constitutivelyactive state without binding to a ligand are not automaticallyconserved within the MC receptors or within the MC1 receptors Therefore, we find it unlikelythat
it is simplythe abilityto bind MSH peptides, the main pharmacological propertyshared bythe MC1 and MC4 receptors, that is the structural feature that automatically allows the E92K switch mechanism to work We find it also unlikelythat the complex structural requirements for single amino acid activation switch were independently developed in chicken and mammals It is thus tempting to speculate that some structural properties evolved once before the divergence of the avian and mammalian lineages The intriguing question is bywhich mechanism this propertycould have been conserved over such long time period One theoretical possibilityis that the specific mutation was ancestral and that an E/K92 heterozygote had a selective advantage over the two homozygotes, therebypreserving both the alleles and also the structural constraints This is, however, veryunlikelydue to the long evolutionarytimes and the divergent lineages involved
Fig 6 Cartoon representation of the adult E locus colour patterns on a wild-type background for the feather colour genes E (dominant extended black) birds are black in all areas in both sexes Males that are e + , e b , e wh or e y (wild-type, partridge or brown, dominant wheaten or recessive wheaten, respectively) all have the black-breasted red feather pattern The only difference is that the wheaten males have a white or cream feather under-colour and the e+and ebmales have a greyunder-colour ER(birchin) males are different in the wings The flight feathers are all black instead of the half-red half-black feathers of the recessive alleles Birchin females have black bodies and gold hackle feathers Both male and female birchins have black melanin in the epidermis of their shanks The brown (e b ) females have brown stippled backs and wings and brown stippled breasts, where the wild-type (e+) females have brown stippled backs and wings and salmon breasts Both females have a greyunder-colour Dominant wheaten (ewh) and recessive wheaten (ey) females have the same phenotype, having the salmon colour of the wild-type breast extended into the plumage of the back and wings Theyhave a white or cream under-colour and black is diluted in the female plumage.
Trang 8There are onlya few examples of the maintenance of specific
alleles byheterozygote advantage (e.g MHC class II alleles,
haemoglobin S) and in these cases the maintenance has been
for onlythousands to at most a few million years [34]
Moreover, it is notable that the E92K mutation in
bananaquit (mentioned above) is believed to have occurred
recentlyin that lineage [21] We believe therefore that it is
most likelythat the structural properties for the E92K
switch mechanism were maintained through evolutionary
pressure on closelylinked structural features These
pro-perties were subsequentlyretained in certain lineages, like
mouse and birds, and lost in others, like humans Further
structural characterization of the protein structure and
studies on the MC receptors in more ancient tetrapod
groups, such as reptiles, mayshed further light into the
mechanism on how structural properties for a single amino
acid switch mechanism mayhave survived such a long
evolutionarydistance
Acknowledgements
We thank Prof D Larhammar, Uppsala Universityfor valuable
criticism The studies were supported bythe Swedish Medical Research
council (MRC), the Swedish Societyfor Medical Research (SSMF),
Svenska La¨karesa¨llskapet, A˚ke Wibergs Stiftelse and Melacure
Therapeutics AB, Uppsala, Sweden to H.S., the Japanese Societyfor
Promotion of Science (Grant-in Aid for Scientific Research) to S.T.
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