Vollum Institute (D.L., R.D.C.) Oregon Health Sciences
University Portland, Oregon 97201
Department of Animal
Science (D.I.V.) Agricultural University of Norway N-1432 As,
Norway
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ABSTRACT |
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INTRODUCTION |
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Two genetic loci known to regulate the eumelanin/phaeomelanin switch are agouti and extension, and most domesticated animals have multiple alleles at each of these loci (36). The agouti and extension alleles in concert determine the distribution of phaeomelanin and eumelanin, both along each hair shaft as well as spatially along the coat of the animal. Dominant alleles at extension result in dark brown or black coat color while animals homozygous for recessive alleles have yellow or red coats; the opposite is true for agouti alleles. The identification of the MC1-R as the extension locus in the mouse led to the finding that a point mutation, E92K, in the dominant Eso-3J allele resulted in a receptor that was constitutively active, defined as being able to significantly elevate adenylyl cyclase activity in the absence of ligand (28). A second allele (Eso, L98P) was found in an independent occurrence of the Sombre phenotype and also produces a constitutively active receptor (5). More recently, a second constitutively active MC1-R has been characterized in the red fox, Vulpes vulpes (42). In this animal, a C125R change in the third membrane-spanning domain produces the dark black morph, known as the Alaska silver fox. Additional dominant mutations in the MC1-R have now been reported in cattle (17) and in chickens (38); however, these mutations have not yet been characterized pharmacologically.
Constitutively active G protein-coupled receptors (GPCRs) were first
identified in chimeras of the 1- and
ß2-adrenergic receptors (6). Ultimately, this effect was
mapped to residues at the C-terminal end of the third intracellular
loop, and, in particular, the replacement of an alanine at position 293
with any other residue was found to increase the basal activity of the
receptor and enhance the affinity for ligand as much as 100 fold (16).
After the identification of naturally occurring constitutively active
MC1-Rs (28) and rhodopsin molecules (29), activating mutations in GPCRs
were found to be responsible for a diverse array of inherited as well
as somatic genetic disorders including hyperfunctioning thyroid
adenomas (21, 23, 43), autosomal dominant hyperthyroidism (40, 41, 44),
familial precocious male puberty (18, 37, 45), metaphyseal
chondrodysplasia (35), familial hypoparathyroidism (22, 39), and
congenital night blindness (8, 25).
While constitutively activating mutations have been found in virtually all domains of the GPCRs, some mechanistic similarities are commonly found. Many constitutively active receptors demonstrate a higher affinity for agonist and lower EC50 for further activation (37, 43). In some cases the increased affinity for agonist, but not antagonist, was dramatic (27), and the correlation between agonist efficacy and increased affinity in the constitutively active mutants (31) led to a proposed modification of the ternary complex model for GPCR activation (7). The established model holds that agonist binding stabilizes the active conformation (R*G) of the receptor in a complex with G protein while antagonists typically bind equally well to R and R*. Based on the identification and characterization of constitutively active GPCRs, an extended or allosteric ternary complex model was proposed in which receptor, independent of ligand binding, is in equilibrium between an inactive and active conformation (19). Mutations that constitutively activate receptors are proposed to disrupt internal constraints in the receptors, make the receptors less conformationally constrained (10), and therefore decrease the energy required to reach the R* state. The model thus explains the increased affinity of agonists for constitutively active receptors, even in the absence of G protein, since constitutive activation results in a higher percentage of receptors in the high-affinity R* state.
Two constitutively activating mutations of the mMC1-R, an E92K change
near the extracellular margin of the second transmembrane domain and a
L98P change approximately two turns in the -helix above E92, were
initially predicted to activate the receptor by directly disrupting an
internal constraint resulting from an electrostatic interaction between
E92 and another unidentified residue (28). The L98P was predicted to
alter the constraint involving E92 by disrupting the
-helical
structure of the second transmembrane domain in which E92 was found. A
similar model was proposed for a K296E mutation found in the seventh
membrane-spanning domain of rhodopsin in a family with retinitis
pigmentosa (29). This mutation, which occurs at the site of covalent
attachment of the retinal chromophore, was found to produce an opsin
that was capable of fully activating transducin in the absence of light
or retinal. Twelve different amino acid substitutions at this position
have been examined, and all but the basic lysine or arginine residues
constitutively activate the receptor (4). Furthermore, a glutamic acid
at position 113 is generally accepted as a counterion of the Schiff
base linkage between K296 and retinal (20, 30), and mutagenesis of this
glutamic acid residue to a Gln also constitutively activates rhodopsin
(29).
Remarkably, however, in contradiction to the allosteric ternary complex model, the E92K, L98P, and C125R mutations constitutively activate the MC1-R but dramatically lower agonist affinity and efficacy. We show here that these mutations do not directly disrupt internal constraints favoring the inactive receptor conformation. Rather, these constitutively activating mutations cluster in an acidic domain of the receptor predicted to interact electrostatically with an arginine residue in the agonist that is required for high-affinity binding. We demonstrate that introduction of a basic residue in this region of the receptor is essential for constitutive activation and propose that these mutations activate the receptor by ligand mimicry.
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RESULTS |
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Insertion of a Proline Residue Adjacent to E92 Constitutively
Activates the MC1-R in Cattle (ED)
and in an Independent Occurrence of the Sombre Mouse
(Eso)
Two proline insertion events, L98P in an independent occurrence of
the Sombre phenotype in mice (28), and L99P in black
Icelandic cattle (17) have been found to be linked to dominant MC1-R
alleles. Preliminary data demonstrated that the murine L98P receptor
was constitutively active (5). The L98P receptor has a basal activity
equal to 30% to 50% of the maximal stimulation level of the wild-type
receptor (Fig. 2E and Table 1
). The L98P mutant receptor can be
slightly activated by
-MSH (data not shown) and nearly fully
activated by NDP-
-MSH (Fig. 2E
). Similar to the E92K and E92R
mutants, the L98P mutant demonstrates constitutive activity and an
increase in EC50 for stimulation by NDP-
-MSH from
0.016 ± 0.005 nM to 3.27 ± 0.19 nM.
The IC50 for the L98P mutant receptor (301 ± 55
nM) was 100-fold higher than the wild-type receptor (Fig. 2F
and Table 1
).
While the L99P change in the bovine receptor was not
characterized pharmacologically, it might be expected to behave
similarly to the murine L98P mutation. To test the limits of the domain
in which a proline insertion will constitutively activate the receptor,
an E100P mutation was also constructed and tested pharmacologically.
This mutant was found to be very similar to the wild-type receptor,
with comparable EC50 and IC50 values (Fig. 2, EF, and Table 1
).
Mutation of Conserved Basic Receptor Residues H183, H258,
and K276 Does Not Constitutively Activate the MC1-R
A hypothesis for constitutive activation of rhodopsin is
that an ionic bridge between K296 and E113 constrains the receptor in
an inactive conformation, since removal of the charge at either
position will activate the receptor in the absence of light or retinal
(3, 29). The requirement of a basic residue at position 92 for
constitutive activation, rather than simple ablation of the negative
charge, argues against a similar model for the MC1-R. Nevertheless, to
test for basic residues that might stabilize the receptor via an
electrostatic interaction with E92, we mutagenized all three conserved
basic residues that might be in a position to interact with E92. In
theory, disruption of such a residue should also result in a
constitutively active receptor.
H258 and K276 were mutagenized to acidic residues, as well as to
other amino acids; however, no significant constitutive activation
resulted (Fig. 3). Similar data were seen
for H183 (data not shown). Interestingly, both the H258E and K276
mutations increased maximal hormone-stimulated activity of the
receptor. The EC50 for NDP-
-MSH stimulation was
unchanged for the receptor with a H258E change. Introduction of
isoleucine or tryptophan at this position increased the
EC50 value for NDP-
-MSH stimulation by 5- and 20-fold,
respectively, and made the receptor nearly resistant to
-MSH
stimulation (data not shown). All three mutations at position 258
increased the IC50 for competition binding of
-MSH by
100-fold or more (Fig. 3B
and Table 1
). Quite differently, the H183E
and K276E mutant receptors showed wild-type activation and binding
properties. The two other K276 mutant receptors, K276A and K276L,
showed about a 10-fold increase in EC50 for
-MSH
stimulation but no change in the EC50 for NDP-
-MSH and
no change in the IC50 (Table 1
). Thus, removal of
potentially basic residues at positions 183, 258, and 276 not only does
not activate the receptor, but suggests that residues 183 and 276 have
no major role in ligand binding or activation.
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In parallel with the E92 and C123 positions, elevated basal level of
cAMP-response ß-galactosidase activity equivalent to about 2040%
of the maximal stimulation level of the mMC1-R was only seen after the
insertion of a basic residue at this position (Fig. 5A). Likewise, neither one of the three
mutant receptors could be activated by
-MSH, while all three were
activated by NDP-
-MSH at greatly reduced efficacy (Fig. 5B
and Table 1
). In contrast with changes at E92 and C123, all three D119 variants
demonstrated a 10- to 100-fold increase in IC50 values for
competition of NDP-
-MSH binding in comparison with the wild-type
receptor, implying a contribution of D119 in high-affinity ligand
binding (Fig. 5C
and Table 1
).
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Removal of All Three Acidic Residues, E92, D115, and D119, Still
Yields a Constitutively Active Receptor
Insertion of a basic residue with a possible positive charge
in the second or third membrane-spanning domain could be argued to
create an electrostatic interaction between the two membrane-spanning
domains that stabilizes the active receptor conformation. To test this
hypothesis, we constructed four different receptors with two or three
basic residue substitutions. Even when all three conserved acidic
residues, E92, D115, and D119, are replaced with lysines, the receptor
remains constitutively activated. Thus, once insertion of a single
basic residue occurs, removal of the remaining acid residues does not
inactivate the receptor. The most potent constitutive activation
appeared to occur when both transmembrane domains 2 and 3 were each
replaced with a single basic residue. Additionally, insertion of
multiple basic residues appeared to remove responsiveness even to the
potent ligand NDP--MSH (Fig. 6
, A and
B).
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DISCUSSION |
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Remarkably, two additional residues, C125 and D119, were identified
with properties very similar to E92. C125R is a mutation found as a
dominant allele of the MC1-R in the darkly pigmented Alaska silver fox
(42). When this mutation was introduced into the mMC1-R (C123R), the
receptor was constitutively activated (Fig. 4, A and B). Again, only
changes to lysine or arginine resulted in constitutive activation. A
change from cysteine to alanine resulted in a receptor that was
identical to the wild type, suggesting that the disulfide-bonding
capability of the cysteine does not play a role in ligand binding or
receptor activation. Similar to the effects seen at E92 and C123, only
the introduction of a positive charge at D119 caused constitutive
activation of the receptor (Fig. 5
, A and B).
E92, D115, and D119 are conserved in all five melanocortin receptors
and are the only conserved acidic residues existing in the presumed
exterior part of the transmembrane domains that would thus be in a
position to readily interact as counterions with the essential arginine
residue in the H-F-R-W pharmacophore of -MSH (9). Independent
modeling studies of the MC1-R have suggested this acidic domain as part
of the ligand-binding pocket (11, 24). Introduction of nonbasic
residues at positions 92 and 115 had no effect on the affinity of
ligand binding as assessed in competition binding studies. In contrast,
all three D119 variants constructed demonstrated a 10- to 100-fold
lower affinity for NDP-
-MSH or
-MSH, pointing to this residue as
a likely site of electrostatic interaction for arginine 8 of ligand
(Fig. 5C
and Table 1
).
Constitutively activating mutations in GPCRs have often been found to
have increased affinity for, and increased sensitivity to, agonist (16, 27, 31, 37, 43). Surprisingly, the opposite was the case for activating
mutations at E92, C123, and D119 of the MC1-R. Insertion of basic
residues at these positions uniformly decreased apparent affinity for
-MSH and NDP-
-MSH and greatly decreased the efficacy of these
agonists (Figs. 2
, 4
, and 5
). Scatchard analysis of one mutation, E92K,
demonstrated a 10-fold decrease in affinity of the receptor for
NDP-
-MSH from 0.62 nM to 6.4 nM (Fig. 2D
)
with little change in receptor number. In some cases, agonist efficacy
at the constitutively activated MC1 receptors could only be
demonstrated with the use of the superpotent agonist NDP-
-MSH.
Two proline mutations near the extracellular margin of the second
membrane-spanning domain have also been found associated with dominant
extension locus alleles, L98P in the sombre mouse (5, 28),
and L99P in darkly pigmented cattle (17). The L98P receptor showed the
same pharmacological properties as the E92K, C123R, and D119K mutant
receptors (Figs. 2, 4
, and 5
). These data suggest that the packing of
the second and third transmembrane domains is critical for activation
of the MC1-R.
Given the potential proximity of E92, D115, and D119 in space, it is
possible that significant repulsion results between TM2 and TM3.
Introduction of a basic residue by mutagenesis or by ligand binding
could thus reduce this repulsion between the transmembrane domains as
part of the conformational change involved in forming R*. Both the
E92K/D115K and E92K/C123R double-mutant receptors have basal levels of
cAMP-dependent ß-galactosidase activity that are greater than that of
either mutation alone, suggesting that addition of basic residues
constitutively activates the receptor via independent and additive
mechanisms (Fig. 6). Furthermore, even replacement of all three
conserved acidic residues, E92, D115, and D119, with lysines produces a
constitutively active receptor that has now lost any detectable
responsiveness, even to the superpotent ligand NDP-
-MSH (Fig. 6B
).
These data make it difficult to support a model in which any specific
interaction between the acidic residues in transmembrane domains 2 and
3 is disrupted as part of the mechanism of constitutive activation.
Rather, the mutations may be independently altering the structures of
the TM2 and TM3 helices.
In contrast to mutations in the acidic domain, at which basic residues
are required for constitutive activation, replacement of the aspartate
at D115 with any other residue will cause constitutive activation (Fig. 5, DF). This observation suggests that the D115 may be involved in an
electrostatic interaction or hydrogen bond that may serve to constrain
the receptor in an inactive conformation. It is likely that D115 is not
in a transmembrane milieu, but rather in the first extracellular loop.
As such, the constraining interaction may involve residues in this or
other extracellular loops that have not yet been studied. Additionally,
constitutively activating mutations at this position did not
consistently decrease ligand affinity or efficacy, suggesting a
different mechanism of action from the L98P, E92K, C123R, and D119K
mutations.
According to the ternary allosteric complex model, constitutively
activating mutations of the GPCRs should have a reduction in the energy
barrier that must be overcome in the R to R* transition (19, 31). Early
studies of adrenergic receptors identified several residues in the
third intracellular loop, known to be a region involved in G protein
coupling (6), that enhanced the basal activity while also demonstrating
increasing agonist affinity and efficacy (6, 27, 31). Many subsequent
activating mutations characterized in the TSH receptor (43) and LH
receptor (37) share this property, and it has been suggested that
increased agonist affinity is a hallmark of constitutive activation.
However, this did not take into account the possibility of mutations
that would be more proximal to ligand binding, decreasing the energy
barrier to R* by directly mimicking the conformational effects of
ligand binding, and disrupting a component of high-affinity ligand
binding in the process. Based on the data presented here, we propose
that constitutive activation of the MC1-R by mutations at E92, C123,
L98, and D119 is occurring by ligand mimicry. According to this model,
the arginine residue at position 8 of -MSH, known to be essential
for high-affinity ligand binding, normally binds in the pocket formed
by these residues, interacting electrostatically with D119. Replacement
of E92, C123, or D119 with a basic residue thus mimics the effects of
ligand arginine on receptor conformation.
Furthermore, introduction of a basic residue with possible positive
charge into the arginine-binding pocket did not appear to release a
specific receptor constraint involving molecular interactions between
E92, D119, and D115. Substitution of acidic residues with lysines in
each transmembrane domain 2 and 3 of the MC1-R appeared to be additive,
and even substitution of all three acidic residues produced a
constitutively active receptor, suggesting that the relative degree of
electrostatic attraction or repulsion of transmembrane domains 2 and 3
is not a critical factor in MC1 receptor activation. Consequently, we
propose that one effect of ligand binding, or constitutive activation
by ligand mimicry, may be the direct transmission of a conformational
change along the TM2 and/or TM3 -helical bundles. This could be
envisioned as a release from a vertical constraint imposed on the TM
-helical domains, perhaps by interaction of the charged E92, D119,
and D115 residues with the negatively charged lipid head groups at the
junctions of the membrane with the aqueous intracellular and
extracellular milieu. This information could be transmitted across the
membrane in a fashion that ultimately affects the environment of the
conserved E/DRY sequence at the beginning of second intracellular loop.
Significant evidence exists for rhodopsin (26), as well as the
1-adrenergic receptor (33, 34), that the protonation state of the
glutamic/aspartic acid within the E/DRY sequence is dramatically
affected by local changes in the milieu, and the protonation state of
this residue in turn appears to have a critical impact on G protein
coupling. Mutations in the MC1-R TM2 may indirectly affect protonation
state of the aspartic acid by altering the positioning of TM2 residues,
such as M71 and D82 of the MC1-R, since comparable residues in the
adrenergic receptors (34) and rhodopsin (26) are known to be involved
in the formation of a polar pocket that affects the protonation state
of the TM3 aspartate. Mutations in TM3 could directly alter the
lipophilic environment of the DRY sequence by directly altering the
packing of this
-helix. In conclusion, we provide data here for a
corollary of the ternary allosteric model; constitutively activating
mutations that are ligand mimetic may increase the fraction of
receptors in R* while decreasing agonist affinity and efficacy by
virtue of disruption of the ligand-binding site.
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MATERIALS AND METHODS |
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Receptor Expression
The BamHI-SalI fragments from the
wild-type mMC1-R and different mMC1-R mutants were cloned into the
eukaryotic expression vector pCDNA3 (Invitrogen, San Diego, CA). Human
embryonic kidney 293 cells were transfected with 20 µg DNA of each
construct using the calcium phosphate method (1). Selection began
72 h posttransfection in DMEM containing 10% newborn calf serum
and 1 mg/ml geneticin.
ß-Galactosidase Activity Assay
HEK 293 stable cell populations expressing the wild-type mMC1
receptor or different mutants were transfected with a
pCRE/ß-galactosidase (pCRE/ß-gal) construct using the calcium
phosphate method (1). Four micrograms of pCRE/ß-gal DNA was used for
transfection of a 10 cm dish of cells. At 1524 h posttransfection,
cells were split into 96-well plates with 20,000 to 30,000 cells per
well and incubated at 37 C in a 5% CO2 incubator until
48 h posttransfection. Cells were then stimulated with different
concentrations of -MSH and NDP-
-MSH diluted in stimulation medium
(DMEM containing 0.1 mg/ml BSA and 0.1 mM
isobutylmethylxanthine) for 6 h at 37 C in a 5% CO2
incubator. The cells were also stimulated by 10 µM
forskolin to normalize for transfection efficiency. After stimulation,
cells were lysed in 50 µl lysis buffer (250 mM Tris-HCl,
pH 8.0, 0.1% Triton X-100), frozen and thawed, and then assayed for
ß-galactosidase activity as described (2). ß-Galactosidase activity
was normalized to protein concentration and displayed as a fold of
forskolin (10 µM)-stimulated level to normalize for
efficiency of transfection of the receptor construct. Previous work has
demonstrated that ß-galactosidase activity is proportional to
intracellular cAMP over a wide range of physiological concentrations
(2). Data represent means and SDs from triplicate data
points, and curves were fitted by nonlinear regression using Prism
software (GraphPAD, San Diego, CA).
Ligand Binding
Competition binding experiments were performed on stable cell
populations containing the wild or mutant receptors. Cells were plated
at 5 x 106 cells per well in 24-well plates the day
before the binding experiment was performed. The cells were then
incubated for 45 min at room temperature in binding medium (1 mg/ml BSA
in Ca2+/Mg2+ PBS) containing 30,000-100,000 cpm
of [125I]NDP--MSH per well. Series concentrations of
unlabeled NDP-
-MSH or
-MSH were used to compete with the labeled
NDP-
-MSH. Controls for nonspecific binding contained 1 or 10
µM unlabeled NDP-
-MSH or
-MSH. After 45 min of
incubation, the medium was aspirated, and the cells were washed once
with 1 ml of BSA/PBS (1 mg/ml BSA in Ca2+/Mg2+
PBS) per well. Later, 0.5 ml versine (GIBCO, Grand Island, NY) was used
to transfer cells to test tubes for counting radioactivity. Data
represent means and SDs from duplicate data points, and
curves were fitted by nonlinear regression using Prism software
(GraphPAD).
Saturation binding experiments were performed on stable cell lines
containing the wild-type or E92K mutant receptors. Increased
concentrations of [125I]NDP--MSH (100,000 cpm to
1,000,000 cpm) in binding medium (1 mg/ml BSA in
Ca2+/Mg2+ PBS) were added to cells in the
absence and presence of 10-4 M unlabeled
-MSH. The cells were incubated at room temperature for 45 min. After
the incubation, cells were washed twice with 1 ml of BSA/PBS (1 mg/ml
BSA in Ca2+/Mg2+ PBS) per well. Later, 0.5 ml
of versine was used twice to transfer cells to test tubes for counting
radioactivity. Data represent means and SDs from duplicate
data points, and curves were fitted by linear regression using Prism
software (GraphPAD).
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FOOTNOTES |
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This work was supported by NIH Grant AR-42415 (R.D.C.) and the Norwegian Fur Breeders Association (D.I.V.).
Received for publication September 2, 1997. Revision received November 28, 1997. Accepted for publication January 13, 1998.
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REFERENCES |
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