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INTRODUCTION |
Studies of the mouse coat color Agouti gene have led to
the identification of a novel pair of secreted signaling molecules which regulate mammalian pigmentation and body weight.
Agouti encodes a 131-amino acid secreted protein that is
expressed in the skin, where it induces the production of yellow
pigment (pheomelanin) in hair follicle melanocytes (Refs. 1 and 2, and
reviewed in Refs. 3 and 4). Agouti protein is secreted but has a small
sphere of action; its localized expression is thought to give rise to
characteristic white or yellow markings found in many different mammals
such as chinchillas and the Doberman breed of domestic dogs (Ref. 5,
and reviewed in Ref. 6). In the dominant mutations
Ay and Avy or in
transgenic mice, ectopic expression of transcripts encoding Agouti
protein results in yellow hair, obesity, hyperinsulinemia, and
increased body length (Refs. 7-9, and reviewed in Refs. 4 and 10). The
nonpigmentary effects of ectopic Agouti expression likely
reflect the normal function of Agouti-related protein
(Agrp),1 a protein expressed
in the hypothalamus and adrenal gland that is similar to Agouti protein
in size, sequence, and biochemical activity (11, 12). Ubiquitous
expression of Agrp transcripts causes obesity and increased
body length but does not alter pigmentation (11, 13).
Agouti protein and Agrp act by antagonism of melanocortin receptors, a
family of G-protein-coupled receptors responsive to endocrine peptides
such as
-melanocyte stimulating hormone (
-MSH) and
adrenocorticotrophic hormone (ACTH) (reviewed in Refs. 14 and 15).
Genetic and biochemical studies indicate that Agouti protein alters
pigmentation by antagonism of the melanocortin 1 receptor (Mc1r)
expressed on melanocytes (16, 17), whereas Agrp affects body weight and
length by antagonism of the Mc4r and/or Mc3r expressed in the
hypothalamus and other regions of the central nervous system (11,
18-21). Most evidence suggests Agouti protein and Agrp act as
competitive antagonists of melanocortin receptors (17, 22, 23), meaning
that their effects are due solely to their ability to inhibit binding
of melanocortin receptor agonists such as
-MSH. In agreement with
this model, Agouti protein and
-MSH inhibit the binding of each
other to the Mc1r (17, 24, 25). Additional findings, however, have
suggested that Agouti protein and possibly Agrp inhibit melanocortin
receptor signaling by mechanisms besides simple competitive antagonism of
-MSH binding (26-33). Using a sensitive bioassay based on
-MSH-induced pigment dispersion in Xenopus melanophores,
we have previously shown that inhibition of melanocortin signaling by
Agouti protein is increased significantly by preincubating melanophores
in Agouti protein for several hours prior to the addition of
-MSH
(25). This observation suggested that Agouti protein induces
melanocortin receptor down-regulation in addition to its ability to
inhibit
-MSH binding.
Our previous studies utilized a full-length
(His23-Cys131) recombinant form of Agouti
protein generated in a baculovirus expression system (11). Here we
demonstrate that the related cysteine-rich carboxyl-terminal domains of
Agouti protein and Agrp are sufficient for competitive antagonism in
Xenopus melanophores. However, the amino-terminal residues
of Agouti protein are required for additional effects likely due to
down-regulation of melanocortin receptor signaling. To investigate
whether Agouti protein was proteolytically cleaved to smaller forms
in vivo, extracts of skin were examined by Western blot
analysis using antisera that detected epitopes in both the amino and
carboxyl termini. We found little, if any, post-translational
proteolysis of full-length (His23-Cys131)
Agouti protein. These findings demonstrate that Agouti protein alters
melanocortin signaling by two mechanisms mediated by distinct domains
within the native protein.
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EXPERIMENTAL PROCEDURES |
Recombinant Proteins--
Production and characterization of
recombinant mouse Agouti protein and Agrp using the baculovirus system
has been previously described (11, 25). In brief, cation followed by
anion exchange chromatography resolves mouse Agouti protein to >99%
purity as determined by analysis of silver-stained PAGE, mass
spectroscopy, and amino-terminal sequencing. Blue-Sepharose (Amersham
Pharmacia Biotech) followed by anion exchange chromatography resolves
multiple forms of Agrp into two pools. One pool contains form A, a
single species processed by signal peptidase cleavage after residue 20, in an approximately equimolar ratio with form B, a mixture of three
species processed by cleavage after residues 46, 48, or 50, present in
a 15:1:4 ratio, respectively. The second pool contains form C, a
mixture of two species processed by cleavage after residues 69 or 71, present in a 1:4 ratio, respectively. Relative purity of Agrp
preparations was estimated by scanning densitometry of a 10-µg sample
loaded on a 10% Tricine gel stained with ProBlue (Integrated
Separation Systems, MA) and was used to calculate 60% purity for forms
A+B and 20% purity for form C.
Generation of Anti-mouse Agouti Antibodies--
Polyclonal
antibodies to full-length (His23-Cys131) mouse
Agouti were generated in rabbits by standard procedures (37); 100-200 µg of protein at approximately 90% purity was used for each of five
injections; antisera were recovered at day 70.
Following SDS-PAGE carried out under nonreducing conditions and Western
blotting, the antiserum detects 1 ng/lane of the immunogen against
which it was raised, as well as 1 ng/lane carboxyl-terminal (Ser73-Cys131) Agouti protein. Disulfide bond
reduction of the antigen prior to SDS-PAGE slightly reduces sensitivity
of the antiserum for detection of the full-length protein (<2-fold)
but reduces sensitivity for detection of the carboxyl-terminal fragment
by more than 100-fold. These findings suggest that the antiserum
contains multiple antibodies: one or more directed at an epitope in the
amino terminus and one or more that recognize an epitope in the
cysteine-rich carboxyl terminus that requires proper disulfide bonding.
Extraction of Agouti Protein from Mouse Skin and Western
Blotting--
Sections of dorsal and ventral skin from 3-day old
at/at mice were
homogenized in ice-cold 1% Nonidet P-40 buffer (25 mM
HEPES, pH 7.4, 50 mM NaCl, 10 mM EDTA) or high
salt buffer (25 mM HEPES, pH 7.4, 750 mM NaCl,
10 mM EDTA) plus protease inhibitors (0.5 µg/ml
leupeptin, 1 mM benzamidine) using a ground glass
homogenizer. Following homogenization, samples were centrifuged in a
microfuge for 25 min at 4 °C. Samples in 1% Nonidet P-40 buffer and
a portion of the high salt extracts were added to an equal volume of
2× SDS loading buffer, boiled 5-10 min, and kept on ice. To
concentrate and partially purify Agouti protein by cation exchange, 1 ml of each high salt buffer extract was added to 9 ml of 25 mM HEPES, pH 7.4, to bring the final [NaCl] to 75 mM. The diluted samples were rocked with 50 µl of a
cation exchange resin (SP-Sepharose FF, Amersham Pharmacia Biotech) for
30 min at 4 °C. The resin was washed in batch mode with 25 mM HEPES, pH 7.4, 50 mM NaCl, 10 mM
EDTA, 0.5 µg/ml leupeptin, 1 mM benzamidine and eluted
with 25 mM HEPES, pH 7.4, 750 mM NaCl, 10 mM EDTA. Eluates were added to an equal volume of 2× SDS
sample buffer, boiled, and loaded on an 18% polyacrylamide gel.
Transfer and incubation with primary antisera at 1:1000 dilution were
carried out according to standard methods (37). Secondary
immunodetection was accomplished with a goat-anti-rabbit IgG conjugated
to horseradish peroxidase (Bio-Rad, Hercules, CA) and the ECL detection
kit (Amersham Pharmacia Biotech).
Production and Purification of Carboxyl-terminal Agouti Protein
(Ser73-Cys131)--
5 mg of recombinant mouse
Agouti protein was dialyzed into 50 mM HEPES, pH 7.1, 50 mM NaCl, 5 mM CaCl2, and incubated
at 30 °C for 6 h with 5000 units/mg Kex-2 protease (38, 39), a
generous gift from Dr. Michael Kay, Stanford University Department of
Biochemistry. To resolve the products of cleavage, the sample was
loaded at 800 µl/min onto a 1-ml HiTrap SP cation exchange column,
washed with 5 ml of 50 mM Bicine, pH 9.0, 100 mM NaCl and then eluted with a 20-ml NaCl gradient
(100-600 mM) in 50 mM Bicine, pH 9.0. Fractions (500 µl) were tested in the melanophore assay for
-MSH-inhibitory activity at a 1:200 dilution, and two peaks of
activity were found to correspond to residual uncleaved full-length
protein and the Ser73-Cys131 fragment at >99%
purity. Active fractions were pooled, dialyzed into 20 mM
PIPES, pH 6.8, 50 mM NaCl, flash frozen, and stored at
70 °C. The data shown utilized a preparation of carboxyl-terminal Agouti protein that was made by cleaving a full-length
hemagglutinin-tagged Agouti protein (25) (the hemagglutinin tag was
inserted in the amino terminus of the Agouti protein and is therefore
not present in the Ser73-Cys131 fragment).
Identical results were observed using a preparation of
carboxyl-terminal Agouti protein made from recombinant Agouti protein
His23-Cys131. To test the effects of the amino
terminus in trans, a mixture of
His23-Arg70 or Arg72 and
Ser73-Cys131 was made by digesting recombinant
mouse Agouti protein (99% purity) with Kex-2 as above. SDS-PAGE, mass
spectroscopy, and amino-terminal sequencing revealed the presence of
equimolar amounts of His23-Arg70 or
Arg72 and Ser73-Cys131, along with
a minor amount (<2% of total protein) of uncut full-length Agouti protein.
Xenopus Melanophore Culture and Pigment Dispersion
Assay--
Xenopus melanophores were grown at 27 °C in 50% L-15
medium (Specialty Media, Lavallete, NJ), supplemented with 20%
heat-inactivated fetal calf serum, 1 mM
L-glutamine, penicillin, and streptomycin; the medium had
been previously conditioned using Xenopus fibroblasts as
described by Potenza and Lerner (48). The pigment dispersion assay
developed by Potenza and Lerner (48) is based on the ability of agents
that cause a decrease or increase in intracellular cAMP levels to
produce a dose-dependent aggregation or dispersion, respectively, of intracellular pigment granules. Because pigment granules are neither fully aggregated nor dispersed in the absence of
any drug, pretreatment of the cells with melatonin to aggregate pigment
granules increases the range and sensitivity of the assay for detecting
agents such as
-MSH that disperse pigment granules. For a typical
assay, cells were plated 24-48 h beforehand in 96-well plates at
25,000 cells/well, washed briefly with 250 µl/well assay buffer (70%
L-15 medium; 0.1% bovine serum albumin), and 40 µl/well assay buffer
was then added, followed by 40 µl/well assay buffer that contained 2 nM melatonin (Sigma) to provide a final melatonin concentration of 1 nM. After a 45-min incubation to
aggregate pigment granules, the optical density of each well was
measured at 650 nm (ODinitial) to provide a base-line
optical density reading. Test samples (Agouti protein, Agrp, or control
buffer) were then added at 40 µl/well, followed by the addition of
various concentrations of
-MSH or NDP-MSH at 40 µl/well. All
additions were made in assay buffer supplemented with 1 nM
melatonin to maintain a constant concentration of melatonin during the
assay. Optical density at 650 nm was then determined at multiple time
points from 30 to 420 min (ODfinal). Unless stated
otherwise, the entire assay was carried out at 22 °C in triplicate,
and assay points represent the mean ± S.E. of the mean. A
unitless parameter, degree of pigment dispersion, was calculated as
described by Potenza and Lerner (48), (ODfinal
ODinitial)/ODfinal, which creates an internal standard for each well (ODinitial) and scales the maximal
degree of pigment dispersion to 1. The effects of 1 mM
melatonin occasionally increase during the course of the assay, which
gives rise to negative values for the degree of pigment dispersion.
Optical density at 650 nM melanophores was measured with a
Vmax kinetic microplate reader (Molecular
Devices, Menlo Park, CA) in end point mode, and data were transferred
electronically to a Microsoft Excel spreadsheet for analysis. Graphing
and curve fitting of dose-response curves were carried out with
DeltaGraph (DeltaPoint, Monterey, CA), using a four-parameter logistic
equation, y = a + ((b
a)/(1 + (10c/10x)d));
where a = minimum, b = maximum,
c = half-maximal value, and d = slope.
Schild Analysis of Dose-response Curves (40)--
Dose ratios
(the amount of
-MSH required for half-maximal response in the
presence of Agouti protein/amount of
-MSH required for half-maximal
response in the absence of Agouti protein) were determined using the
four-parameter logistic equation described above. Within each
experiment, individual slopes of the
-MSH dose-response curves were
not significantly different and therefore were fixed at the average
slope to provide the data for dose-ratio calculations. Schild plots
[log(dose ratio
1)] versus
log([Ser73-Cys131Agouti or Agrp]) were
created and analyzed in DeltaGraph (DeltaPoint, Monterey, CA). Linear
curve fitting was used to determine the slope and the correlation
coefficient. To estimate KB, a second linear
curve fitting was performed with the slope fixed at 1.
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RESULTS |
Agouti Protein Is Present in Mouse Skin in the Full-length
Form--
The similar genomic structure of Agouti and
Agrp suggests evolution from a common ancestral gene, yet
the sequence similarity of the two proteins is confined entirely to the
carboxyl-terminal region (Fig. 1).
Twenty-one of the final forty-seven residues in Agouti protein and Agrp
are identical, of which ten residues are cysteines in a spacing similar
to that found in conotoxins and plectoxins from the venoms of cone
snails (34) and spiders (35), respectively. In addition, the amino
terminus of the Agouti protein contains several paired basic residues
that could serve as potential proteolytic cleavage sites (Fig.
1A). These observations suggested that the Agouti protein
might be processed in vivo into an active fragment spanning
the cysteine-rich region. In support of this hypothesis, proteolytic
fragments containing the carboxyl terminus of either protein retain
-MSH-inhibitory activity (11, 23), and a short deletion
(Arg64-Lys77) in the amino-terminal half of the
mouse Agouti protein does not disrupt activity (36).

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Fig. 1.
Alignment of mouse Agouti protein with mouse
(M) and human (H) Agrp and production
of carboxyl-terminal
Ser73-Cys131 mouse Agouti
protein. A, Agouti and Agrp have similar genomic
structures, but sequence similarity is confined to the cysteine-rich
carboxyl-terminal region; in the same region, plectoxin XI (35) shows a
nearly identical pattern of cysteine spacing. Arrowheads
mark signal sequence cleavage sites, and arrows designate
peptide fragments used in this study. Agrp forms B and C are
heterogeneous and likely to result from proteolysis during secretion
and/or purification from conditioned media of baculovirus-infected
cells as described under "Experimental Procedures." B,
treatment of full-length Agouti protein with Kex-2 protease
(Kex-2 digest) yields a mixture of amino-terminal and
carboxyl-terminal fragments that comigrate at 7.5 kDa plus a small
amount of residual uncleaved full-length protein. The conditions used
for Kex-2 digestion are described under "Experimental Procedures";
full-length Agouti protein subjected to the same conditions in the
absence of protease addition (mock-digest) shows no change
in electrophoretic mobility.
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Recombinant mouse Agouti protein produced in insect cells is secreted
as a mature form with the signal sequence removed
(His23-Cys131) (17, 23, 25). However,
recombinant proteins secreted by insect cells may undergo altered or
incomplete post-translational processing (41, 42). To investigate
whether the active form of Agouti protein in mouse skin underwent
proteolytic cleavage in vivo, we raised polyclonal
antibodies against full-length recombinant mouse Agouti protein
(His23-Cys131). Western blotting experiments
determined that the antiserum detects epitopes in both the amino- and
carboxyl-terminal regions of Agouti protein with a sensitivity of <1
ng/lane provided that SDS-PAGE was carried out under nonreducing
conditions (see "Experimental Procedures").
We used the anti-Agouti antiserum on nonreducing Western blots of
dorsal and ventral skin extracts from 3-day-old black and tan
(at/at) mice. These animals express
Agouti transcripts in ventral, but not in dorsal skin (43),
providing a control for antiserum specificity. As shown in Fig.
2, Agouti protein is detected in ventral,
but not dorsal, at/at skin extracted
with 1% Nonidet P-40 or a buffer containing 750 mM NaCl.
In addition, we concentrated and partially purified Agouti protein from
the 750 mM NaCl extracts using a cation exchange resin
known to bind either full-length Agouti protein or the
Ser73-Cys131 carboxyl-terminal fragment (25).
Agouti protein extracted from mouse skin has a mobility of 21.5 kDa on
a nonreducing gel, similar to that of the full-length
(His23-Cys131) recombinant form. The intensity
of the 21.5-kDa band and the lack of any smaller fragments demonstrates
that the full-length form predominates in mouse skin and indicates that
Agouti protein undergoes little, if any, proteolytic processing
in vivo after cleavage of the signal sequence.

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Fig. 2.
Detection of Agouti protein in skin extracts
of black and tan
(at/at) mice
by Western blotting. As described under "Experimental
Procedures," dorsal and ventral skin samples from
at/at mice were extracted in 1%
Nonidet P-40 buffer or 750 mM NaCl buffer; a portion of the
750 mM NaCl extracts were concentrated and partially
purified by cation exchange. Samples were separated by SDS-PAGE under
nonreducing conditions and transferred to a nylon membrane. Agouti
protein was detected using a polyclonal antisera and a chemiluminescent
detection system as described under "Experimental Procedures."
Exposure time, extraction buffer, and skin source (dorsal
(D) or ventral (V)) are indicated above
each lane. A 21.5-kDa band is detected in ventral, but not dorsal,
skin extracts. Pre-immune serum taken from the rabbit prior to antibody
production does not detect this band (lower panel).
Arrows represent the sizes of full-length and
carboxyl-terminal (Ser73-Cys131) Agouti protein
run on the same gel.
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Production and Analysis of an Active Carboxyl-terminal Agouti
Protein Fragment--
To investigate the potential function of the
amino terminus of the Agouti protein, we used the Xenopus
melanophore pigment dispersion assay to compare the activity of
full-length Agouti protein and a carboxyl-terminal Agouti protein
fragment. We produced a carboxyl-terminal fragment,
Ser73-Cys131, by treating recombinant Agouti
protein with Kex-2, a protease that cleaves specifically after paired
Lys-Arg residues (39). Two such sites are present at residues 69 and 70 and at 71 and 72 (Fig. 1A). Treatment of Agouti protein with
Kex-2 produces a 48- or 50-residue amino-terminal fragment,
His23-Arg70 or Arg72, and a
59-residue carboxyl-terminal fragment,
Ser73-Cys131, that comigrate at 7.5 kDa on
SDS-PAGE carried out under reducing conditions (Fig. 1B).
Cation exchange chromatography resolved the carboxyl-terminal fragment
at >99% purity (determined by amino-terminal sequencing, SDS-PAGE,
and mass spectrometry) from both the amino-terminal fragment and a
small amount of uncleaved full-length protein.
Like full-length Agouti protein, the carboxyl-terminal fragment
inhibits
-MSH-induced pigment dispersion in Xenopus
melanophores but has no effect in the absence of
-MSH or other
melanocortin peptides (data not shown). By analyzing the effect of the
two Agouti peptides on melanocortin receptor signaling over time, however, we discovered a difference in their activities. Addition of
-MSH to melanophores causes a rapid increase in pigment dispersion that reaches equilibrium in 15-30 min. When full-length Agouti protein
is added simultaneously or immediately prior to
-MSH, a gradual
inhibition of pigment dispersion ensues that increases for several
hours (Fig. 3A). By contrast,
under the same conditions, the carboxyl-terminal fragment of Agouti
protein maximally inhibits the effects of
-MSH within 15-30 min,
after which pigment dispersion gradually increases (Fig.
3A). As described previously (25) and as shown in Fig.
3B, these effects can be studied in more detail by an
experimental protocol where melanophores are preincubated with Agouti
protein alone for varying periods of time (during which there is no
effect on pigment dispersion) and then exposed to
-MSH for a
relatively brief and uniform period. Under these conditions,
-MSH
dose-response curves display a progressive rightward shift as a
function of time preincubated with full-length Agouti protein (Fig.
3B). By contrast, the same protocol carried out with
Ser73-Cys131 yields
-MSH dose-response
curves that shift slightly to the left as a function of preincubation
time (Fig. 3B). Thus, while the effectiveness of full-length
Agouti protein increases during preincubation, the effectiveness of the
carboxyl-terminal fragment decreases. A quantitative estimate of
relative activity can be derived by comparing the
-MSH
concentrations required for half-maximal stimulation of pigment
dispersion under different sets of experimental conditions. Following a
10-min preincubation in 15 nM full-length Agouti protein,
for example, 15 nM
-MSH is required for half-maximal stimulation of pigment dispersion. After a 420-min preincubation in 15 nM full-length Agouti protein, the amount of
-MSH
required for half-maximal stimulation is increased 2-fold to 31 nM. A complete analysis for both Agouti peptides at various
preincubation intervals is shown in Fig. 3C and reveals that
a 420-min preincubation increases the effectiveness of full-length
Agouti protein 2-fold while decreasing the effectiveness of the
carboxyl-terminal fragment 1.5-fold.

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Fig. 3.
Kinetics of -MSH
inhibition by full-length and carboxyl-terminal Agouti protein.
A, the indicated concentrations of full-length or
Ser73-Cys131 Agouti protein were added to
melanophores immediately prior to addition of 0.5, 2.0, or 8 nM -MSH or a buffer control, and pigment dispersion was
measured at 5-min intervals for 4 h. For clarity, only the results
obtained with 2 nM -MSH are shown, and error
bars have been removed; the standard error of all assay points was
<5%. Treatment with full-length or carboxyl-terminal Agouti protein
in the absence of -MSH had no effect on pigment dispersion.
B, full-length or Ser73-Cys131
(C-term) Agouti protein at a concentration of 15 nM or buffer control was added to melanophores for 10, 210, or 420 min, then -MSH at the indicated concentrations was added to
all assay wells simultaneously. Pigment dispersion was measured at
45-300 min; for clarity only the 50-min time point is shown. Also,
different times of preincubation with buffer control had no effect on
pigment dispersion, and for clarity, only the 210-min time point is
shown. The -MSH dose-response curves for full-length Agouti protein
exhibit a progressive rightward shift and depression of maximal
signaling as the time of preincubation with full-length Agouti protein
increases; the same effect is not evident for carboxyl-terminal Agouti
protein. C and D, relative activity of
carboxyl-terminal or full-length Agouti protein was estimated by first
calculating the dose ratio for individual -MSH dose-response curves,
defined as the concentration of -MSH required for half-maximal
pigment dispersion in the presence of Agouti protein, divided by the
concentration of -MSH required for half-maximal pigment dispersion
in the absence of Agouti protein. The dose ratios for carboxyl-terminal
and full-length Agouti protein after 210 or 420 min of preincubation
were then compared with the corresponding dose ratios after 10 min of
preincubation. Thus, relative activity corresponds to the degree to
which individual -MSH dose-response curves are shifted; the plot
depicted in panel C corresponds to the data from panel
B. In panel D, relative activity data are shown for
carboxyl-terminal or full-length Agouti protein solutions that were
first preincubated, then transferred to, and tested on, new
melanophores during a 50-min incubation with various concentrations of
-MSH. This transfer data therefore reflects the specific
activity of Agouti protein solutions over time, whereas the
preincubation data reflects a combination of changes in
specific activity and melanophore response.
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The effect of preincubation on full-length Agouti protein and the
carboxyl-terminal fragment could result from a change in the specific
activity of the peptides in solution over time or from a change in the
response of melanophores to
-MSH following prolonged exposure to
Agouti peptides. To distinguish between these possibilities, we asked
how preincubation of full-length or carboxyl-terminal Agouti protein
solutions on melanophores affected the activity of these solutions when
they were transferred to and tested on a new plate of melanophores. As
above, melanophores in a 96-well plate were preincubated in full-length
or carboxyl-terminal Agouti protein for 10, 210, or 420 min. Prior to
adding
-MSH, however, the solution in each well was transferred to a
new plate of melanophores. Addition of
-MSH to this "transfer"
plate revealed that full-length and carboxyl-terminal Agouti peptides
in solution exhibit a parallel decline in their relative activity (Fig.
3D). In summary, preincubation of melanophores in
full-length Agouti protein reduces their responsiveness to
-MSH
despite the fact that relative activity of the Agouti protein solution
declines. This paradoxical result is not observed with the
carboxyl-terminal peptide and implicates amino-terminal residues of the
protein in the preincubation effect.
Full-length Agouti Protein and the Carboxyl-terminal
Ser73-Cys131 Fragment Have Different Effects on
the Slope and Maximum of the
-MSH Dose-response Curve in
Melanophores--
In previous studies (25), we have shown that the
effects of full-length Agouti protein on melanophores differ from those of a competitive antagonist because, as above, its effectiveness increases during preincubation and because
-MSH dose-response curves
carried out at different Agouti protein concentrations do not exhibit
the same slope. Another characteristic of competitive antagonism is the
ability of increasing concentrations of agonist to overcome the effects
of antagonist regardless of its concentration, i.e. high
concentrations of antagonist should have no effect on maximal
-MSH
signaling (40, 44). In the conditions used for our previous
experiments, full-length Agouti protein did not affect the maximal
level of pigment dispersion induced by
-MSH; however, we
subsequently found that such an effect can be revealed by carrying out
the melanophore assay at higher temperatures.
Fig. 4, A and B,
depicts
-MSH dose-response curves carried out with different
concentrations of full-length Agouti protein at 16 and 27 °C,
respectively, where Agouti protein is added immediately prior to (<10
min)
-MSH. The slopes of individual dose-response curves vary at
both temperatures, but an effect of Agouti protein on the maximal level
of
-MSH-induced pigment dispersion is apparent only at 27 °C. A
similar experiment with carboxyl-terminal Agouti protein at 27 °C
shows no effect on the slope or maximal
-MSH signaling (Fig.
5A).

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Fig. 4.
Effect of temperature and different forms of
Agouti protein and Agrp on depression of -MSH
maximal signaling. A and B, assays were
carried out in parallel at 16 or 27 °C with the indicated
concentrations of full-length Agouti protein added immediately prior to
various concentrations of -MSH, and pigment dispersion was measured
from 45 min to 6 h later. For clarity, only results from the
85-min time point are shown, which reveal depression of -MSH maximal
signaling that depends on Agouti protein concentration at 27 °C but
not 16 °C. B, at 85 min, a greater effect on depression
of -MSH maximal signaling is observed for 10 nM
full-length Agouti protein than for 1 or 100 nM; as
described under "Results," this effect varies according to the time
of incubation and is eventually lost after 6 h. C-F,
assays were carried out in parallel at 22 °C for full-length Agouti
protein, Ser73-Cys131 (C-term)
Agouti protein, Agrp forms A + B, or Agrp form C, all at a
concentration of 15 nM; each solution was preincubated for
20, 85, or 330 min, then various concentrations of -MSH were added,
and pigment dispersion was measured after 50 min. A buffer control was
also included for each preincubation time point, but there was no
effect on pigment dispersion so only the 85-min time point is shown for
clarity.
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Fig. 5.
Schild analysis of
Ser73-Cys131 Agouti
protein (A), Agrp forms A + B (B),
and Agrp form C (C). In all cases, various
concentrations of -MSH or NDP-MSH were added immediately after the
indicated concentrations of antagonist. Data are shown for only a
single incubation time as indicated; the effects of varying incubation
time are reported in Table I. Note that Schild analysis only yields
meaningful results when the shapes of individual agonist dose-response
curves at different antagonist concentrations are completely parallel,
which applies for the three antagonists shown here but not for
full-length Agouti protein.
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Depression of maximal
-MSH signaling is also observed when
melanophores are preincubated with 15 nM full-length Agouti
protein (Fig. 4C, also Fig. 3B), and the degree
of depression is proportional to the time of preincubation. By
contrast, a parallel experiment carried out with 15 nM
carboxyl-terminal Agouti protein shows no effect on maximal
-MSH
signaling (Fig. 4D). The difference between full-length and
carboxyl-terminal Agouti proteins is not because of independent
activity of the amino terminus because a mixture of carboxyl-terminal
and amino-terminal Agouti protein (after Kex-2 cleavage) behaves
identically to the purified carboxyl-terminal protein (data not shown).
Several hours after the addition of
-MSH, the effect of full-length
Agouti protein on depression of maximal signaling is lost (data not
shown). This raises the possibility that depression of maximal
signaling might be because of failure to reach equilibrium between
Agouti protein and
-MSH binding, i.e. a competitive
antagonist with a very slow disassociation rate would fail to come to
equilibrium with agonist binding and might effectively block the
receptor even in the presence of high agonist concentrations. However, this is unlikely to explain our results because depression of maximal
signaling is lost most rapidly at higher Agouti protein concentrations.
For example, in the "snapshot" of pigment dispersion at 85 min
after
-MSH addition (Fig. 4B), depression of maximal
-MSH signaling is greater for 10 nM Agouti than for 100 nM Agouti; however, pigment dispersion measured at earlier
times revealed a greater depression of maximal
-MSH signaling for
100 nM Agouti protein than for 10 nM Agouti
protein (not shown). These observations indicate that the decrease in
maximal
-MSH signaling induced by full-length Agouti protein is not
because of a failure to reach equilibrium between agonist and
antagonist binding. Instead, these effects are likely explained by an
effect of full-length Agouti protein on receptor down-regulation that
is saturable and reversible (see "Discussion").
The Ser73-Cys131 Agouti Protein Fragment
Acts as a Competitive Antagonist of Melanocortin Receptors in Xenopus
Melanophores--
As described above, the characteristics of
full-length Agouti protein differ in several ways from those predicted
for a competitive antagonist. However, the carboxyl-terminal fragment
of Agouti protein meets all expectations for competitive antagonism. As depicted in Fig. 5A, increasing concentrations of
carboxyl-terminal Agouti protein cause a progressive rightward shift in
the
-MSH dose-response curve without altering the slopes of
individual curves (e.g. compare with Fig. 4, A or
B). An additional prediction for a competitive antagonist is
that displacement of the agonist dose-response curve should be
proportional to changes in antagonist concentration (40). These
criteria are commonly analyzed by a Schild regression plot, log(dose
ratio
1) versus log[antagonist], which should
yield a straight line with a slope near unity for competitive
antagonists, and allows an estimation of KB, the
antagonist dissociation constant. A Schild regression for the data
depicted in Fig. 5A is linear (r2 = 0.998) with a slope = 1.3 and an estimated
KB of 8.6 nM.
Effects of Agrp on Melanophores--
In parallel with our studies
of full-length and carboxyl-terminal Agouti protein, we examined
different forms of recombinant Agouti-related protein (Agrp). Agrp can
be partially purified into two pools (11) (Fig. 1A). The
first pool contains full-length Agrp with the signal sequence removed
(Fig. 1A, form A) along with three fragments
cleaved after residues 46, 48, and 50 (referred to collectively as
"form B"). The second pool contains carboxyl-terminal fragments cleaved after residues 69 and 71 (referred to collectively as
"form C"). We first investigated the kinetics of Agrp
using a preincubation protocol as described above for Agouti protein. As shown in Fig. 4, E and F, preincubation does
not increase the apparent effectiveness of 15 nM Agrp forms
A + B or form C. Maximal
-MSH-induced pigment dispersion is also
unchanged by treatment with Agrp. Thus, the different pools of Agrp,
even the full-length protein "form A," exhibit pharmacologic
characteristics consistent with competitive antagonism, similar to
those displayed by carboxyl-terminal Agouti protein.
In previous studies with Agrp forms A + B, we found its effects on
melanophores were consistent with competitive antagonism of
melanocortin receptors, with Schild plots providing an estimated KB of 6.9 nM for Agrp forms A + B. Fig. 5, B and C, depict similar experiments
carried out with Agrp forms A + B, Agrp form C, and the potent
melanocortin analog [Nle4,
D-Phe7]
-MSH (NDP-MSH). At equilibrium, a
competitive antagonist should yield similar Schild plots for agonists
with similar binding sites, such as
-MSH and NDP-MSH. To assure
analysis at equilibrium, data was obtained at several time points from
1-4 h after the addition of Agrp and
-MSH or NDP-MSH. Increased
incubation time has little or no effect on the Schild plot and
estimated KB (Table I). The slope of the Schild plot remains
slightly greater than unity in experiments carried out with NDP-MSH,
possibly because this melanocortin analog dissociates very slowly from
the receptor and may prevent complete equilibrium between agonist and
antagonist binding. Regardless, the effects of all forms of Agrp
conform to predictions for a competitive antagonist yielding estimated dissociation constants of 10.8 ± 1.5 nM for Agrp
forms A + B and 1.1 ± .09 nM for Agrp form C (Table
I).
 |
DISCUSSION |
The molecular mechanism by which Agouti protein and Agrp inhibit
melanocortin signaling has been controversial. Many studies support
competitive antagonism (17, 22, 23) in which all of the effects of
Agouti protein and Agrp result from inhibition of agonist binding,
whereas other findings suggest more complicated interactions of Agouti
protein/Agrp and melanocortin receptors (25-28, 32, 33, 45, 46). Our
studies in Xenopus melanophores indicate that Agouti protein
has effects that appear to be a combination of two mechanisms:
competitive antagonism of melanocortin receptors and down-regulation of
melanocortin signaling. Removal of amino-terminal residues from Agouti
protein, however, yields a peptide
(Ser73-Cys131) that acts solely as a
competitive antagonist of melanocortin receptors, without the time- and
temperature-dependent down-regulation of melanocortin
signaling observed with full-length Agouti protein. Agrp also acts as a
competitive antagonist of melanocortin receptors in the melanophore
assay. Conservation between Agrp and Agouti protein is confined to the
cysteine-rich regions in their carboxyl termini (11, 12), providing
further evidence that this region is sufficient for competitive
antagonism of melanocortin receptors, whereas residues unique to the
amino terminus of Agouti protein are required for down-regulation of
melanocortin receptor signaling in melanophores.
Schild Analysis and the Xenopus Melanophore Assay--
Although
there is no sequence similarity between Agouti protein and the
13-residue peptide
-MSH, each inhibits the binding of the other to
melanocortin receptors (17, 25). To help distinguish between
competitive antagonism (interaction with identical or neighboring sites
such that binding of one ligand physically blocks binding of the other)
and allosteric antagonism (interaction with nonoverlapping sites that
induces reciprocal conformational changes in the receptor), we used
Schild analysis to examine agonist dose-response curves carried out at
different antagonist concentrations (40, 44). For a competitive
antagonist, these curves should remain completely parallel and, in
addition, should exhibit a progressive rightward displacement
proportional to the change in antagonist concentration. Both criteria
were fulfilled for the Ser73-Cys131 fragment of
Agouti protein and for all forms of Agrp tested, which suggests that
the carboxyl-terminal fragments of these proteins contains a domain or
subdomains that physically block
-MSH binding. A more detailed
understanding of these interactions will probably require
three-dimensional structural studies, but given the differences in size
and probable tertiary structure between Agouti protein and
-MSH,
individual subdomains within the cysteine-rich regions of Agouti
protein and Agrp may mediate receptor binding separately from
-MSH
competition. In this regard, Wilkison and colleagues (47) have recently
demonstrated that substitution of alanine for Arg116 and
Phe118 causes large decreases in the ability of Agouti
protein to inhibit
-MSH binding, and it will be interesting to
determine the effect of these substitutions on binding of Agouti
protein to melanocortin receptors.
Our results are based on the endogenous melanocortin receptor of
Xenopus melanophores, which exhibits agonist and antagonist profiles in between those of the mammalian Mc1r and Mc4r (48, 49), but
whose sequence is not known. Compared with most assays for
Gs-coupled receptors in mammalian cells, a principle
advantage of the melanophore assay is the ability to examine multiple
time points to assure that our analyses are performed at equilibrium. In addition, because hundreds of individual assays can be carried out
simultaneously, the shapes of individual dose-response curves can be
determined and compared with a precision typically not approached by
most mammalian assay systems.
A Unique Role for the Amino Terminus of Agouti Protein--
Unlike
the Ser73-Cys131 fragment, full-length Agouti
protein exhibits several pharmacologic characteristics that are
inconsistent with competitive antagonism, including a time- and
temperature-dependent potentiation of
-MSH inhibition,
agonist dose-response curves that exhibit different slopes depending on
antagonist concentration, and depression of maximal
-MSH signaling.
Although depression of maximal
-MSH signaling might be explained by
a very slow antagonist disassociation rate in some systems, it is
unlikely to account for our results because it was only observed at
temperatures 22 °C or above, and is eventually lost at a rate
proportional to Agouti protein concentration. Taken together, these
observations suggest that full-length Agouti protein has a direct
effect on melanocortin receptor down-regulation distinct from of its
ability to block
-MSH binding. By analogy to mammalian systems,
down-regulation could be mediated by receptor internalization or
post-translational modifications such as phosphorylation or
palmitoylation that alter the ability of the receptor to couple to
second messenger systems (reviewed in Ref. 50). Both types of mechanism
are likely to be temperature-sensitive and occur over a time course
longer than that required for antagonist binding. In this regard,
Siegrist et al. (27) have reported that treatment of B16
melanoma cells with Agouti protein causes a loss of
-MSH binding
sites on the cell surface. Regardless of the exact mechanism, the
differences we observe between full-length and carboxyl-terminal Agouti
protein demonstrate a unique role for a domain or domains in the amino terminus of Agouti protein. Furthermore, arguments based on
evolutionary conservation, genomic structure, and secondary structure
predictions suggest that the amino terminus and carboxyl terminus of
Agouti protein constitute distinct protein modules. However, the amino terminus of Agouti protein cannot act in trans,
i.e. full-length Agouti protein digested with Kex-2 protease
behaves identically to the purified carboxyl-terminal fragment (data
not shown); therefore effects of the amino terminus are likely to
require high affinity binding to melanocortin receptors mediated by a
domain or domains in the carboxyl-terminal region.
Results from other groups also support a role for the amino terminus of
Agouti protein. Kiefer et al. (52) have shown that deletion
of Lys57-Arg85 impairs the ability of Agouti
protein to inhibit
-MSH binding and adenylate cyclase activation (a
5-15-fold increase in apparent Ki). Site-directed
mutagenesis has also revealed amino acids outside of the cysteine-rich
region which are important for Agouti protein activity, including
Val83, Arg85, Pro86,
Pro89, and the glycosylation site Asn39 (36,
52). None of these residues are conserved in Agrp, which suggests that
they are not crucial for antagonist binding and instead may play a role
in protein folding and/or stability or, as described here, a signaling
role distinct from inhibition of
-MSH binding.
In pharmacologic studies apparently at odds with those described here,
Willard et al. (23) reported that a
Val83-Cys131 carboxyl-terminal Agouti protein
fragment was identical to full-length Agouti protein in its ability to
inhibit melanocortin binding and adenylate cyclase activation in B16
melanoma cells. In addition, dose-response analysis of full-length
Agouti protein in melanoma cells was consistent with competitive
antagonism (22). It is possible that the noncompetitive actions of
full-length Agouti protein we have described are unique to
melanophores; alternatively, the increased accuracy and sensitivity of
the melanophore assay may reveal aspects of Agouti protein function
that are difficult to detect in some mammalian cell culture systems.
In vivo, the pigmentary effects of Agouti protein differ
from those caused by deficiency for the Mc1r (25), which points to a
mechanism or mechanisms of action other than simple competitive
antagonism. Regardless, our results demonstrate that Agouti protein
normally produced in vivo contains the amino-terminal
portion largely intact and highlights a potential role for these
residues in its biologic function.
Agouti Protein and Agrp Similarities and Differences--
The
pharmacologic behavior of Ser73-Cys131 Agouti
protein is very similar to that displayed by a carboxyl-terminal
fragment of Agrp (form C); both ligands act as simple competitive
antagonists. Although Agrp form C is approximately 10-fold more potent
than Ser73-Cys131 Agouti protein in the
melanophore assay, estimated antagonist dissociation constants for both
ligands are in the nanomolar range, which lends further support to the
notion that the domain or domains required for melanocortin receptor
binding are contained entirely within the cysteine-rich
carboxyl-terminal fragments of Agouti protein or Agrp and implies that
these domains have been conserved since divergence from a common
ancestor. Orthologs for Agouti protein and Agrp have been found in all
mammals examined (12, 33, 53), but there is, as yet, no molecular
evidence for endogenous melanocortin receptor antagonists in
nonmammalian vertebrates. Primary sequence similarity at the level of
cysteine spacing between Agouti protein and Agrp is also shared by
several of the plectoxins (Fig. 1A) and, to a lesser extent,
-conotoxins, which suggests the possibility of a more distantly
related common ancestor. Calcium channels are the direct targets for
most of the
-conotoxins (reviewed in Ref. 34), but the same is
unlikely to be true for Agouti protein because it binds the Mc1r and
its pigmentary effects are completely blocked by deficiency for the
Mc1r (25). Nonetheless, an ancestral pattern of protein folding common
to the endogenous melanocortin receptor antagonists and invertebrate
toxins may serve as a scaffold for interaction with a diverse group of
cell surface proteins, and binding of the Mc1r by Agouti protein may lead indirectly to changes in calcium metabolism that have been reported in certain cell culture systems.
In melanophores, longer forms of Agrp (A + B) are approximately 10-fold
less potent than form C but, unlike full-length Agouti protein, still
display pharmacologic behavior expected for a competitive antagonist.
This difference is unlikely to be because of impurities in the
preparations because full-length Agouti protein is >99% pure. Effects
of the Agrp amino terminus on
-MSH signaling analogous to those
displayed by Agouti protein might be apparent in other assay systems
and are consistent with our earlier studies (25) in which Agrp forms
A+B were found to depress basal levels of intracellular cAMP and
-MSH maximal signaling in Mc4r-expressing cells.
Concluding Remarks--
The Mc1r and Mc4r play important roles in
the regulation of pigmentation and body weight, respectively (16, 19),
but signaling through these receptors seems to be controlled primarily
by alterations in Agouti protein or Agrp rather than melanocortin
peptides. For example, in altered metabolic states induced by fasting
or leptin deficiency, changes in the levels of transcripts encoding
-MSH (Pomc) are relatively modest, 1.5- to 2-fold (54, 55) compared with 10- to 12-fold alterations in Agrp (11, 12). Similarly, altered
expression of Agouti protein rather than Pomc is the primary determinant of the balance between pheomelanin and eumelanin synthesis in pigment cells (56, 57). In part, regulation of melanocortin receptor
signaling by Agouti or Agrp offers more precise temporal and spatial
control than would be possible by altering levels of circulating
melanocortin peptides. In addition, the ability of Agouti protein to
signal via two separate mechanisms may allow a wider phenotypic range
than would be possible by varying levels of
-MSH. As described here,
these mechanisms require separate regions of the Agouti protein: the
similar cysteine-rich regions of Agouti protein and Agrp are sufficient
for competitive antagonism, while amino-terminal residues in Agouti
protein are necessary for the time- and
temperature-dependent down-regulation of melanocortin receptor signaling we have observed. We anticipate that studies examining receptor trafficking and post-translational receptor modifications will provide additional insight into the underlying biochemical and cell biologic mechanisms and may lead to a deeper understanding of Agouti protein and Agrp signaling in several physiologic processes.