Common Requirements for Melanocortin-4 Receptor Selectivity
of Structurally Unrelated Melanocortin Agonist and Endogenous
Antagonist, Agouti Protein*
Julia
Oosterom
,
Keith M.
Garner
,
Wijnand K.
den Dekker
,
Wouter A. J.
Nijenhuis
,
Willem Hendrik
Gispen
,
J. Peter
H.
Burbach
,
Greg S.
Barsh§, and
Roger A. H.
Adan
¶
From the
Department of Medical Pharmacology, Rudolf
Magnus Institute for Neurosciences, University Medical Center
Utrecht, P. O. Box 85060, 3508 AB Utrecht, The Netherlands and the
§ Howard Hughes Medical Institute, Beckman Center B271A,
Stanford University School of Medicine, Stanford, California
94305-5323
Received for publication, August 10, 2000
 |
ABSTRACT |
The activity of melanocortin receptors (MCR) is
regulated by melanocortin peptide agonists and by the endogenous
antagonists, Agouti protein and AgRP (Agouti-related protein). To
understand how the selectivity for these structurally unrelated
agonists and antagonist is achieved, chimeric and mutants MC3R and MC4R were expressed in cell lines and pharmacologically analyzed. A region
containing the third extracellular loop, EC3, of MC4R was essential for
selective Agouti protein antagonism. In addition, this part of MC4R,
when introduced in MC3R, conferred Agouti protein antagonism. Further
mutational analysis of this region of MC4R demonstrated that
Tyr268 was required for the selective interaction
with Agouti protein, because a profound loss of the ability of Agouti
protein to inhibit 125I-labeled
[Nle4,D-Phe7]
-melanocyte-stimulating
hormone (MSH) binding was observed by the single mutation of
Tyr268 to Ile. This same residue conferred selectivity for
the MC4R selective agonist,
[D-Tyr4]MT-II, whereas it inhibited
interaction with the MC3R-selective agonist,
[Nle4]Lys-
2-MSH. Conversely, mutation of
Ile265 in MC3 (the corresponding residue of
Tyr268) to Tyr displayed a gain of affinity for
[D-Tyr4]MT-II, but not for Agouti protein,
and a loss of affinity for [Nle4]Lys-
2-MSH
as compared with wild-type MC3R. This single amino acid mutation thus
confers the selectivity of MC3R toward a pharmacological profile like
that observed for MC4R agonists but not for the antagonist, Agouti
protein. Thus, selectivity for structurally unrelated ligands with
opposite activities is achieved in a similar manner for MC4R but not
for MC3R.
 |
INTRODUCTION |
Melanocortin (MC)1
receptors are activated by the POMC
(pro-opiomelanocortin)-derived ACTH (adrenocorticotropic hormone) and MSH (melanocyte-stimulating hormone) peptides. MC3R and MC4R are the
main MC receptors in the brain, and MC4R is thought to play a prominent
role in the regulation of body weight in both rodents and human (1-3).
The identification of the endogenous MC receptor antagonists, Agouti
protein and Agouti related-protein (AgRP), gave rise to an additional
level of regulation of MC receptors, in which ligands with opposite
activities control MCR activity (4, 5). It is unknown, however, whether
the molecular interaction with the receptor of these oppositely acting
ligands is achieved in a similar manner.
Mouse Agouti protein is a 131-amino acid protein normally expressed in
hair follicles, which acts on MC1R-expressing melanocytes to regulate
pigmentation (6, 7). In mice that ectopically overexpress Agouti
protein, chronic MC1R blockade by Agouti protein results in a yellow
coat color. In addition, these mice display severe obesity,
hyperphagia, and increased plasma levels of the adipose derived satiety
factor, leptin (8, 9). In these mice, obesity is thought to be the
result of continuous blockade of the hypothalamic MC4R, since
recombinant murine Agouti protein has been demonstrated to act as a
high affinity antagonist for both mouse MC1R and MC4R in
vitro but not for MC3R and MC5R (10-12). This
determination is in agreement with the finding that
MC4R
/
mice recapitulate the obesity phenotype as
observed in yellow obese mice (2).
In wild-type mice, Agouti protein is not expressed in the brain. But
its homologue, AgRP, is expressed in the hypothalami of mice, rats,
primates, and humans (13-16). Recombinant human AgRP acts as a high
affinity antagonist for the MC3R and MC4R, and to a lesser extent for
MC5R, but not for MC1R and MC2R (17). Transgenic mice that overexpress
AgRP display an obesity phenotype similar to that found in
MC4R
/
mice (4). Thus, pharmacological, histochemical,
and genetic studies suggest that hypothalamic AgRP is an important
endogenous stimulator of feeding and exerts this function by inhibiting
MCR signaling.
Even though an important role of MC4R in body weight homeostasis is
evident, the role of MC3R in the control of body weight and other
processes is ill-defined because of the absence of MC3R selective
ligands. Knowledge of the interaction between MCR and its ligands at
the level of molecular detail would contribute to the design of
selective MC3R and MC4R ligands. This is important not only for
the understanding of MCR subtype-specific functions, but in addition,
ligands that selectively activate or block MC4R may be therapeutically
useful in the treatment of obesity (18) and anorexia (19, 20).
To investigate whether MCR agonists and the structurally unrelated
antagonist, Agouti protein, are regulated in a similar manner, the aim
of this study was to identify which part of the human MC4R is important
for selective Agouti protein interaction and antagonism. To this end,
chimeric and mutant MC3R and MC4R were expressed in cell lines and were
tested for their interaction with full-length human Agouti
protein, [Nle4]Lys-
2-MSH, and
[D-Tyr4]MT-II. The results indicate that a
single residue in MC4R is required for the selective interaction with
Agouti protein. In addition, the results show that selectivity for
structurally unrelated agonists and antagonist is achieved in a similar manner.
 |
MATERIALS AND METHODS |
Peptides--
NDP-
-MSH
([Nle4,D-Phe7]-
-MSH) and
-MSH were purchased from Bachem Feinchemicalien, Bubendorf,
Switzerland. [D-Tyr4]MT-II and
[Nle4]Lys-
2-MSH were synthesized using
solid phase Fmoc chemistry and purified as described previously (21).
Full-length human Agouti protein and AgRP were prepared as described
previously (17, 22).
Radioiodination of NDP-
-MSH--
Iodination was performed
exactly as described previously (23). In short, 4 µg of NDP-
-MSH
was mixed with 1.2 IU bovine lactoperoxidase (Calbiochem) and 1 mCi of
Na125I (ICN) in a final volume of 100 µl of 0.05 M phosphate buffer (pH 6.5). Then, 5 µl of 0.003%
H2O2 was added every 60 s. After 4 min, 50 µl of 1 mM dithiothreitol was added to stop the reaction. The sample was purified by high pressure liquid chromatography with a
µBondapak C18 column, 3.9 × 300 mm (Waters, Div. of
Millipore) by elution with a 22-52% acetonitrile gradient in 10 mM ammonium acetate (pH 5.5) in 40 min. The specific
activity of 125I-NDP-
-MSH was 2.25 × 106 Ci/mol.
Expression of Chimeric and Mutant Receptors--
All chimeric
and MCR mutants used in this study were generated by polymerase chain
reaction, and the sequences were verified as described previously (23).
The chimeric receptors were named after the extracellular receptor
domain that was replaced. With "A" being the N-terminal domain
(corresponding to residues 1-60 of MC4R), "B" the first
extracellular loop (EC1, residues 67-142), "C" the second
extracellular loop (EC2, residues 149-195), and "D" the third
extracellular loop (EC3, residues 202-332). These receptors include
chimera 3AB (MC4R with N-terminal through TM3 of MC3R), 3B (MC4R with
IC1 through TM3 of MC3), 3C (MC4R with IC2 through EC2 of MC3),
3D (MC4R with TM5 through C-terminal of MC3R), 4D (the reverse of
chimera 3D; MC3R with TM5 through C-terminal of EC3 of MC4R),
"Loop" (MC4R with EC3 of MC3R), "1st half" (MC4R with
first half of EC3 of MC3R), "2nd half" (MC4R with second half of
EC3 of MC3R), and mutant MC4 (Tyr268
Ile). In
these constructs, EC3 of MC4R runs from residue Phe267
through Ala282, the first half of EC3 runs from
Phe267 through Gln273, and the second half of
EC3 runs from Val278 through Ala282 (Fig.
1). For the radioligand and adenylate
cyclase assays, wild-type rat MC3R (24), human MC4R (25), and chimerae
3AB, 3B, 3C, 3D, and 4D were stably expressed in B16G4F mouse melanoma
cells. These cells were grown in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% fetal calf serum. In addition, human MC4R, loop,
1st half, 2nd half, and MC4 (Tyr268
Ile) were expressed
in 293 HEK cells and used in the radioligand binding assay. 293 HEK
cells were grown in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) supplemented with 10% fetal calf serum. An MC3R
mutant was generated in which Ile265 of MC3R was mutated
into Tyr (MC3(Ile265
Tyr)). This mutant was made using
the QuikChangeTM Site-Directed Mutagenesis Kit
(Stratagene). The rat MC3R cloned in pcDNA3.1 was used as template.
The sequence of the mutagenic oligonucleotides was
5'-TTCCTCCACCTGGTCCTGTACATCACCTGCCCCACCAACC-3' and its
complementary oligonucleotide. For the pharmacological analysis, the
rat MC3R (wild type) and MC3(Ile265
Tyr) were expressed
in BHK (baby hamster kidney) and 293 HEK cells with similar results.
These cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum.

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Fig. 1.
Model of the human MC4R. The gray
circles indicate a stretch of residues with complete homology
between MC3R and MC4R that were used as boundaries for construction of
the chimeric MC3/MC4 receptors. The larger circles indicates
residues belonging to the third extracellular loop (EC3, residues
267-282). The black circles with double-residue
symbols (for example, Y/I at position 268) indicate
that the first residue (Y) is present in MC4R and the second
residue (I) is present in MC3R at the corresponding
position. The white circles in EC3 are homologous in MC4R
and MC3R. The numbering corresponds to the human MC4R amino
acid sequence. The N- and C-terminal residues are not shown.
|
|
Adenylate Cyclase Assay--
B16G4F cells, stably expressing
human MC4R, rat MC3R, and chimerae 3AB, 3B, 3C, 3D, or 4D were grown in
24-well plates (Corning Costar). Agonist-stimulated adenylate cyclase
activity was measured as described previously (26). In short, after
prelabeling with 500 µl of [3H]adenine (PerkinElmer
Life Sciences) in a concentration of 2 µCi/ml, the cells were
incubated for 30 min at 37 °C in Dulbecco's modified Eagle's
medium containing 0.1 mM isobutylmethylxanthine,
-MSH in
a concentration ranging from 0.3 nM to 3 µM,
with and without 40 nM Agouti protein. The cells were
harvested, and [3H]cAMP formation was calculated as the
percentage of [3H]ATP converted into
[3H]cAMP. For each curve, 12 duplicate data points were
collected. EC50 values were determined by fitting the data
to a sigmoidal curve with variable slope using GraphPad Prism 2.01 for
Windows 95/NT (GraphPad Software Inc., San Diego, CA). Experiments were performed two times with the same results.
Receptor Binding Assay--
MC receptor expressing B16G4F, 293 HEK cells, or BHK cells were grown in 24-well plates (Corning Costar).
The cells were incubated with 100,000 cpm (
0.1 nM) of
125I-NDP-
-MSH and various concentrations of
nonradioactive peptides diluted in binding buffer consisting of Ham's
F-10 medium (Life Technologies, Inc.) (pH 7.4), 2.5 mM
calcium chloride, 0.25% bovine serum albumin, 10 mM Hepes,
and 50 µg/ml aprotinin (Sigma). After a 30-min incubation at room
temperature, the cells were washed twice with ice-cold Tris-buffered
saline containing 2.5 mM calcium chloride and lysed in 1 M sodium hydroxide. Radioactivity of the lysates was
counted in a Packard Cobra
-counter. Competition curves were fitted
from 12 duplicate data points with GraphPad Prism 2.01 for Windows
95/NT, nonlinear regression, one-site competition. IC50
values were calculated with 99% confidence interval. Experiments were
repeated at least two times with the same results.
 |
RESULTS |
The affinities (IC50 values, using
125I-NDP-
-MSH as radioligand) of full-length human
Agouti protein, AgRP, and NDP-
-MSH were determined for the wild-type
MC4R and MC3R expressed in B16G4F cells. NDP-
-MSH and AgRP possessed
comparable IC50 values for both MC3R and MC4R (Fig.
2). In contrast, Agouti protein displayed an almost 40-fold lower affinity for MC3R than AgRP and NDP-
-MSH, whereas Agouti protein displayed high affinity for MC4R. The
IC50 of Agouti protein was 10-fold higher for MC3R than for
MC4R.2

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Fig. 2.
Affinity of
NDP- -MSH, Agouti protein, and AgRP for the
human MC4R and rat MC3R expressed in B16G4F cells. The
graphs show competition of 125I-NDP- -MSH by NDP- -MSH
( ), Agouti protein ( ) and AgRP ( ). Data points represent the
mean of duplicate measurements ± S.D. Binding curves were fitted
with GraphPad Prism, one-site competition. The IC50 values
for MC3R and MC4R were, respectively: NDP- -MSH, 2.4 and 11 nM; Agouti protein, 101 and 12 nM; and AgRP,
2.7 and 9.7 nM.
|
|
Next, the antagonistic properties of Agouti protein were analyzed.
Agouti protein at a concentration of 40 nM was a potent antagonist for MC4R (Fig. 3) and was able
to increase the EC50 of
-MSH almost 20-fold (Table
I). In contrast, the same concentration of Agouti protein was not able to significantly alter the
EC50 of
-MSH for MC3R.

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Fig. 3.
Agouti protein antagonism for MC4R, MC3R, and
chimeric receptors. -MSH-stimulated adenylate cyclase activity
in the absence ( ) and presence ( ) of 40 nM Agouti
protein at MC4R, MC3R, and chimerae 3AB, 3B, 3C, 3D, and 4D expressed
in B16G4F cells. Data points represent the mean of duplicate
measurements ± S.D. Curves were fitted with GraphPad Prism,
sigmoidal dose-response curve fitting, variable slope.
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Table I
EC50 value (in nM) of -MSH in the absence and
presence of 40 nM human Agouti protein for MC3R, MC4R, and
chimerae 3AB, 3B, 3C, 3D, and 4D expressed in B16G4F cells
EC50 values were calculated using GraphPad Prism, sigmoidal
dose-response curve fitting, variable slope.
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|
In addition, it was investigated as to which region of MC4R is required
for the antagonistic properties of Agouti protein. Therefore, the
ability of Agouti protein to antagonize
-MSH-stimulated adenylate
cyclase activity of chimeric MC3/MC4 receptors was analyzed. Agouti
protein was able to effectively antagonize
-MSH-stimulated adenylate
cyclase activity for all chimeric receptors except chimera 3D, which
only has the C-terminal portion of MC3R starting at TM5 (Fig. 3 and
Table I). Furthermore, the reverse chimera, 4D, which is an MC3R with
only the C-terminal portion (starting at TM5) of MC4R, was able to
confer Agouti protein antagonism to MC3R. Agouti protein was unable to
significantly increase the EC50 of
-MSH for chimera 3D
as observed for MC3R.
To assess which region of MC4R determined the high affinity of Agouti
protein for MC4R, the IC50 values of Agouti protein for
chimeric MC3/MC4 receptors was analyzed. Table
II summarizes the IC50 values
of Agouti protein for all chimeric receptors. Chimerae 3AB, 3B, and 3C
all displayed a high affinity for Agouti, which was not significantly
different from the IC50 for MC4R. However, chimera 3D (MC4R
with the C-terminal portion of MC3R, starting at TM5), displayed a
significant reduction in the affinity for Agouti protein. The
affinities of Agouti protein for chimera 3D and MC3R were comparable
(Fig. 4). Conversely, chimera 4D (the reverse of 3D, MC3R with the C-terminal portion of MC4R) displayed high
affinity for Agouti protein, which was not different from wild-type MC4R.
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Table II
Inhibition constants (IC50 in nM) of human Agouti
protein for MC3R, MC4R, and chimerae 3AB, 3B, 3C, 3D, and 4D
All receptors were stably expressed in B16G4F cells. IC50
values were determined from competition binding curves using
125I-NDP- -MSH as radioligand, derived from 12 duplicate data
points using GraphPad Prism, one site competition.
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Fig. 4.
Affinity of Agouti protein for MC4R, MC3R,
and chimerae 3D and 4D. The graph shows competition of
125I-NDP- -MSH by Agouti protein on MC4R ( ), MC3R
( ), and chimerae 3D ( ) and 4D ( ) expressed in B16G4F cells.
Data points represent the mean of duplicate measurements ± S.D.
Binding curves were fitted with GraphPad Prism, one-site
competition.
|
|
Next, the role of EC3 of MC4R in interaction with Agouti protein was
further specified. To this end, wild-type MC4R, mutant MC4(Tyr268
Ile), and chimeric MC4R with small parts of
EC3 of MC3R were expressed in 293 HEK cells. The chimera were: loop
(MC4R with EC3 from MC3R), 1st half (MC4R with first
half of EC3 from MC3R), and 2nd half
(MC4R with second half of EC3 from MC3R). Fig.
5 and Table
III show that the affinities of
NDP-
-MSH for the MC4R, chimeric, and mutant receptors were
comparable. However, when EC3 of MC4R was replaced by MC3R sequence
(loop), the IC50 for Agouti protein increased
more than 4-fold as compared with MC4R. This loss of affinity was also
observed when only the first half of EC3 was replaced (1st
half). Moreover, when only Tyr268 in the first half of
EC3 of MC4R was mutated to Ile, the corresponding residue of MC3R, a
more than 10-fold drop in Agouti protein affinity was still observed.
In contrast, replacement of the second half of EC3 (2nd half
chimera) did not alter the affinity for Agouti protein as compared with
wild-type MC4R. Next, MC3(Ile265
Tyr), the reverse
mutant of MC4 (Tyr268
Ile), was analyzed by determining
the IC50 values of NDP-
-MSH, Agouti
protein,
[Nle4]Lys-
2-MSH, and
[D-Tyr4]MT-II affinity (Fig.
6 and Table
IV). The latter two peptides were shown
previously to display increased and decreased affinity, respectively,
for MC4 (Tyr268
Ile) (21). The data show that the
IC50 of NDP-
-MSH and Agouti protein for MC3R and
MC3(Ile265
Tyr) did not significantly differ. However,
the affinity of [Nle4]Lys-
2-MSH was ~40
times lower for the mutant as compared with MC3R. In contrast, the
affinity of [D-Tyr4]MT-II was significantly
higher for MC3(Ile265
Tyr).

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Fig. 5.
Affinity of NDP- -MSH
and Agouti protein for the human MC4R, loop, 1st
half, 2nd half, and MC4 (Tyr268 Ile)
expressed in 293 HEK cells. The graphs show the competition of
125I-NDP- -MSH by NDP- -MSH ( ) and Agouti protein
( ). The data points represent the mean of duplicate
measurements ± S.D. Curves were fitted with GraphPad Prism,
one-site competition.
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Table III
Inhibition constants (IC50 in nM) of human Agouti
protein for the MC4R, chimerae loop, 1st half, 2nd half, and mutant MC4
(Tyr268 Ile) expressed in 293 HEK cells
For each ligand, IC50 values were determined from competition
binding curves derived from 12 duplicate data points. Curves were
fitted with GraphPad Prism, one-site competition using
125I-NDP- -MSH as radioligand (see Fig. 3).
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Fig. 6.
Affinity of NDP- -MSH
and Agouti protein for the MC3R and MC3 (Ile265 Tyr) expressed in BHK cells. The graphs show the competition
of 125I-NDP- -MSH by NDP- -MSH ( ), Agouti protein
( ), [D-Tyr4]MT-II ( ), and
[Nle4]Lys- 2-MSH ( ). The data points
represent the mean of duplicate measurements ± S.D. Curves were
fitted with GraphPad Prism, one-site competition.
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Table IV
Inhibition constants (IC50 in nM) of human Agouti
protein for the MC3R and MC3(Ile265 Tyr) expressed in BHK
cells
For each ligand, IC50 values were determined from competition
binding curves derived from 12 duplicate data points. Curves were
fitted with GraphPad Prism, one-site competition using
125I-NDP- -MSH as radioligand (see Fig. 4).
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 |
DISCUSSION |
The melanocortin system is unique in the sense that MCR activity
is regulated by two structurally unrelated endogenous peptides with
opposing activities: on the one hand, POMC-derived
melanocortin agonists, and on the other hand, Agouti protein and AgRP,
which act as antagonists for MCR. Currently, selective MCR ligands are necessary to obtain a better understanding of the role of brain MC3R
and MC4R in processes like the regulation of body weight. Because
Agouti protein is a high affinity antagonist for the MC4R, this study
was aimed at characterizing the selective interaction of human Agouti
protein with the human MC4R. The pharmacological analysis of chimeric
and mutant MC3R and MC4R suggested that a single amino acid residue in
the first half of the third extracellular loop of MC4R conferred
selectivity for Agouti protein.
The results confirm that human Agouti protein is a high affinity
antagonist for MC4R but not for MC3R. In contrast, AgRP does not
discriminate between MC3R and MC4R. The selective interaction of Agouti
protein with MC4R was studied using chimeric and mutant MC3R and MC4R.
Apparently, the lower affinity of Agouti protein for chimera 3D (MC4R
with EC3 and surrounding domains of MC3R) and MC3R wild type accounts
for the inability of Agouti protein to antagonize
-MSH action on
these receptors. Chimera 4D was tested for a gain of function for
Agouti binding and antagonism to test whether this region (EC3 and
surrounding domains of MC4R) alone is sufficient to obtain an
MC4R-specific pharmacological profile. Indeed, this EC3-containing
region of MC4R fully conferred Agouti protein antagonism to MC3R. Thus,
this region changes the MC3R pharmacological profile to that of MC4R.
A more detailed analysis of the role of EC3 in Agouti protein binding
showed that the first half of EC3, in particular position Tyr268 of MC4R, was critical for high affinity Agouti
protein binding because, when it was replaced by Ile, a loss of
affinity for Agouti protein occurred. Previously, it was shown that
Tyr268 was also required for the selective interaction of
the agonist [D-Tyr4]MT-II with MC4R, whereas
it hindered interaction with the MC3R selective agonist
[Nle4]Lys-
2-MSH (21). Thus,
Tyr268 is required for selective agonist and antagonist
binding of the MC4R. This is a striking observation because there
exists no obvious amino acid homology between melanocortins and Agouti
protein (Fig. 7).

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Fig. 7.
Amino acid sequences of
-MSH,
[Nle4]Lys- 2-MSH,
[D-Tyr4]MT-II, and
Agouti-(116-123)*. The amino acid
residues present in a loop defined by disulfide-bridged
Cys116 and Cys123 were suggested to be involved
in the interaction with MCR. Presumably, Phe118 is crucial
for selective interaction with MC4R (27).
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|
To test whether Ile265 of MC3R hindered interaction with
Agouti protein, Ile265 of MC3R was mutated into Tyr. MC3
(Ile265
Tyr) did not show a gain of affinity for Agouti
protein, indicating that Tyr268, in the context of the MC3R
conformation, does not confer selectivity for antagonists.
Surprisingly, MC3 (Ile265
Tyr) displayed increased
affinity for [D-Tyr4]MT-II and decreased
affinity for [Nle4]Lys-
2-MSH. Thus, this
residue is able to change the pharmacological profile of MC3R toward
that of MC4R for agonists but not for antagonists. These data imply
that it may not be feasible to design MC3R-selective antagonists based
upon MC3R selective agonists (such as
-MSH) and MC4R-selective
antagonists. This approach, however, may be applicable for MC4R because
the structural requirements for selective agonists and antagonists
interaction with MC4R is more alike.
In conclusion, through pharmacological analysis of chimeric and mutant
MC3R and MC4R, it was demonstrated that Tyr268 in EC3 of
MC4R is critical for selective Agouti protein interaction. This study
and previous data show that the mutation of MC4R Tyr268 to
Ile decreases affinity for MC4R-selective ligands but increases affinity for the MC3R-selective ligands. Moreover, Tyr268
in MC4R determined both agonist and antagonist selectivity. This is the
first report describing details of the molecular recognition of the
endogenous MCR antagonist, Agouti protein, by MC4R. This report
demonstrates that the selectivity of structurally unrelated peptide
ligands with opposite activities (melanocortin peptide agonist
versus Agouti protein antagonist) is achieved in a similar manner, strongly suggesting that they use the same binding pocket. An understanding of the molecular basis governing selective
ligand interaction with MC receptors contributes to the rational design of new selective ligands.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of Medical
Pharmacology, Rudolf Magnus Institute for Neurosciences, University Medical Center Utrecht, P. O. Box 85060, 3508 AB Utrecht, The Netherlands. Tel.: 31(0)30-2538817; Fax: 31(0)30-2539032; E-mail: r.a.h. adan@med.uu.nl.
Published, JBC Papers in Press, October 6, 2000, DOI 10.1074/jbc.M007261200
2
In this study, the terms "binding" and
"affinity" always refer to the ability of a ligand to compete for
125I-NDP-
-MSH binding.
 |
ABBREVIATIONS |
The abbreviations used are:
MC, melanocortin;
MCR, MC receptor;
MSH, melanocyte-stimulating hormone;
125I-NDP-
-MSH, 125I-labeled
[Nle4,D-Phe7]
-MSH;
AgRP, Agouti-related protein;
EC, extracellular loop;
BHK cells, baby hamster
kidney cells;
MT-II, cyclic
[Nle4,Asp5,D-Phe7,
Lys10]
-MSH-(4-10).
 |
REFERENCES |
1.
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