Conformation of the Core Sequence in Melanocortin Peptides
Directs Selectivity for the Melanocortin MC3 and MC4 Receptors*
Julia
Oosterom,
Wouter A. J.
Nijenhuis,
Wim M. M.
Schaaper
,
Jerry
Slootstra
,
Rob H.
Meloen
,
Willem Hendrik H.
Gispen,
J. Peter H.
Burbach, and
Roger A. H.
Adan§
From the Rudolf Magnus Institute for Neurosciences, Department of
Medical Pharmacology, Utrecht University, P.O. Box 80040, 3508 TA
Utrecht and the
Institute for Animal Science and Health,
P.O. Box 65, 8200 AB Lelystad, The Netherlands
 |
ABSTRACT |
Melanocortin peptides regulate a variety of
physiological processes. Five melanocortin receptors (MC-R) have been
cloned and the MC3R and MC4R are the main brain MC receptors. The aim
of this study was to identify structural requirements in both ligand and receptor that determine
-melanocyte-stimulating hormone (MSH) selectivity for the MC3R versus the MC4R. Substitution of
Asp10 in [Nle4]Lys-
2-MSH for
Gly10 from [Nle4]
-MSH, increased both
activity and affinity for the MC4R while the MC3R remained unaffected.
Analysis of chimeric MC3R/MC4Rs and mutant MC4Rs showed that
Tyr268 of the MC4R mainly determined the low affinity for
[Nle4]Lys-
2-MSH. The data demonstrate that
Asp10 determines selectivity for the MC3R, however, not
through direct side chain interactions, but probably by influencing how
the melanocortin core sequence is presented to the receptor-binding
pocket. This is supported by mutagenesis of Tyr268 to Ile
in the MC4R which increased affinity and activity for [Nle4]Lys-
2-MSH, but decreased affinity
for two peptides with constrained cyclic structure of the melanocortin
core sequence, MT-II and [D-Tyr4]MT-II, that
also displayed lower affinity for the MC3R. This study provides a
general concept for peptide receptor selectivity, in which the major
determinant for a selective receptor interaction is the conformational
presentation of the core sequence in related peptides to the
receptor-binding pocket.
 |
INTRODUCTION |
The melanocortins, adrenocorticotropic hormone,
-,
-, and
-melanocyte-stimulating hormone
(MSH),1 are derived from the
precursor protein pro-opiomelanocortin. Pro-opiomelanocortin is
expressed in the pituitary gland, in two brain nuclei (1), and in
several peripheral tissues (2). Effects of melanocortins have been
described on behavior (3), metabolism (4), fever, inflammation (5),
analgesia (6), addiction (7), nerve regeneration (8), and the
cardiovascular system (9, 10). Since virtually all
[125I]NDP-
-MSH-binding sites overlap with expression
of either MC3R and/or MC4R mRNA, these are the main MC receptors in
the brain mediating the variety of effects of melanocortin peptides
(11-13). The melanocortin (MC) receptor subtypes, MC1R, MC2R, MC3R,
MC4R, and MC5R (14-20) constitute a subfamily within G-protein-coupled receptor superfamily. MC receptors differ in ligand binding specificity as well as in tissue distribution. Insight into these two aspects is
essential to understand the physiological functions of the pro-opiomelanocortin/MC receptor system. Therefore, it is essential to
identify ligands that can discriminate between the MC3R and the MC4R.
The development of selective ligands for the MC receptors has been
hampered by the absence of detailed knowledge about the structural
requirements of peptide ligands for selective MC receptor binding and
activation. Nevertheless, it has been demonstrated that HFRW (MSH
(6-9)) forms the core sequence of melanocortins, which is necessary to
bind to all MC receptors (21-23). Ligand selectivity may therefore be
determined by residues outside the core region either through a
selective interaction with different receptor subtypes, by altering
folding of the core sequence, or by a combination of both.
Although the MC3R and MC4R both recognize
-MSH, the affinity of
-MSH is 50-fold higher for the MC3R. Of the three forms of
-MSH,
1- and
2-MSH (11 and 12 amino acid
residues, respectively) are most related, while
3-MSH
has an extended C terminus. In vivo amidation of the
C-terminal Gly12 residue of
2-MSH results in
the formation of
1-MSH with an C-terminal
Phe11-amide. In mammals, the natural forms of
-MSH
contain an additional N-terminal Lys residue (3).
-MSH and
Lys-
2-MSH both contain the core sequence, HFRW, a Tyr
residue at position 2 and a Met residue at position 4, while N- and
C-terminal residues and the residue at position 5 differ (Fig.
1). Recently it was shown that Asp10 in Lys-
2-MSH determined MC3R selective
activation (24). However, it is not clear whether replacement of
Asp10 in Lys-
2-MSH increases binding
affinity or only increases efficacy for the MC4R. Moreover, it remains
to be determined whether there exists a direct selective interaction of
Asp10 with the MC3R, or whether Asp10 induces a
peptide structure that is favorable for the MC3R. To solve this problem
a more detailed analysis is required regarding the contribution of each
individual amino acid in the ligand to receptor binding and activation.
Therefore, the aim of this study was to gain insight into the molecular
mechanism of the selectivity of
2-MSH for the MC3R
versus the MC4R. Using a gain of function approach, we
tested [Nle4]
-MSH and
[Nle4]Lys-
2-MSH and derivatives with
exchanged amino acid residues on in vitro binding and
activation of wild type and chimeric MC3R/MC4R and mutant MC4Rs. We
demonstrate here that the selectivity of
2-MSH for the
MC3R versus the MC4R is determined by a single amino acid
residue in the ligand and, to a large extend, a single residue in the
receptor. A new concept is proposed in which MC3R/MC4R selectivity is
determined by how the melanocortin core sequence is presented to the
receptor-binding pocket.
 |
MATERIALS AND METHODS |
Peptides--
NDP-
-MSH
([Nle4,D-Phe7]
-MSH),
-MSH,
and
2-MSH were purchased from Bachem Feinchemicalien,
Bubendorf, Switzerland. Lys-
2-MSH was synthesized by the
National Institute of Public Health and Environmental Protection
(RIVM), Bilthoven, The Netherlands (9).
1-MSH was a gift
from Organon, Oss, The Netherlands. MT-II was a gift from V. Hruby
(Tucson, AZ). [D-Tyr7]MT-II,
[Nle4]
-MSH,
[Nle4,Gly5,Asp10]
-MSH,
[Nle4,Phe12]
-MSH,
[Nle3]
2-MSH,
[Nle4]Lys-
2-MSH,
[Nle4,Gly10]Lys-
2-MSH,
[Nle4,Pro12]Lys-
2-MSH, and
[Nle4,Gly10,Pro12]Lys-
2-MSH
were synthesized using solid phase N-(9-fluorenyl)methoxycarbonyl chemistry and purified as described in Schaaper et al. (25). The products were analyzed using liquid chromatography mass
spectrometry. Ion spray mass spectrometry performed on a Micromass
Quattro sq confirmed the expected molecular weights. All other peptides
mentioned in Table II were synthesized using pepscan and their
concentration and purity were verified by high pressure liquid
chromatography (26). All peptides in Table II were N-terminal
acetylated and C-terminal amidated. Peptides were dissolved in 1 mM hydrogen chloride and diluted in phosphate-buffered
saline or binding buffer (see below).
Radioiodination of NDP-
-MSH--
Iodination was performed as
described by Tatro et al. (27). In short, 4 µg of
NDP-
-MSH was mixed with 1.2 IU of 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. Then, the sample was high pressure liquid
chromatography purified with a µBondapak C18 column 3.9 × 300 mm (Waters, Division of Millipore) by elution with a 22-52%
acetonitrile gradient in 10 mM ammonium acetate (pH 5.5) in
40 min. Specific activity of the [125I]NDP-
-MSH was
determined in the
-galactosidase activation assay and was
approximately 0.4 pmol/100,000 cpm.
Construction of Chimeric and Mutant Receptors--
Chimeric
receptors were made with polymerase chain reaction using overlapping
primers directed against the region coding for identical residues in
the rat MC3R and human MC4R in TM1, the junction of TM3-IC2, and in TM5
(Fig. 2). Thus, within the junction sites
of the chimeric receptor the amino acid sequence was not changed. Both
chimeric and mutant receptors were made with a similar stategy. In the
first polymerase chain reaction (Cloned Pfu DNA Polymerase, Stratagene)
separate fragments were obtained with primer T7 combined with the 3'
mutagenic/chimeric primer and with SP6 combined with the 5'
mutagenic/chimeric primer (complete overlap with the 3' primer).
Fragments were isolated and purified from agarose gel (QIAEX Gel
Extraction Kit, QIAGEN) and a second polymerase chain reaction
(Taq DNA polymerase, Perkin-Elmer) was performed with the
two purified bands as template for primers SP6 and T7. The polymerase
chain reaction products were digested with BamHI and
XhoI, separated through agarose gel electrophoresis, and
purified. Chimera 3B, MC4(267-282 MC3), MC4(267-273 MC3),
MC4(278-282 MC3), and MC4R mutants, F267L/Y268I (double mutant),
F267L, Y268I, S270T, Q273T, M281T were cloned into pcDNA3.1.
Chimera 3AB, 3C, 3D, and 4D were cloned into pcDNA1/Neo). The
inserts of the clones were sequenced completely (T7 sequencing kit,
Pharmacia Biotech) and only mutations introduced by the
mutagenic/chimeric primers were found.

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Fig. 2.
Model of the human MC4R. Gray
circles indicate residues identical in the rat MC3R and the human
MC4R. Black circles indicate regions with complete homology
that were used as boundaries for construction of the chimeric
receptors. These were named after the extracellular domain of the MC3R
that was placed into the MC4R. Thus, chimera 3AB is the MC4R containing
the N terminus through TM3 (third transmembrane domain) of the MC3R,
chimera 3B is the MC4R containing the IC1 (first intracellular loop)
through TM3 of the MC3R, chimera 3C is the MC4R containing IC2, TM4,
EC2 (second extracellular loop) and part of TM5 of the MC3R, chimera 3D
is the MC4R containing part of TM5 through the C terminus of the MC3R
and vice versa, chimera 4D is the MC3R containing part of TM5 through
the C terminus of the MC4R.
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|
Cells and Transfection--
HEK 293 cells were grown in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% fetal calf serum. Optimal amounts of receptor
cDNA used for transfection of cells for the activation assay were
determined empirically. Approximately 7 × 106 cells
were transiently transfected with 15-200 ng of the rat MC3R, the human
MC4R, chimeric or mutant receptor cDNA constructs, combined with 7 µg of the pCRELacZ for the
-galactosidase activation assay using
the calcium phosphate precipitation method. For the receptor binding
assay, cells were transfected with 7 µg of the receptor cDNA constructs.
-Galactosidase Activation Assay--
The assay makes use of
the
-galactosidase (lacZ) gene fused to five copies of the cyclic
AMP response element (CRE) to detect the activation of CRE-binding
protein resulting from increased intracellular cAMP and
Ca2+ (28). Twenty-four hours after transfection 293 HEK
cells were distributed into 96-well plates (Primaria). The next day the
cells were treated for 6 h with various concentrations of peptides
in Dulbecco's modified Eagle's medium supplemented with 0.5% bovine serum albumin, 25 mM Hepes (pH 7.4), and 50 µg/ml (150 KIU/ml) aprotinin (Sigma). After treatment the cells were lysed,
frozen, thawed, and assayed for
-galactosidase activity. For each
peptide 12 data points were measured in quadruplicate. EC50
values were then calculated with a 95% confidence interval using
GraphPad Prism software (sigmoidal dose-response curve fitting,
variable slope). Experiments were repeated at least twice with the same results.
Adenylate Cyclase Assay--
293 HEK cells stably expressing
human MC4R, rat MC3R, mutant MC4(Y268I), and chimera MC4(267-282 MC3)
were grown in poly-L-lysine-coated (Sigma) 24-well Costar
plates. Agonist stimulated adenylate cyclase activity was measured as
described by Salomon (29, 30). In short, after prelabeling with 500 µl of [3H]adenine (NEN Life Science Products Inc.) in a
concentration of 2 µCi/ml, the 293 cells were incubated for 20 min at
37 °C in phosphate-buffered saline containing 0.1 mM
isobutylmethylxanthine (IBMX), 1 µM forskolin, and
agonist in a concentration ranging from 10
11 to
10
5 M. The cells were harvested and
[3H]cAMP formation was determined. For each peptide 12 duplicate data points were measured. EC50 values were then
calculated with a 95% confidence interval using GraphPad Prism
software (sigmoidal dose-response curve fitting, variable slope).
Receptor Binding Assay--
Transfected HEK 293 cells were grown
in poly-L-lysine-coated 24-well Costar plates. Two days
after transfection, the cells were incubated with 100,000 cpm of
[125I]NDP-
-MSH and various concentrations of peptides
diluted in binding buffer consisting of Ham's F-10 medium (Life
Technologies, Inc.) (pH 7.4) containing 2.5 mM calcium
chloride, 0.25% bovine serum albumin, 10 mM Hepes, and 50 µg/ml (150 KIU/ml) aprotinin. After incubation for 30 min 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 11 duplicate data points with GraphPad Prism software, nonlinear
regression, one site competition. Ki values were
calculated using the Cheng and Prusoff equation with 95% confidence
interval. Experiments were repeated at least twice with the same results.
 |
RESULTS |
Characterization of the Reference Peptides--
In order to
delineate
/
-MSH selectivity we first excluded influence of
peptide length, N- and C-terminal modifications, and oxidation of the
Met residue, and therefore used synthetic [Nle4]
-MSH
and [Nle4]Ac-Lys-
2-MSH-NH2 as
reference peptides (the latter will be referred to as
[Nle4]Lys-
2-MSH).
Table I shows that, using displacement of
[125I]NDP-
-MSH, the affinity for
[Nle4]
-MSH, as compared with
-MSH, was increased
about 2-fold for both the MC3R and the MC4R. Also
[Nle4]Lys-
2-MSH showed increased affinity
for both the MC3R and the MC4R as compared with
1-MSH,
2-MSH, Lys-
2-MSH, and
[Nle3]
2-MSH. On the MC4R,
[Nle4]Lys-
2-MSH displayed a 2-fold lower
affinity and 20-fold lower activity of cAMP-dependent
-galactosidase activity than [Nle4]
-MSH (Table
II). On the MC3R, however,
[Nle4]Lys-
-MSH exhibited a 3.5-fold higher affinity
and similar activity as compared with [Nle4]
-MSH. Even
though [Nle4]
-MSH had a 6-fold lower affinity for the
MC4R than for the MC3R, [Nle4]Lys-
2-MSH
maintained selectivity for the MC3R, since the affinity is almost 50 times higher for the MC3R than for the MC4R. Thus, [Nle4]
-MSH and
[Nle4]Lys-
2-MSH appeared to be valid as
reference peptides in studies aimed at delineating amino acid residues
important for MC3R and MC4R selectivity.
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Table I
Inhibition constants (Ki in nM) of natural
occurring and synthetic MSH peptides for the MC3R and MC4R
The rat MC3R and the human MC4R were transiently transfected in 293 cells. The Ki values were determined by displacement
of [125I]NDP- -MSH. Data are given as Ki
values derived from 11 duplicate data points. Values are representative
of at least two independent experiments.
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Table II
Inhibition constants (Ki in nM) and EC50
values (in nM) of synthetic - and -MSH analogues for
the MC3R and MC4R
All peptides were N-terminal and C-terminal amidated and contain a
Nle4. The rat MC3R and the human MC4R were transiently
transfected in 293 cells. Ki values were determined
by displacement of [125I]NDP- -MSH and calculated with 95%
confidence interval derived from 11 duplicate data points. EC50
values were determined in the -galactosidase assay in the same batch
of transfected cells to exclude influence of receptor expression
levels. For each peptide 12 data points were measured in quadruplicate
and EC50 values were calculated with a 95% confidence
interval. Both Ki and EC50 values are
representative of at least two independent experiments.
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|
Role of [Nle4]
-MSH and
[Nle4]Lys-
2-MSH Residues in Binding and
Activation of the MC3R and MC4R--
First, each amino acid residue in
[Nle4]
-MSH was substituted separately for the
corresponding residues of [Nle4]Lys-
2-MSH.
When Ser1, Ser3, Glu5,
Pro12, or Val13 were substituted for
Lys1, Val3, Gly5,
Phe12, and Gly13, respectively, no significant
effect was observed on affinity or activity for the MC3R nor the MC4R
as compared with reference peptide [Nle4]-
-MSH (upper
panel of Table II). The only exception was
[Nle4,Phe12]
-MSH, which exhibited a 2-fold
decrease in activity for the MC4R and a 2-fold increase in affinity for
the MC3R. However, when Gly10 or Lys11 of
[Nle4]
-MSH were substituted for Asp10 or
Arg11 a clear loss of affinity and activity was observed
for both the MC3R and MC4R. When the Glu5 to
Gly5 substitution was combined with the Gly10
to Asp10 substitution in [Nle4]
-MSH, the
affinity and activity for the MC4R decreased more than 3-fold, whereas
the affinity for the MC3R remained the same as for
[Nle4]
-MSH.
Next, each amino acid residue in
[Nle4]Lys-
2-MSH was substituted for
corresponding residues of [Nle4]
-MSH (lower panel of
Table II). When Lys1 was substituted for Ser1 a
slight decrease in affinity was observed for both MC3R and MC4R but the
activity remained unaffected. Substitution of Arg11 for
Lys11 slightly increased activity for both receptors.
Substitution of Val3 for Ser3 or
Gly13 for Val13 decreased affinity for only the
MC4R, while the activity remained unaffected. With respect to binding
affinity, [Val13]Lys-
2-MSH displayed the
largest difference between MC3 and MC4 as did
[Ser3]Lys-
2-MSH in activation.
Interestingly, when Asp10 was substituted for
Gly10, a 5-fold increase in both affinity and activity for
the MC4R was observed, while the MC3R was unaffected.
[Nle4,Gly10]Lys-
2-MSH had an
even higher affinity for the MC4R than [Nle4]
-MSH. The
Phe12 for Pro12 substitution also gave an
increase in affinity and activity of more than 3-fold for the MC4R, but
not for the MC3R. Strikingly, the Asp10 to
Gly10 substitution combined with the Phe12 to
Pro12 substitution further increased the affinity to almost
13-fold for the MC4R, but there was no additive effect of these two
substitutions on MC4R activation.
Receptor Domains Involved in
[Nle4]Lys-
2-MSH Selective
Binding--
The MC3R and the MC4R share 58% overall amino acid
identity and 76% similarity. The transmembrane regions (TM) show the
highest degree of homology while the intra- and extracellular loops (IC and EC) have lower homology (Fig. 2). To identify regions of the MC3R
responsible for [Nle4]Lys-
2-MSH
selectivity a series of MC3R and MC4R chimeric receptors were generated
with boundaries in stretches of amino acid residues with complete
homology in order to minimize effects on receptor folding. These
chimeric receptors were designed to determine the contribution of each
of the extracellular domains to ligand recognition. Thus, the first
extracellular loop with or without the N-terminal domain (named 3B and
3AB, respectively), the second (3C) and third (3D) extracellular loop
of the MC4R were swapped individually with the corresponding domain of
the MC3R. These chimeric receptors were transfected into 293 HEK cells
and the affinities of NDP-
-MSH, [Nle4]
-MSH,
[Nle4]Lys-
2-MSH, and
[Nle4,Gly5,Asp10]
-MSH were
determined (the latter two peptides displayed MC3R selectivity).
Fig. 3 summarizes ligand affinity for the
MC3R, MC4R, and five chimeric receptors. All chimera bound
[125I]NDP-
-MSH, demonstrating that they were all
expressed on the plasma membrane. In general, the affinities of MSH
peptides for MC3R and chimera 3AB, 3B, and 4D were higher than for MC4R
and chimeras 3C and 3D. For example, the affinity of NDP-
-MSH on these chimera was 6-20-fold higher than for the MC4R, but the same as
for the MC3R. All chimeric receptors, except 3D, had the same affinity
profile as the MC4R, in which the affinity of NDP-
-MSH
[Nle4]
-MSH > [Nle4]Lys-
2-MSH = [Nle4,Gly5,Asp10]
-MSH. The
Ki values of
[Nle4,Gly5,Asp10]
-MSH (data
not shown in Fig. 3) for 3AB (1.9 nM), 3B (5.0 nM), 3C (131 nM), and 4D (11 nM),
were not different from [Nle4]Lys-
2-MSH,
but were significantly lower than [Nle4]
-MSH
(p < 0.05). 3D was the only chimera with the same
affinity profile as the MC3R, in which
[Nle4]Lys-
2-MSH exhibited higher affinity
than [Nle4]
-MSH. However, the absolute
Ki values were more than 10-fold higher than for the
MC3R. The affinity of
[Nle4,Gly5,Asp10]
-MSH for 3D
(Ki of 170 nM) was lower but not
significantly different from [Nle4]
-MSH.

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Fig. 3.
Ki values of
NDP- -MSH ( ),
[Nle4] -MSH ( ), and
[Nle4]Lys- 2-MSH
( ) for the MC3R, MC4R, and chimera 3AB, 3B, 3C, 3D, and 4D.
Error bars indicate 95% confidence interval.
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No disparity was observed between activity and affinity of
[Nle4]Lys-
2-MSH,
[Nle4]
-MSH, and
[Nle4,Gly5,Asp10]
-MSH for the
chimeric receptors (data not shown). Thus, the region from TM5 through
the C-terminal portion of the MC3R (containing EC3) determined
selectivity for [Nle4]Lys-
2-MSH. Since
peptide hormones presumably interact with their ligands via the
extracellular regions and with the binding pocket formed by the
transmembrane helices of the receptor (31), we concentrated on the
third extracellular loop for
[Nle4]Lys-
2-MSH selectivity.
The radioligand receptor binding assay was used to further analyze the
chimeric and mutant receptors described below. In order to determine in
more detail which residues are important for selectivity we first
substituted residues 267 to 282 of the MC4R (containing EC3), hereafter
named MC4(267-282 MC3), for corresponding residues of the MC3R (Fig.
4). Fig.
5A shows that this chimeric
receptor displayed gain of affinity for
[Nle4]Lys-
2-MSH as compared with
[Nle4]
-MSH. Therefore, this region was subdivided into
two smaller parts. MC4(278-282 MC3), containing the second half of EC3
of the MC3R, did not display gain of affinity for
[Nle4]Lys-
2-MSH. In contrast, MC4(267-273
MC3), containing the first half of EC3 of the MC3R, displayed equal
affinity for [Nle4]Lys-
2-MSH and
[Nle4]
-MSH. Thus,
[Nle4]Lys-
2-MSH selectivity resides within
the first half of EC3. The Ki value of
[Nle4,Gly5,Asp10]
-MSH for
chimera MC4(267-282 MC3) and MC4(267-273 MC3), 128 and 76 nM, respectively, was significantly lower than
[Nle4]
-MSH (p < 0.05, data not shown
in Fig. 4).

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Fig. 4.
Alignment of the rat MC3R and the human MC4R
focused on the EC3 and adjacent TM domains. Blocks
indicate homologous residues and residue numbering corresponds to the
human MC4R amino acid sequence.
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Fig. 5.
A, Ki values of
NDP- -MSH ( ), [Nle4] -MSH ( ), and
[Nle4]Lys- 2-MSH ( ) and
[Nle4,Gly10]Lys- 2-MSH ( ) on
the MC4R and chimera. Error bars indicate 95% confidence
interval. For experimental details see "Materials and Methods" and
the legend of Table II. B, Ki values of NDP- -MSH
( ), [Nle4] -MSH ( ),
[Nle4]Lys- 2-MSH ( ), and
[Nle4, Gly10]Lys- 2-MSH ( )
on the MC4(267-273 MC3) and MC4R mutants. Error bars
indicate 95% confidence interval. For experimental details see
"Materials and Methods" and the legend of Table II.
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Four residues in this region of the MC4R were mutated for the
corresponding MC3R residue. Like MC4(267-273 MC3), the double MC4R
mutant F267L/Y268I had the same affinity for
[Nle4]Lys-
2-MSH as for
[Nle4]
-MSH (Fig. 5B). Therefore, F267L and
the Y268I were also mutated separately. The affinity for
[Nle4]Lys-
2-MSH appeared to be the same as
for [Nle4]
-MSH on the Y268I mutant. However, the F267L
mutant displayed lower affinity for
[Nle4]Lys-
2-MSH than for
[Nle4]
-MSH. The affinity of
[Nle4,Gly5,Asp10]
-MSH (124 nM) was significantly lower than
[Nle4]
-MSH for the Y268I mutant (p < 0.05). [Nle4,Gly10]Lys-
2-MSH
exhibited similar affinity for the Y268I mutant and the MC4R (8.9 and
7.6 nM, respectively), demonstrating there is no further
gain of affinity for [Nle4]Lys-
2-MSH in
the absence of Asp10. The S270T, Q273T, and M281T mutants
did not gain affinity for [Nle4]Lys-
2-MSH
as compared with [Nle4]
-MSH. Thus, mutation of the
single residue, Y268I, of the MC4R altered
[Nle4]Lys-
2-MSH selectivity.
Affinity of NDP-
-MSH Compared with the Cyclic Melanocortin
Peptides, MT-II, and [D-Tyr4]MT-II, on the
MC4R, MC4(Y268I), and the MC3R--
The lactam-bridged molecules,
MT-II (melanotan II) and [D-Tyr4]MT-II, are
conformationally constrained heptapeptide analogs of
-MSH. The
affinity of these peptides was determined on the MC4R and MC3R and the
MC4(Y268I) mutant. The competition binding curves in Fig.
6A show that
[D-Tyr4]MT-II has equal affinity for the MC4R
as NDP-
-MSH, but 10-fold lower affinity than MT-II. Thus, the order
in affinity is, MT-II > NDP-
-MSH = [D-Tyr4]MT-II. In contrast, the competition
binding curves for [D-Tyr4]MT-II and MT-II on
MC4(Y268I) were both shifted to the right, whereas the affinity of
NDP-
-MSH was not statistically significantly different from MC4R.
Thus, for MC4(Y268I) and the MC3R the order in affinity is,
NDP-
-MSH = MT-II > [D-Tyr4]MT-II.
Fig. 6B summarizes the relative affinity (NDP-
-MSH = 1) of MT-II and [D-Tyr4]MT-II on MC4(Y268I)
and the MC3R as compared with MC4R.

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|
Fig. 6.
Affinity of
NDP- -MSH, MT-II, and
[D-Tyr4]MT-II on the MC3R, MC4(Y268I), and
MC4R expressed in HEK 293 cells. a, the
graphs show competition of [125I]NDP- -MSH
by NDP- -MSH ( ), MT-II ( ), and
[D-Tyr4]MT-II ( ). Data points represent
mean of duplicate measurements ± S.D. Graphs were fitted with
GraphPad Prism, one site competition. Curves are representative of two
independent experiments. b, the table shows the relative
affinity's (footnote a indicates a statistically significant
difference (p < 0.05) with the affinity of
NDP- -MSH). The IC50 values of NDP- -MSH for MC3R,
MC4(Y268I), and MC4R were 2.6, 8.3, and 17 nM,
respectively.
|
|
Activity of [Nle4]
-MSH compared with
[Nle4]Lys-
2MSH and
[D-Tyr4]MT-II on MC4R, MC4(267-282 MC3),
MC4(Y268I), and MC3R--
In order to determine whether differences in
binding affinity are reflected by similar differences in activity we
determined adenylate cyclase activity in response to two peptides that
showed the most pronounced differences in the binding affinity. Thus, we tested [Nle4]Lys-
2-MSH and
[D-Tyr4]MT-II on the MC4R, MC3R, and the key
chimeric and mutant receptor MC4(267-282 MC3) and MC4(Y268I) and
compared the EC50 values with [Nle4]
-MSH
(Table III). The EC50 values
of [Nle4]
-MSH for MC4R, MC4(267-282 MC3), MC4(Y268I),
and MC3R were 24, 113, 113, and 2.2 nM, respectively. Like
for MC3R, [Nle4]
-MSH and
[Nle4]Lys-
2-MSH have equal activity on
both MC4(267-282 MC3) and MC4(Y268I), whereas
[Nle4]Lys-
2-MSH has a 15-fold lower
activity compared with [Nle4]
-MSH on MC4R. Moreover,
the selective MC4R agonist, [D-Tyr4]MT-II,
displayed significantly lower activity on MC4(267-282 MC3),
MC4(Y268I), and MC3R compared with [Nle4]
-MSH while
these peptides have similar activity on MC4R.
View this table:
[in this window]
[in a new window]
|
Table III
Adenylate cyclase activity (relative efficacy) of
[Nle4] -MSH (set at 1),
[Nle4]Lys- 2-MSH, and
[D-Tyr4]MT-II on 293 cells stably expressing
MC4R, MC4(267-282 MC3), MC4(Y268I) and MC3R
|
|
 |
DISCUSSION |
Using a gain of function approach in which systematic exchanges of
peptides and receptors were used, we identified the importance of
Asp10 in Lys-
2-MSH in determining MC3R
selectivity. Furthermore, Tyr268 in the MC4R hindered
interaction with [Nle4]Lys-
2-MSH.
Tyr268 mutated toward the corresponding residue of the
MC3R, Ile, not only increased
[Nle4]Lys-
2-MSH affinity but, in addition,
decreased affinity for two cyclic melanocortin peptides, MT-II and
[D-Tyr4]MT-II. Thus, this receptor residue is
critical in determining MC3/MC4 receptor selectivity.
Independent substitutions of Gly10 and Lys11 in
[Nle4]
-MSH for Asp10 and
Arg11, respectively, led to a significant decrease in
activity and affinity for both MC3R and MC4R. This indicates that
introduction of these residues in [Nle4]
-MSH
prohibited interaction with both receptors. In a previous study (32)
the affinity of [Asp10]
-MSH also appeared to be much
lower than that of
-MSH for MC3R and MC1R. Still, Gly10,
and also Lys11, of [Nle4]
-MSH are probably
not important for a direct interaction with the receptor, since Ala
substitution on positions 10 and 11 of
-MSH did not affect affinity
on the MC3R nor the MC1R (23).
Substitution of Pro12 of [Nle4]
-MSH for
Phe12 slightly increased MC3R affinity while decreasing
activity, but not affinity, for the MC4R. This is in agreement with the
observation that substitution of Pro12 for Ala in
-MSH
did not affect binding to the MC3R (22). This implies that the residue
at this position is not essential for receptor binding but may be
important for MC4R activation only. Our data and others (33) indicate
that Phe12 in [Nle4]Lys-
2-MSH
may hinder interaction with MC4R. Nevertheless, this residue is
probably not involved in a direct interaction with MC3R, since it may
substituted for Pro without loss of affinity.
The results show that Asp10 in
[Nle4]Lys-
2-MSH determined selectivity for
the MC3R. First, because
[Nle4,Gly5,Asp10]
-MSH had
similar affinity and activity as [Nle4]
-MSH on the
MC3R, but lower on the MC4R. Second, the large increase in affinity and
activity of [Nle4,Gly10]Lys-
-MSH for the
MC4R clearly indicates that Asp10 either hinders
interaction with MC4R, or influences the peptide conformation to become
unfavorable for interacting with the MC4R. Since
[Nle4,Gly10]Lys-
2-MSH and
[Nle4]Lys-
2-MSH had similar affinity and
activity on MC3R, it is unlikely that Asp10 interacts
directly with the binding pocket of MC3R. Based upon the data presented
in this study we predict that guinea pig
-MSH, which has a Gly
instead of Asp at position 10 (34), activates the MC3R and MC4R equally
well. This implies that, if guinea pig
-MSH has a different
physiological role than
-MSH, there might be an as yet unidentified
-MSH specific receptor.
With respect to the receptor we showed that
[Nle4]Lys-
2-MSH selectivity resides in the
region from TM5 through the C terminus of the MC3R. Analysis of this
region in more detail revealed that [Nle4]Lys-
2-MSH and
[Nle4]
-MSH had similar affinity and activity for
MC4(Y268I). Besides Tyr268, components in the rest of the
EC3 loop (residues 267 through 282) of the MC3R further increased the
affinity for [Nle4]Lys-
2-MSH.
[Nle4,Gly10]Lys-
2-MSH had the
same affinity for the MC4R as for the Y268I mutant, confirming that
Asp10 in [Nle4]Lys-
2-MSH
determined MC3/MC4R selectivity.
Interestingly, it was found that all chimera containing the 3B segment
(TM2, EC1, and TM3) of the MC3R displayed a high affinity for MSH
peptides, similar to MC3R. In this same region of the MC1R, Yang
et al. (35) suggested an important role for residues Glu94 in TM2 and Asp117 or Asp121
in TM3 of the MC1R in forming an ionic binding pocket for the Arg8 residue of NDP-
-MSH,
-MSH,
2-MSH
(Arg7), and MT-II (35). Although these residues are
conserved in all MC receptors, other, non-conserved residues may
influence the strength of the receptor-ligand interaction, explaining
the overall lower affinity of MSH peptides, used in this study, for the
MC4R versus the MC3R.
A selective receptor-ligand interaction could occur through a mechanism
of exclusion rather than a specific recognition. In that respect,
Tyr268 of the MC4R may hinder
[Nle4]Lys-
2-MSH interaction and the
following model may apply. His264 of the MC4R is the
equivalent of the His260 in the MC1R and may, as was
suggested for the MC1R (36, 37), also be important for interaction with
-MSH. His264 has been demonstrated to be essential for
melanocortin peptide activation of MC4R as well (38).
His264 of the MC4R is located only four residues lower in
TM6 than Tyr268. Therefore, Tyr268 may mask
His264 and thereby exclude an interaction with
[Nle4]Lys-
2-MSH (Fig.
7).

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|
Fig. 7.
Helical wheel model of the MC4 receptor.
Residues in black indicate MC receptor conserved residues.
The bulky aromatic Tyr268 of the MC4R may hinder
interaction of -MSH with His264 which is positioned
lower in TM6. Figure template used with permission from Dr. T. W. Schwartz, Laboratory of Molecular Pharmacology, Copenhagen,
Denmark.
|
|
Using NDP-
-MSH containing Asp10, Arg11, and
Phe12 of
-MSH, Schioth et al. (39) suggested
that the
-MSH C terminus hinders MC4R binding. However, this peptide
showed an equal loss of affinity for the MC3R. Nevertheless, this loss
of affinity agrees with our data for
[Nle4,Asp10]
-MSH. This suggests that the
presence of two acidic amino acid residues on positions 5 and 10 (Glu
and Asp, respectively), lead to a significant decrease in MC3R and MC4R
affinity, possibly because of repulsion of these two negatively charged
residues. This is in agreement with the high affinity of cyclic lactam- (cyclization between residues 5 and 10) and disulfide-bridged (cyclization between residues 4 and 10) melanocortin derivatives (40-44). Taken together, these data suggest that in the active conformation the residues in positions 4/5 and 10 are in close proximity. We here propose that the presence of Asp10 in
[Nle4]Lys-
2-MSH decreased the chance of
the core sequence to be in the optimal conformation necessary to bind
to the MC4R.
To investigate whether presentation of a constraint core sequence
determined MC4R selectivity, the affinity of cyclic melanocortin peptides with a structurally constrained core sequence was tested. MT-II displayed marked increased and
[D-Tyr4]MT-II equal affinity as compared with
NDP-
-MSH for the MC4R, but not for the MC3R and MC4(Y268I). These
differences in ligand activity appeared to be represented by
differences in affinity. Thus, mutation of Tyr268 in MC4R
toward the Ile residue present on the corresponding position in MC3R
increased both affinity and activity of
[Nle4]Lys-
2-MSH and decreased affinity and
activity for the high affinity MC4R agonist,
[D-Tyr4]MT-II. Thus, a single amino acid
residue determined MC3/MC4R selectivity for different ligands and MC4R
binding and activation increases when the core sequence is presented in
a constraint conformation.
Recently, it was shown that cyclic peptides have higher affinity than
-MSH on all MC-Rs (45). Strikingly, for the MC4R, all cyclic
peptides displayed higher or similar affinity than for MC3R (45-47),
while linear peptides always seemed to exhibit lower affinity on the
MC4R than on the MC3R (this study). Therefore, for design of new MC-R
selective ligands, cyclization may be appropriate for the MC4R. In
contrast, modification in linear MSH peptides may be more valuable for
MC3R selectivity, as was suggested by Haskell-Luevano et al.
(41). A similar model was proposed for the opioid receptors in which
linear dynorphin A analogues were generally more selective for the
µ-opioid receptor while cyclic constrained dynorphin A peptides
demonstrated slight selectivity for
- versus
-opioid
receptors or were nonselective (48). Taken together, these data
suggests that the message sequence residues do not interact with
conserved neuropeptide receptor residues in the same manner. Indeed, it
has been shown that the HFRW pharmacophore interacts differently with
all MC-R (49).
Here, we propose a general concept for selective receptor-ligand
interaction that may apply to all peptide receptors. Ligand residues
outside the peptide core sequence direct the conformation of the
receptor interacting core sequence presented to the receptor-binding pocket, and thereby determine selectivity. There are several examples that emphasize the critical role of residues positioned outside the
core of true contact residues, in determining selectivity of ligands
to, for instance, opioid (36) and neuropeptide Y receptors (50).
This study provides, for the first time, a detailed analysis of the
structural requirements for selective MC3R versus MC4R recognition. Using a gain of function approach we have demonstrated that the Asp10 of
[Nle4]Lys-
2-MSH and Tyr268,
near the extracellular face of TM6, of the MC4R, determine the low
affinity and activity of [Nle4]Lys-
2-MSH
for the MC4R. The large aromatic residue Tyr268 of the MC4R
may change the MC4R-binding pocket unfavorable for [Nle4]Lys-
2-MSH but favorable for the
cyclic peptides MT-II and [D-Tyr4]MT-II.
 |
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: Rudolf Magnus Institute
for Neurosciences, Medical Faculty, Utrecht University, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands. Fax:
31-302539032; E-mail: adan{at}med.uu.nl.
 |
ABBREVIATIONS |
The abbreviations used are:
-MSH,
-melanocyte-stimulating hormone;
MC, melanocortin;
TM, transmembrane;
CRE, cAMP response element;
MT, melanotan.
 |
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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.