Contribution of Melanocortin Receptor Exoloops to Agouti-related
Protein Binding*
Ying-kui
Yang
,
Chris J.
Dickinson§,
Qun
Zeng¶,
Ji-Yao
Li
,
Darren A.
Thompson
, and
Ira
Gantz
¶**
From the
Departments of General Surgery, University
of Michigan Medical School and ¶ Ann Arbor Veterans Administration
Hospital, Ann Arbor, Michigan 48109, § Department of
Pediatrics, University of Michigan Medical School, Ann Arbor,
Michigan 48109, and
Gryphon Sciences, South San Francisco,
California 94080
 |
ABSTRACT |
Agouti-related protein (AGRP) is an endogenous
antagonist of melanocortin action that functions in the hypothalamic
control of feeding behavior. Although previous studies have shown that AGRP binds three of the five known subtypes of melanocortin receptor, the receptor domains participating in binding and the molecular interactions involved are presently unknown. The present studies were
designed to examine the contribution of extracytoplasmic domains of the
melanocortin-4 receptor (MC4R) to AGRP binding by making chimerical
receptor constructs of the human melanocortin-1 receptor (MC1R; a
receptor that is not inhibited by AGRP) and the human MC4R (a receptor
that is potently inhibited by AGRP). Substitutions of the
extracytoplasmic NH2 terminus and the first extracytoplasmic loop (exoloop) of the MC4R with homologous domains of
the MC1R had no effect on AGRP (87-132) binding affinity or inhibitory
activity (the ability to inhibit melanocortin-stimulated cAMP
generation). In contrast, cassette substitutions of exoloops 2 and 3 of
the MC4R with the homologous exoloops of the MC1R resulted in a
substantial loss of AGRP binding affinity and inhibitory activity.
Conversely, the exchange of exoloops 2 and 3 of the MC1R with the
homologous exoloops of the MC4R was found to confer AGRP binding and
inhibitory activity to the basic structure of the MC1R. Importantly,
these substitutions did not affect the ability of the
-melanocyte
stimulating hormone analogue
[Nle4,D-Phe7] melanocyte
stimulating hormone to bind or activate the chimeric receptors. These
data indicate that exoloops 2 and 3 of the melanocortin receptors are
important for AGRP binding.
 |
INTRODUCTION |
The melanocortin peptides,
-,
-, and
-melanocyte
stimulating hormone (MSH)1
and adrenocorticotropic hormone, are a group of peptides derived from
the pro-opiomelanocortin prohormone that share a common message sequence (His-D-Phe-Arg-Trp). These peptides have been
implicated in diverse physiological processes but are most widely
recognized for their roles in melanogenesis, steroidogenesis, and, more
recently, feeding behavior (1-4). There are five known seven
transmembrane G-protein-coupled melanocortin receptors that couple to
the stimulatory G-protein, Gs (Refs. 5-9). In addition,
two endogenous melanocortin antagonists, Agouti and AGRP, have been
discovered that function as modifiers of melanocortin action.
The most well-documented function of Agouti is its action as a
paracrine signaling molecule that modifies melanocortin action at the
MC1R (the melanocyte MCR) (10, 11). Agouti is temporally produced by
cells adjacent to the hair follicle melanocyte and is responsible for
the Agouti phenotype (dark hair with a sub-apical yellow band).
In vitro studies have demonstrated that recombinant Agouti
is a potent antagonist of melanocortin action at MCR subtypes 1, 2, and
4 (11-13).
AGRP was originally identified from its sequence similarity to Agouti
(14-16). Both AGRP and Agouti have a COOH-terminal cysteine-rich motif. In contrast to Agouti, AGRP is a potent antagonist of the melanocortin-3 receptor and the MC4R and has also been shown to have a
lesser degree of inhibitory action at the melanocortin-5 receptor.
Although a full understanding of the biological spectrum of AGRP action
remains to be determined, its expression in the hypothalamus, its
ability to inhibit the action of
-MSH at the MC4R, and its ability
as a transgene (under the control of a
-actin promoter) to cause
obesity in mice indicate that it is involved in the regulation of
feeding behavior. In the hypothalamus,
-MSH is believed to act a
satiety-inducing factor that mediates its action through the MC4R,
whereas AGRP is one of several opposing orexigenic agents.
AGRP is transcribed as 132 amino acids in man (131 amino acids in
mouse), and although it is not presently known whether it is
post-translationally processed in mammals, we have recently shown that
a 46-amino acid COOH-terminal AGRP variant has the same ability to
selectively bind MCR subtypes and functionally inhibit melanocortins as
the full-length molecule (17). Pharmacological studies using this
chemically synthesized truncated AGRP variant, AGRP (87-132), indicate
that AGRP is a competitive antagonist of
-MSH at MCR subtypes 3, 4, and 5.
AGRP has very little amino acid sequence similarity to melanocortins.
Even in its artificially truncated form, AGRP is 46 amino acids and
contains 10 cysteine residues capable of forming five disulfide bonds.
In contrast, the predominant melanocortin in the hypothalamus,
-MSH,
is only 13 amino acids in length and has no cysteine residues. These
observations suggest that the members of this agonist-antagonist pair
have significantly different tertiary structures, despite their use of
the same receptors. It is also important to note that with respect to
their competitive interaction,
-MSH is capable of activating MCR
subtypes 1, 3, 4, and 5, whereas AGRP can only inhibit the action of
-MSH action at MCR subtypes 3, 4, and 5. In view of their apparent
structural dissimilarity and subtype specificity, it is likely that
-MSH and AGRP have receptor binding determinants that are not identical.
To date, little is known about the molecular forces and receptor
domains involved in AGRP-MCR binding. Based on assumptions about its
structure and presently held concepts about the way larger peptides
bind seven transmembrane G-protein coupled receptors, we hypothesized
that one potential binding determinant for AGRP might be the
extracytoplasmic domains of the MCRs.
 |
EXPERIMENTAL PROCEDURES |
Construction of MCR Chimeras--
The amino acid sequences of
the MC1R and MC4R were examined by a hydrophobicity plot (Genetics
Computer Group, Inc., Madison, WI) and examined manually by comparing
their sequences to a previously published alignment of seven
transmembrane G-protein-coupled receptor
-helices (18). The chimeras
utilized in these studies are schematically diagrammed in Fig.
1. The chimeras were constructed by
polymerase chain reaction (PCR) using Pfu polymerase (Stratagene, La
Jolla, CA). The human MC1R and MC4R served as a template. During an
initial round of PCR, partial-length receptor fragments were generated. The sequence of one of the PCR primer oligonucleotides consisted of an
extracytoplasmic domain of interest coupled to a portion of the
transmembrane domain required to form a chimeric receptor. The second
oligonucleotide primer consisted of either the 5' or 3' end of the MC1R
or MC4R. Receptor fragments were separated by agarose gel
electrophoresis and used in a second round of PCR in which full-length
chimeric receptor constructs were assembled by cycling the appropriate
fragments together for 10 cycles before adding both 5' and 3' receptor
primers. The chimeric receptors were subcloned into the M13 vector for
single-stranded dideoxynucleotide sequencing to check that the desired
sequences were present and that no sequence errors had been introduced
by PCR. The constructs were then subcloned into the eukaryotic
expression vector pcDNA 3.1 (Invitrogen, Carlsbad, CA). A
comparison of the exchanged amino acid sequences of the
NH2-terminal, first, second, and third exoloops of the MC1R
and MC4R is shown in Fig. 2. The sequence of the wild-type MC4R used in these studies can be found in
GenBankTM under accession number L08603 (8). The sequence
of the wild-type MC1R used in these studies can be found in
GenBankTM under accession number X65634 (5), except that
position 163 is Arg, and position 164 is Gln. This genetic polymorphism in transmembrane 4 does not cause any apparent change in either basal
or stimulated cAMP or 125I-NDP-MSH binding compared with
the published MC1R sequence.

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Fig. 1.
Schematic representation of the chimeric
human melanocortin receptors utilized in these studies.
A schematically depicts the seven transmembrane structure of
the wild-type (WT) MC4R (drawn with thick lines)
and MC1R (drawn with thin lines). B depicts the
structure of the chimeric MC4R with the substituted NH2
terminus (NH2) of the MC1R. C depicts the
structure of the chimeric MC4R with the substituted first
extracytoplasmic loop (1e) of the MC1R. D depicts
the chimeric MC4R with the substituted second extracytoplasmic loop
(2e) of the MC1R (left) and the chimeric MC1R
with the substituted 2e of the MC4R (right). E
depicts the chimeric MC4R with the substituted third extracytoplasmic
loop (3e) of the MC1R (left) and chimeric MC1R
with substituted 3e of the MC4R (right). F
depicts the chimeric MC4R with simultaneous substitutions of the 2e and
3e (2e,3e) of the MC1R (left) and the chimeric
MC1R with the simultaneous substitution of 2e,3e of the MC4R
(right). The amino acid sequences of the NH2,
1e, 2e, and 3e of the MC1R and MC4R are shown for comparison. Only
exoloops 2e and 3e appear to contain determinants for AGRP
binding.
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Fig. 2.
Displacement of the radioligand
125I-NDP-MSH from MC4R chimeras by
(A) NDP-MSH and (B) AGRP
(87-132). C depicts the displacement of
125I-AGRP (87-132) by AGRP (87-132) from the chimeric
MC4R. This set of chimeric receptors consists of the basic structure of
the MC4R with substitutions of various extracellular portions of the
MC1R. MC4RWT, wild-type MC4R; MC1RWT, wild-type
MC1R; MC4R/NH2MC1R, MC4R containing the NH2
terminus of the MC1R; MC4R/1eMC1R, MC4R containing the first
exoloop of the human MC1R; MC4R/2eMC1R, MC4R containing the
second exoloop of the MC1R; MC4R/3eMC1R, MC4R containing the
third exoloop of the MC1R; MC4R/2e,3eMC1R, MC4R containing
second and third exoloops of the MC1R.
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cAMP Assays--
cAMP assays were performed on transiently
transfected HEK-293 cells as described previously (13) using a
competitive binding assay (Amersham Pharmacia Biotech cAMP assay kit
TRK 432). NDP-MSH was obtained from Peninsula Laboratories (Belmont,
CA), and human AGRP (87-132) was provided by Gryphon Sciences (South
San Francisco, CA). Data was analyzed using Graphpad Prism (Graphpad
Software, San Diego, CA). All experiments represent n
3 ± S.E.
Binding
Assays--
[125I](Iodotyrosyl2)-NDP-MSH and
125I-AGRP (87-132) were prepared by simple oxidative
methods using chloramine T and Na125I (Amersham Pharmacia
Biotech) as described previously (13). NDP-MSH was purchased from
Peninsula Laboratories, and AGRP (87-132) was provided by Gryphon
Sciences. Binding experiments were performed on transiently transfected
HEK-293 cells using conditions described previously (13), with some
modification. Briefly, 12 h before the experiments, 0.2 million
cells were plated on 24-well plates. Before initiating the binding
experiments, cells were washed twice with minimum Eagle's medium.
Cells were then incubated with different concentrations of unlabeled
ligand containing 0.2% bovine serum albumin and either 1 × 105 cpm of 125I-NDP-MSH or 1 × 105 cpm of 125I-AGRP. After a 1-h incubation,
the cells were again washed twice with minimum Eagle's medium, and the
experiment was terminated by lysing the cells with 0.1 N
NaOH and 1% Triton X-100. Radioactivity present in the lysate was
quantified using an analytical gamma counter. Nonspecific binding was
determined by measuring the amount of 125I label remaining
bound in the presence of 10
5 M unlabeled
ligand, and specific binding was obtained by subtracting the
nonspecific bound radioactivity from the total bound radioactivity. The
binding displacement curves were drawn using Graphpad Prism. The
agonist-antagonist complex dissociation constant was calculated using
the equation Kb = [AGRP]/(EC50
ratio
1). All experiments represent n
3 ± S.E.
 |
RESULTS |
Characterization of MC4R/MC1R Chimeras with
125I-NDP-MSH and 125I-AGRP (87-132)
Binding--
Fig. 2A demonstrates that cassette
substitutions of the NH2 terminus, first, second, and third
exoloop of the MC4R (alone or in combination) with homologous regions
of the MC1R did not alter 125I-NDP-MSH binding. Data in
Figs. 2 and 4 are expressed as total counts per minute (cpm) to
emphasize the point that the various HEK-293 cell lines expressed
roughly the same numbers of receptors.
Fig. 2B summarizes the ability of AGRP (87-132) to displace
125I-NDP-MSH from the MC4R/MC1R chimeras. IC50
values are listed in Table I. As shown,
AGRP (87-132) had only a minute ability to displace
125I-NDP-MSH binding from the wild-type MC1R. In contrast,
AGRP (87-132) dose-dependently displaced all
125I-NDP-MSH from the MC4R. These data are consistent with
the known MCR subtype specificity of AGRP. Substitution of the
NH2 terminus or the first exoloop of the MC4R with
homologous loops of the MC1R had no effect on the ability of AGRP
(87-132) to displace 125I-NDP-MSH. However, substitution
of the second or third exoloop of the MC4R with those of the MC1R
resulted in a significantly reduced IC50 of AGRP (87-132)
in inhibiting 125I-NDP-MSH binding to chimeric receptor
MC4R/2eMC1R or MC4R/3eMC1R. Simultaneous substitution of the second and
third exoloops of the MC4R with those of the MC1R (chimera MC4R/2e,
3eMC1R) led to further inhibition of AGRP binding.
To further examine AGRP binding to the chimerical receptors, binding
studies were performed with 125I-AGRP (87-132) (Fig.
2C). As expected, the wild-type MC1R demonstrated little if
any specific 125I-AGRP (87-132) binding. In contrast,
significant 125I-AGRP (87-132) binding was observed at the
MC4R. Notably, there was a progressive loss in specific
125I-AGRP (87-132) binding at chimeric MC4R with
substitutions of the second and third exoloop with homologous exoloops
of the MC1R (chimeras MC4R/2eMC1R, MC4R/3eMC1R, and MC4R/2e,3eMC1R).
Substitution of the third exoloop had a greater effect than
substitution of the second loop, and simultaneous substitution of
exoloops 2 and 3 had an additive effect. These data are entirely
consistent with the ability of AGRP to displace
125I-NDP-MSH binding presented in Fig. 2B. The
chimerical MC4R containing both exoloops 2 and 3 of the MC1R (MC4R/2e,
3eMC1R) retained only approximately 38% of the specific
125I-AGRP (87-132) binding of the wild-type MC4R.
Characterization of MC4R/MC1R Chimeras with cAMP Assays--
To
study the functional effects of exoloop substitution in more detail,
the ability of AGRP (87-132) to inhibit melanocortin action at the
MC4R/MC1R chimeras was examined in cAMP assays (Fig. 3). Consistent with the known effects of
AGRP, 3 × 10
7 M AGRP (87-132) potently
inhibited NDP-MSH action at the MC4R (Fig. 3A) but had no
effect on NDP-MSH-stimulated cAMP generation at the MC1R (Fig.
3E). Fig. 3, B and C, reveals that
there was a loss in the ability of AGRP to inhibit NDP-MSH-stimulated
cAMP generation at the chimeric MC4R/MC1R containing the second or third exoloops of the MC1R. When compared with the wild-type MC4R, simultaneous substitution of exoloops 2 and 3 of the MC4R with those of
the MC1R resulted in a greater loss in the ability of AGRP (87-132) to
inhibit NDP-MSH-stimulated cAMP than substitution of either individual
exoloop (Fig. 3D). EC50 and
Kb values are reported in Table
II.

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Fig. 3.
Comparison of AGRP (87-132) inhibition on
NDP-MSH-stimulated cAMP generation at (A) the
wild-type MC4R, (E) wild-type MC1R, and
(B-D) MC4R chimeras containing various exoloops of
the MC1R.
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Table II
Effect of AGRP on cAMP formation stimulated by NDP-MSH on HEK cells
transfected with chimeras of the human MC4R
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Characterization of MC1R/MC4R Chimeras with
125I-NDP-MSH and 125I-AGRP (87-132)
Binding--
Because replacement of exoloops 2 and 3 of the MC4R with
the homologous exoloops of the MC1R reduced AGRP binding affinity and
inhibitory activity, we sought to ascertain the effects of the
reciprocal substitutions. We hypothesized that if exoloops 2 and 3 were
important to AGRP binding, then cassette substitution of those MC1R
exoloops with those homologous exoloops of the MC4R would result in a
chimeric MC1R that, unlike the wild-type MC1R, would be able to
interact with AGRP. Fig. 4A
demonstrates that MC1R chimeras with substitutions of their second and
third exoloops of the MC4R retained a 125I-NDP-MSH binding
affinity comparable to that of the wild-type MC1R.

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Fig. 4.
Displacement of the radioligand
125I-NDP-MSH from MC1R chimeras by
(A) NDP-MSH and (B) AGRP
(87-132). C depicts the displacement of
125I-AGRP (87-132) by AGRP (87-132) from MC1R chimeras.
These chimeric receptors consist of the basic structure of the MC1R
with substitutions of various extracytoplasmic portions of the MC4R.
MC1RWT, wild-type MC1R; MC4RWT, wild-type MC4R;
MC1R/2eMC4R, MC1R containing the second exoloop of the MC4R;
MC1R/3eMC4R, MC1R containing the third exoloop of the MC4R;
MC1R/2e,3eMC4R, MC1R containing the second and third
exoloops of the MC4R.
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Fig. 4B shows that AGRP (87-132) had only a minimal ability
to displace 125I-NDP-MSH binding at the wild-type MC1R. A
small displacement of 125I-NDP-MSH binding was observed
only at very high concentrations of AGRP (87-132). In contrast, AGRP
(87-132) dose-dependently displaced all bound
125I-NDP-MSH from the MC4R. Remarkably, substitution of the
second or third exoloop of the MC4R into the sequence of the MC1R
caused a dramatic increase in the ability of AGRP (87-132) to displace 125I-NDP-MSH from the basic structure of the MC1R, whose
nonmutated form lacks responsiveness to AGRP. A stepwise increase in
the ability of AGRP ability to displace 125I-NDP-MSH
binding from chimeras MC1R/2eMC4R, MC1R/3eMC4R, and MC1R/2e,3eMC4R was
observed. Simultaneous substitution of both the second and third
exoloops of MC4R had an additive effect on this parameter. Notably,
substitution of MC4R exoloop 3 had a greater effect on the ability of
AGRP (87-132) to displace 125I-NDP-MSH binding than did
MC4R exoloop 2. The IC50 values are reported in Table
III.
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Table III
Effect of NDP-MSH and AGRP on 125I NDP-MSH binding on cells
transfected with chimeras of the human MC1R
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Fig. 4C shows that individual or simultaneous substitutions
of the second and third exoloops of the MC4R into the sequence of the
MC1R (chimeras MC1R/2eMC4R, MC1R/3eMC4R, and MC1R/2e,3eMC4R) conferred
upon the MC1R a dramatically increased ability to bind 125I-AGRP (87-132). These data are consistent with the
ability of AGRP to displace 125I-NDP-MSH binding presented
in Fig. 4B. As shown, substitution of the third exoloop had
a greater effect than substitution of the second loop, and simultaneous
substitution of MC4R exoloops 2 and 3 had an additive effect. The
chimerical MC1R containing both exoloops 2 and 3 of the MC4R
(MC1R/2e,3eMC4R) demonstrated approximately 76% of the specific
125I-AGRP (87-132) binding of the wild-type MC4R.
Characterization of MC1R/MC4R Chimeras with cAMP Assays--
As in
the case of the MC4R/MC1R chimeras, we sought to more fully examine the
functional effects of exoloop substitutions by examining the ability of
AGRP (87-132) to inhibit melanocortin-stimulated cAMP generation. Fig.
5A demonstrates that 3 × 10
7 M AGRP (87-132) had no ability to
inhibit melanocortin-stimulated cAMP generation at the wild-type MC1R.
In contrast, AGRP (87-132) is a potent antagonist of NDP-MSH action at
the wild-type MC4R (Fig. 5E). As shown in Fig. 5,
B and C, placement of MC4R exoloop 2 or exoloop 3 into the sequence of the MC1R led to a chimerical receptor that, unlike
the wild-type MC1R, was inhibited by AGRP. Substitution of the third
exoloop of the MC1R with the third exoloop of the MC4R had a greater
effect on establishing functional AGRP inhibition than did substitution
of the second exoloop of the MC4R. Simultaneous substitution of
exoloops 2 and 3 of the MC1R with the homologous exoloops of the MC4R
led to a chimerical MC1R that could be significantly inhibited by AGRP
(Fig. 5D). The cAMP data of the MC1R/MC4R chimeras are
completely consistent with radioligand binding studies from the same
chimeric receptors (Fig. 4). EC50 and Kb
values are reported in Table IV.

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Fig. 5.
Comparison of AGRP (87-132) inhibition of
NDP-MSH-stimulated cAMP generation at the (A)
wild-type MC1R, (E) wild-type MC4R, and
(B-D) MC1R chimeras containing various
extracytoplasmic portions of the MC4R.
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Table IV
Effect of AGRP on cAMP formation stimulated by NDP-MSH on HEK cells
transfected with chimeras of the human MC1R
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DISCUSSION |
Recent insights into the hypothalamic control of feeding behavior
indicate that
-MSH, which is produced by neurons in the arcuate
nucleus, purveys a satiety signal that is mediated through the MC4R
(the role of the melanocortin-3 receptor within the arcuate nucleus has
not yet been clearly defined). Produced by adjacent neuropeptide
Y-containing neurons, AGRP is believed to act as an opposing orexigenic
agent. In its role as an important mediator of satiety, the MC4R has
become a prime target for anti-obesity drug development. An
understanding of the molecular basis underlying the ability of
-MSH
and AGRP to bind the MC4R is of potential importance to those
pharmaceutical discovery efforts.
Previous three-dimensional modeling of the MC1R, extensive point
mutagenesis of the human MCR subtypes 1, 3, and 4, and
structure-activity studies using substituted and truncated
adrenocorticotropic hormone 1-13 fragments have led us to formulate a
structural model of MCR in which agonists bind to the MCRs in a
relatively shallow pocket formed by the transmembrane
-helices of
those receptors (19, 20). In general, this model of melanocortin
binding to the MCRs is consistent with current concepts regarding the
molecular interactions of small peptide hormones and seven
transmembrane G-protein-coupled receptors (21). A model for AGRP
binding to the MCRs remains to be developed.
Our previous characterization of AGRP (87-132) described a molecule
that consisted only of the COOH-terminal cysteine motif of AGRP (17).
Although this molecule contained no NH2-terminal amino
acids proximal to the first cysteine residue present in the AGRP
sequence (Cys-87), it retained full biological activity. Whereas it is
plausible to assume that the deletion of cysteine residues would
disrupt the AGRP tertiary structure by removing disulfide bonds and
would result in a molecule with markedly diminished functional
capabilities, those experiments did not test this point, and it cannot
definitely be said that AGRP (87-132) is a truly "minimized" AGRP molecule.
Based on the probable size of AGRP, we hypothesized that AGRP was
likely to bind to receptor domains other than or in addition to
transmembrane domains. Extracytoplasmic receptor domains constitute a
logical site that could be involved in AGRP binding. Seven
transmembrane receptors have four potential extracytoplasmic domains
including the NH2 terminus, first, second, and third
extracytoplasmic loops (exoloops) at which binding might occur.
Importantly, the NH2 terminus of the MCRs is not long,
unlike the glycoprotein, metabotropic glutamate, and ion-sensing
receptors in which lengthy NH2 termini have been found to
play a crucial role in ligand binding.
In the present experiments, we demonstrate that exoloops 2 and 3 of the
MC4R are crucial determinants of AGRP binding affinity and inhibitory
activity. Substitution of MC4R exoloops 2 and 3 with those homologous
exoloops of the MC1R resulted in a loss of AGRP binding affinity and
inhibitory activity, whereas placement of those MC4R exoloops into the
sequence of the MC1R led to the establishment of significant AGRP
activity at the MC1R. A comparison of the relative degree of change in
AGRP binding affinity and inhibitory activity displayed by the MCR
chimeras indicates that substitution of exoloop 3 induced more profound
effects than substitutions of exoloop 2. In this regard, it is
interesting to note that the amino acid sequence of exoloop 3 of the
MC1R and MC4R is longer than that of exoloop 2 and that exoloop 3 has a
greater degree of dissimilarity in amino acid side chain charge and
hydrophobicity than exoloop 2 (Fig. 1). Importantly, the observation
that similar receptor numbers were expressed suggests that receptor
expression is not the basis of the observed changes in cAMP assays.
Whereas the present studies have clearly identified receptor domains
(exoloops 2 and 3) that are involved in antagonist binding, they do not
conclusively reveal the mechanism underlying those changes. Inherent to
most mutagenesis studies is the inability to unequivocally discern
whether pharmacological effects result from a direct disruption of
atomic interactions involving the ligand and receptor or whether they
result from conformational changes imposed on the receptor that only
indirectly affect ligand binding. In other words, it is difficult to
state with absolute certainty that the changes in AGRP binding affinity
and inhibitory activity observed in these studies truly resulted from
the addition or removal of specific amino acids (present in the
exoloops) that have direct atomic interactions with the amino acids of
AGRP. Instead, it is possible that the observed pharmacological changes simply resulted from an alteration in chimeric receptor structure unrelated to the pharmacophore binding pocket that permitted or discouraged AGRP binding, despite being uncharacteristic of the wild-type receptor on which they were based.
Several observations favor an interpretation that specific sites of
interaction were identified in these studies. First, all chimeras
retained a 125I-NDP-MSH binding affinity similar to that
observed at the wild-type MCRs, and all chimeras were activated by
nanomolar concentrations of NDP-MSH. If the tertiary structure of the
chimeric receptors had been drastically altered, one would expect that
the ability of NDP-MSH to bind and activate the chimeric receptors
should have been altered. Second, exchange of exoloops between the MC1R and MC4R led not only to a loss of the ability of AGRP to bind chimeric
MCRs and inhibit NDP-MSH action (MC4R/MC1R chimeras) but also to a gain
in the functional ability of AGRP to bind chimeric MCRs and inhibit
NDP-MSH action (MC1R/MC4R chimeras).
Despite these salient points, the possibility that small, localized
conformational changes in the chimeric receptors are responsible for
the observed pharmacological effects lingers. However, regardless of
the distinction between direct and indirect causation, it is still
possible to conclude that the present studies have identified an
important intrinsic property of the MCRs residing in exoloops 2 and 3 that facilitates AGRP binding.
The present studies should not be interpreted to mean that the only
receptor domains involved in AGRP binding are exoloops 2 and 3. In
fact, the observation that exoloops 2 and 3 of the MC4R were not
sufficient to confer full AGRP binding affinity and inhibitory activity
to the MC1R (chimera MC1R/2e,3eMC4R) could be viewed as evidence that
other MCR domains also participate in AGRP binding. Similarly, removal
of exoloops 2 and 3 from the MC4R (MC4R/2e,3eMC1R) was not sufficient
to lead to a complete loss of AGRP (87-132) binding affinity and
inhibitory activity.
The studies reported herein primarily address the binding of AGRP to
the MCRs. Nonetheless, some additional comment and a specific word of
caution are warranted regarding the implications of these studies to
NDP-MSH binding. Existing literature is conflicting regarding the
contribution of exoloops to the binding of melanocortin agonists to the
MCRs. Chhajlani et al. (22) reported that mutating several
hydrophilic residues in the NH2 terminus and exoloops 1, 2, and 3 of the MC1R resulted in significant shifts in
125I-NDP-MSH binding. In contrast, Schiöth et
al. (23) performed mutagenesis studies on exoloop 2 of the human
melanocortin-3 receptor and concluded that this exoloop was not
involved in NDP-MSH binding. At the time of publication, neither of
these groups had the advantage of using radioligand
125I-AGRP (87-132), which is of a distinctly different
nature from 125I-NDP-MSH, to check the structural integrity
of their mutated receptors. Unfortunately, the present studies are not
capable of resolving the controversy over whether exoloops are
significantly involved in NDP-MSH binding. The fact that changing
exoloops did not alter NDP-MSH binding affinity or potency could
support the notion that exoloops are not critical to its binding. On
the other hand, because both the MC1R and MC4R bind NDP-MSH, any
binding determinants in exoloops 2 and 3 could simply have been
switched in the construction of the chimeras.
These data also make it tempting to hypothesize that a component of the
subtype specificity of the MCRs for Agouti resides in the second and
third receptor exoloops. It is not unreasonable to speculate that the
relative affinity of Agouti for selected MCR subtypes could be
increased or decreased by altering exoloops 2 and 3. Cassette
mutagenesis experiments could be used to address this question
(e.g. by exchanging the second and third exoloops between
the melanocortin-3 receptor or the melanocortin-5 receptor and the MC1R
and observing the increases or decreases in the affinity or inhibitory
activity of Agouti).
In summary, the present data have identified an important MCR
structural property involved in AGRP binding that resides in exoloops 2 and 3. Whereas these exoloops appear to have a preeminent effect on
AGRP binding, it will be important in the future to determine whether
other MCR domains are also involved in AGRP binding and to define the
relative importance of those domains compared with these exoloops.
 |
ACKNOWLEDGEMENTS |
We thank Dr. L. H. T. Van der
Ploeg and Dr. T. M. Fong for helpful discussions and critical
evaluation of this work.
 |
FOOTNOTES |
*
This work was supported by a Veterans Administration Merit
Review Award and National Institutes of Health Grant 1RO1 DK54032-01 (to I. G.), by National Institutes of Health Grant RO1 DK47398 (to
C. J. D.), and by funds from the University of Michigan
Gastrointestinal Peptide Research Center (National Institutes of Health
Grant P30DK34933).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: 6504 MSRB I, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0682. Tel.: 734-647-2942; Fax:
734-763-2535; E-mail: IGantz{at}UMich.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
MSH, melanocyte
stimulating hormone;
AGRP, Agouti-related protein;
MC4R, melanocortin-4
receptor;
MC1R, melanocortin-1 receptor;
MCR, melanocortin receptor;
PCR, polymerase chain reaction;
NDP-MSH, [Nle4,D-Phe7] MSH.
 |
REFERENCES |
-
Tatro, J. B.
(1996)
Neuroimmunomodulation
3,
259-284[Medline]
[Order article via Infotrieve]
-
Eberle, A. N.
(1988)
The Melanotropins: Chemistry, Physiology and Mechanisms of Action, pp. 210-319, Karger, Basel, Switzerland
-
Huszar, D.,
Lynch, C. A.,
Fairchild-Huntress, V.,
Dunmore, J. H.,
Fang, Q.,
Berkemeier, L. R.,
Gu, W.,
Kesterson, R. A.,
Boston, B. A.,
Cone, R. D.,
Smith, F. J.,
Campfield, L. A.,
Burn, P.,
and Lee, F.
(1997)
Cell
88,
131-141[Medline]
[Order article via Infotrieve]
-
Fan, W.,
Boston, B. A.,
Kesterson, R. A.,
Hruby, V. J.,
and Cone, R. D.
(1997)
Nature
385,
165-168[CrossRef][Medline]
[Order article via Infotrieve]
-
Mountjoy, K. G.,
Robbins, L. S.,
Mortrud, M. T.,
and Cone, R. D.
(1992)
Science
257,
1248-1251[Medline]
[Order article via Infotrieve]
-
Chhajlani, V.,
and Wikberg, J. E. S.
(1992)
FEBS Lett.
309,
417-420[CrossRef][Medline]
[Order article via Infotrieve]
-
Gantz, I.,
Konda, Y.,
Tashiro, T.,
Shimoto, Y.,
Miwa, H.,
Munzert, G.,
Watson, S. J.,
DelValle, J.,
and Yamada, T.
(1993)
J. Biol. Chem.
268,
8246-8250[Abstract/Free Full Text]
-
Gantz, I.,
Miwa, H.,
Konda, Y.,
Shimoto, Y.,
Tashiro, T.,
Watson, S. J.,
DelValle, J.,
and Yamada, T.
(1993)
J. Biol. Chem.
268,
15174-15179[Abstract/Free Full Text]
-
Gantz, I.,
Shimoto, Y.,
Konda, Y.,
Miwa, H.,
Dickinson, C. J.,
and Yamada, T.
(1994)
Biochem. Biophys. Res. Commun.
200,
1214-1220[CrossRef][Medline]
[Order article via Infotrieve]
-
Bultman, S. J.,
Michaud, E. J.,
and Woychik, R. P.
(1992)
Cell
71,
1195-1204[Medline]
[Order article via Infotrieve]
-
Lu, D.,
Willard, D.,
Patel, I. R.,
Kadwell, S.,
Overton, L.,
Kost, T.,
Luther, M.,
Chen, W.,
Woychik, R. P.,
Wilkison, W. O.,
and Cone, R. D.
(1994)
Nature
371,
799-802[CrossRef][Medline]
[Order article via Infotrieve]
-
Blanchard, S. G.,
Harris, C. O.,
Ittoop, O. R. R.,
Nichols, J. S.,
Parks, D. J.,
Truesdale, A. T.,
and Wilkison, W. O.
(1995)
Biochemistry
34,
10406-10411[Medline]
[Order article via Infotrieve]
-
Yang, Y.-K.,
Ollmann, M. M.,
Barsh, G. S.,
Yamada, T.,
and Gantz, I.
(1997)
Mol. Endocrinol.
11,
274-280[Abstract/Free Full Text]
-
Shutter, J. R.,
Graham, M.,
Kinsey, A. C.,
Scully, S.,
Lthy, R.,
and Stark, K. L.
(1997)
Genes Dev.
11,
593-602[Abstract]
-
Fong, T. M.,
Mao, C.,
MacNeil, T.,
Kalyani, R.,
Smith, T.,
Weinberg, D.,
Tota, M. R.,
and Van der Ploeg, L. H. T.
(1997)
Biochem. Biophys. Res. Commun.
237,
629-631[CrossRef][Medline]
[Order article via Infotrieve]
-
Ollmann, M. M.,
Wilson, B. D.,
Yang, Y.-K.,
Kerns, J. A.,
Chen, Y.,
Gantz, I.,
and Barsh, G. S.
(1997)
Science
278,
135-138[Abstract/Free Full Text]
-
Yang, Y.-K.,
Thompson, D.,
Dickinson, C. J.,
Wilken, J.,
Barsh, G. S.,
Kent, S. B. H.,
and Gantz, I.
(1999)
Mol. Endocrinol.
13,
148-155[Abstract/Free Full Text]
-
Baldwin, J. M.
(1993)
EMBO J.
12,
1693-1703[Abstract]
-
Haskell-Luevano, C.,
Sawyer, T. K.,
Trumpp-Kallmeyer, S.,
Bikker, J.,
Humblet, C.,
Gantz, I.,
and Hruby, V. J.
(1996)
Drug Des. Discov.
14,
197-211[Medline]
[Order article via Infotrieve]
-
Yang, Y.,
Dickinson, C.,
Haskell-Luevano, C.,
and Gantz, I.
(1997)
J. Biol. Chem.
272,
23000-23010[Abstract/Free Full Text]
-
Ji, T. H.,
Grossmann, M.,
and Ji, I.
(1998)
J. Biol. Chem.
273,
17299-17302[Free Full Text]
-
Chhajlani, V.,
Xu, X.,
Blauw, J.,
and Sudarshi, S.
(1996)
Biochem. Biophys. Res. Commun.
219,
521-525[CrossRef][Medline]
[Order article via Infotrieve]
-
Schiöth, H. B.,
Muceniece, R.,
Szardening, M.,
Prussi, P.,
and Wikberg, J. E. S.
(1996)
Biochem. Biophys. Res. Commun.
229,
687-692[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.