Download Characterization of Agouti-Related Protein

Characterization of Agouti-Related
Protein Binding to Melanocortin
Receptors
Ying-kui Yang, Darren A. Thompson, Chris J. Dickinson,
Jill Wilken, Greg S. Barsh, Stephen B. H. Kent, and Ira Gantz
Departments of Surgery (Y-k.Y., I.G.) and Pediatrics (C.J.D.)
University of Michigan Medical Center
Ann Arbor, Michigan 48109-0682
Howard Hughes Medical Institute (G.S.B.)
Stanford University School of Medicine
Stanford, California 94305
Gryphon Sciences (D.A.T., J.W., S.B.H.K.),
South San Francisco, California 94080
Agouti-related protein (AGRP) is a naturally occurring antagonist of melanocortin action that is
thought to play an important role in the hypothalamic control of feeding behavior. The exact mechanism of AGRP and Agouti protein action has been
difficult to examine, in part because of difficulties
in producing homogeneous forms of these molecules that can be used for direct binding assays. In
this report we describe the application of chemical
protein synthesis to the construction of two novel
AGRP variants. Examination of the biological activity of the AGRP variants demonstrates that a
truncated variant, human AGRP(87–132), a 46amino acid variant based on the carboxyl-terminal
cysteine-rich domain of AGRP, is equipotent to an
111-amino acid variant, mouse [Leu127Pro]AGRP
(mature AGRP minus its signal sequence), in its
ability to dose dependently inhibit a-MSH-generated cAMP generation at the cloned melanocortin
receptors. Furthermore, deletion of the amino-terminal portion of the full-length variant did not alter
the MCR subtype specificity of AGRP(87–132). Finally, iodination of human AGRP(87–132) provided
a useful reagent with which the binding properties
of AGRP could be analyzed. In both conventional
and photoemulsion binding studies [125I]AGRP(87–
132) was observed only to bind to cells expressing
melanocortin receptors MC3R, MC4R, and MC5R.
These results demonstrate that the residues critical for receptor binding, a-MSH inhibition, and
melanocortin receptor subtype specificity are all
located in the carboxyl terminus of the molecule.
Because [Nle4, D-Phe7] (NDP)-MSH displaces the
binding of [125I]AGRP(87–132) to MCRs and
AGRP(87–132) displaces the binding of [125I]NDP-
MSH, we conclude that these molecules bind in a
competitive fashion to melanocortin receptors.
(Molecular Endocrinology 13: 148–155, 1999)
INTRODUCTION
Agouti-related protein (AGRP) is a recently discovered
neuropeptide that has generated intense interest because a growing body of evidence indicates it has a
major role in the regulation of mammalian feeding
behavior (1, 2). AGRP was identified by virtue of its
sequence similarity to the product of the Agouti coat
color gene, a paracrine signaling molecule normally
expressed in skin whose transient expression during
hair growth leads to the barring of coat fur in rodents
(e.g. dark hair with a subapical yellow band) (3).
Ubiquitous expression of Agouti, which occurs in
mice that carry mutations in the 59-flanking region of
the Agouti gene, gives rise to pleiotropic effects including a yellow coat, obesity, insulin resistance, increased body length, and premature infertility (4). The
recent identification of AGRP indicates that the obesity
and diabetes caused by ectopic Agouti expression are
likely explained by its ability to mimic AGRP, since
ubiquitous expression of AGRP in transgenic mice
causes an increased weight gain and body length
phenotype identical to that produced by ubiquitous
expression of Agouti (2). Structurally, however, the
similarity between Agouti and AGRP is confined almost entirely to their 40-residue carboxyl termini
where a total of 20 residues, including 10 cysteine
residues, are identical (Fig. 1).
Both Agouti and AGRP have been shown to antagonize the action of melanocortin peptides such as
a-MSH and ACTH at specific melanocortin receptor
subtypes. Agouti potently antagonizes the action of
melanocortins at the melanocyte melanocortin recep-
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Molecular Endocrinology
Copyright © 1999 by The Endocrine Society
148
AGRP Binding
Fig. 1. Amino Acid Sequence Alignments of Mouse and Human AGRP and Mouse and Human Agouti Protein
Conserved C-terminal cysteine residues are enclosed in
boxes. The presumed signal sequence cleavage position is
denoted by , which also denotes the N terminus of mouse
[Leu127Pro]AGRP. 2 Denotes the start of the C-terminal
sequence that is AGRP(87–132). Note that the C-terminal
portion of mouse and human AGRP are identical except for
position 127 in the human sequence (126 of the mouse
sequence) where Pro is present in the human sequence and
Leu in the mouse sequence. m, Mouse; h, human.
tor (MC1R), adrenocortical ACTH receptor (MC2R),
and the MC4R (5, 6). In contrast, we recently demonstrated that AGRP primarily antagonizes the MC3R
and MC4R (2), each of which is expressed in areas of
the hypothalamus implicated in feeding behavior (7, 8).
Some or all of the growth and weight gain phenotypes
caused by Agouti or AGRP are likely mediated via the
MC4R, since mice carrying an MC4R knockout mutation display obesity and metabolic abnormalities similar to those caused by ubiquitous expression of
Agouti or AGRP (2, 9).
Several explanations have been proposed to explain
the mechanism of Agouti or AGRP action, including
simple competitive antagonism (10), inverse agonism
(11), or activation of an effector other than adenylate
cyclase (12). Distinguishing among these alternatives
has been complicated, in part, by the absence of
direct assays for Agouti or AGRP binding, since a
hallmark of competitive antagonism is the ability of
agonist to displace labeled antagonist. We have recently demonstrated that an epitope-tagged form of
Agouti protein interacts directly with the MC1R in an
overlay experiment, but these assays are not quantitative and so do not allow a comparison among different receptors or antagonists (13).
Our previous studies were carried out with preparations of recombinant AGRP that were partially purified
and heterogeneous in length. In contrast to recombinant techniques, chemical protein synthesis can be
used to make a homogeneous preparation of defined
molecular structure that can be chemically labeled and
derivatized and that can serve as a substrate for structure-function analyses. Here we describe the application of two novel chemically synthesized AGRP variants to studies directed at understanding the
mechanism of AGRP action. The first, AGRP(87–132),
is a 46-residue peptide containing five disulfide bonds
formed by folding the C-terminal portion of human
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AGRP. We demonstrate that radioiodinated AGRP(87–
132) can be used as a high-affinity tracer to directly
quantitate cell surface AGRP binding. The second
variant, mouse [Leu127Pro]AGRP, is a 111-amino acid
AGRP molecule (mature AGRP minus its 20-amino
acid signal sequence) that was made by joining the
N-terminal residues 21–85 of mouse AGRP to human
AGRP(87–132) by native chemical ligation (14). In
these studies we address three important issues: 1)
the bioactivity of the synthetic AGRPs; 2) the binding
properties of AGRP; and 3) the demonstration that
AGRP(87–132) maintains the activity of full-length
AGRP.
RESULTS
Effect of Mouse [Leu127Pro]AGRP and Human
AGRP(87–132) on a-MSH-Stimulated cAMP
Generation in Cell Lines Transfected with
Melanocortin Receptors
To help verify the biological activity of the chemically
synthesized proteins, we examined the ability of the
proteins to inhibit a-MSH-stimulated cAMP generation. We have shown previously that partially purified
recombinant human AGRP is a potent antagonist of
the hMC3R and hMC4R, but has little or no effect on
the hMC1R, hMC2R, or hMC5R. Figure 2, A–E, demonstrates that chemically synthesized mouse
[Leu127Pro]AGRP potently inhibits the action of
a-MSH at the hMC3R and hMC4R. With increasing
concentrations of mouse [Leu127Pro]AGRP, a progressive rightward shift of the a-MSH dose-response
curves is observed. Mouse [Leu127Pro]AGRP was
completely devoid of activity at the hMC1R and
hMC2R. However, at higher concentrations, mouse
[Leu127Pro]AGRP had a modest inhibitory effect on
a-MSH action at the hMC5R. Schild analysis performed by plotting a linear regression of the log concentration of AGRP (x-axis) and log (DR-1) (y-axis)
revealed a slope of 0.94 and 0.96 for mouse
[Leu127Pro]AGRP at the hMC3R and hMC4R, respectively (Fig. 2, D and E, insets) (15). Slopes approaching
unity indicate that mouse [Leu127Pro]AGRP has the
characteristics of a competitive antagonist of a-MSH
action at the hMC3R and hMC4R. Inhibitory constants
(Ki) for mouse [Leu127Pro]AGRP derived from this
Schild analysis revealed a Ki of 4.3 6 0.6 nM at
the hMC3R and a Ki of 2.5 6 0.25 nM at the hMC4R
(Table 1).
We next compared the pharmacological effects of
the 111-residue mouse [Leu127Pro]AGRP to those
displayed by the 46-residue AGRP(87–132) at the various hMCR subtypes. AGRP(87–132) had no effect at
hMC1R or hMC2R and only a minimal effect at the
hMC5R. Schild analysis revealed that AGRP(87–132),
like mouse [Leu127Pro]AGRP, is a competitive antagonist (data not shown) with Ki values for inhibition of
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Fig. 2. Mouse [Leu127Pro]AGRP Inhibition of a-MSH-Stimulated cAMP Generation at the Five Melanocortin Receptor Subtypes
(A–E)
Shown in F are the effects of recombinant AGRP Form A 1 B on a-MSH-stimulated cAMP generation at the hMC4R. Inset
graphs in D, E, and F represent Schild analysis linear regression plots. f, No AGRP; Œ, AGRP 1029 M; , AGRP 1028 M; l, AGRP
1027 M; F, AGRP 1026 M.
Table 1. Comparison of AGRP Ki and IC50 Values on hMC3R and hMC4R
hMC3R
Ki (nM)
Mouse [Leu 127Pro] AGRP
Human AGRP(87–132)
Recombinant Form A 1 B
IC50 (nM)
[125I]NDP-MSHa
[125I]AGRP(87–132)b
4.3 6 0.6
3.3 6 0.28
ND
17.4 6 3.7
11.2 6 3.1
hMC4R
2.5 6 0.25
2.6 6 0.21
1.2 6 0.17
15.7 6 4.1
9.0 6 1.7
ND, Not done.
a 125
[ I]NDP-MSH was displaced by mouse [Leu 127Pro] AGRP.
b 125
[ I]AGRP(87–132) was displaced by unlabeled AGRP(87–132).
a-MSH at the hMC3R and hMC4R of 3.3 6 0.28 nM
and 2.6 6 0.21 nM, respectively. We also examined the
effect of recombinant human AGRP Form A 1 B on
a-MSH-stimulated cAMP generation at the hMC4R
(Fig. 2F). Although the dose-response curves for recombinant human AGRP Form A 1 B were not parallel, a linear regression of the data revealed a slope of
0.94 and Ki of 1.2 6 0.17 nM (Fig. 2F, inset). In contrast
to chemically synthesized mouse [Leu127Pro]AGRP,
the Emax of a-MSH in the presence of recombinant
human AGRP Form A 1 B was about 10% below that
observed in the absence of this antagonist. Neither
chemically synthesized nor recombinant AGRP had an
effect on cAMP accumulation when applied to cells in
the absence of agonist.
[125I][Nle4, D-Phe7] (NDP)-MSH Binding to Cell
Lines Transfected with Melanocortin Receptors
Because the effects of AGRP on cAMP generation are
an indirect measure of antagonism, we measured the
ability of chemically synthesized or recombinant
AGRP to inhibit the binding of NDP-MSH to hMCRexpressing cells. Figure 3A reveals that chemically
synthesized mouse [Leu127Pro]AGRP dose dependently displaces [125I]NDP-MSH from the hMC3R,
hMC4R, and hMC5R. The displacement curve of
[125I]NDP-MSH from the hMC5R was shifted to the
right as compared with the hMC3R and hMC4R. No
significant displacement was observed at the hMC1R
(data not shown). These binding studies are consistent
AGRP Binding
151
Fig. 3. Displacement of Radioligand Binding from the Human Melanocortin Receptors Stably Expressed in HEK-293 Cells
A, Displacement of [125I]NDP-MSH binding from the hMCR 3, 4, and 5 by chemically synthesized mouse [Leu127Pro]AGRP.
B, Comparison of the displacement of [125I]NDP-MSH binding from the hMC4R by recombinant human AGRP Form A 1 B,
chemically synthesized mouse [Leu127Pro]AGRP, and AGRP(87–132). C, Displacement of [125I]AGRP(87–132) binding from the
hMCR 3, 4, and 5 by AGRP(87–132). D, Displacement of [125I]AGRP(87–132) binding from the hMCR 3, 4, and 5 by NDP-MSH.
[125I]AGRP(87–132) does not bind the hMC1R or hMC2R.
with the actions of mouse [Leu127Pro]AGRP and
AGRP(87–132) in the cAMP assays. IC50 values for
[125I]NDP-MSH displacement are hMC1R.1026 M,
hMC3R 5 17.4 6 3.7 nM, hMC4R 5 15.7 6 4.1 nM,
hMC5R 5 310.6 6 18.7 nM. Figure 3B compares the
ability of mouse [Leu127Pro]AGRP, AGRP(87–132),
and recombinant human AGRP Form A 1 B to displace [125I]NDP-MSH from the hMC4R. The displacement curves of baculovirus-produced human AGRP
Form A 1 B and chemically synthesized mouse
[Leu127Pro]AGRP are identical, while the curve of
chemically synthesized AGRP(87–132) is slightly
shifted to the left (3 times more potent). The IC50
for recombinant human AGRP Form A 1 B was 13.4 6
2.9 nM.
[125I]AGRP(87–132) Binding to Cell Lines
Transfected with Melanocortin Receptors
Previous biochemical studies of Agouti protein have
been complicated by the lack of a radiolabeled derivative. In contrast, AGRP has two tyrosine residues,
both of which are present in its carboxyl-terminal sequence. We were therefore able to take advantage of
the purity of chemically synthesized human AGRP(87–
132) to use standard oxidative chemistries to generate
a radiolabeled molecule that exhibited specific binding
to hMCR-expressing cells. In initial autoradiographic
experiments using slides coated with photoemulsion,
we asked whether tracer amounts of [125I]AGRP(87–
132) would bind to HEK 293 cells that expressed
equivalent levels of the different melanocortin receptors (;2.5 3 105 per well). As shown in Fig. 4,
[125I]AGRP(87–132) only binds to cells expressing the
hMC3R, hMC4R, and hMC5R. No specific radioligand
binding was observed in wild-type cells or at the
hMC1R or hMC2R (Fig. 4 and data not shown). The
intensity of the binding studies is consistent with the
rank order of AGRP inhibition noted in our functional
studies (hMC4R 5 hMC3R . hMC5R).
As a quantitative measure of binding, we examined
the ability of unlabeled AGRP(87–132) to displace
[125I]AGRP(87–132) from the hMC3R, hMC4R, and
hMC5R-expressing cells (Fig. 3C). Notably, the displacement curves of [125I]AGRP(87–132) from the
hMC3R and hMC4R are overlapping, which is consistent with our previous functional and photoemulsion
studies and indicate that AGRP(87–132) is essentially
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Fig. 4. Representative Photomicrographs of [125I]AGRP(87–132) Binding to HEK 293 Cells Transfected with hMCR 3, 4, and 5
Under bright field illumination (right) cells are seen as outlines on a light background. Under dark field illumination (left) the
identical cells are seen. Under dark field, cells binding [125I]AGRP(87–132) are seen as white elements in a surrounding dark
background. Because of the absence of binding, wild-type cells only appear as faint outlines. No binding was observed in similar
experiments with cells expressing the hMC1R and hMC2R.
equipotent at the two receptor subtypes. The IC50
values of [125I]AGRP(87–132) displacement by
AGRP(87–132) at the hMC3R 5 11.2 6 3.1 nM,
hMC4R 5 9.0 6 1.7 nM, and hMC5R 5 25.6 6 4.3 nM.
A hallmark of competitive binding is the ability of one
ligand to displace the other, and vice versa. As indicated
in Fig. 3A, AGRP(87–132) can displace [125I]NDP-MSH
from the hMC3R, hMC4R, and hMC5R. The converse is
also true as shown in Fig. 3D, which examines the ability
of NDP-MSH to displace [125I]AGRP(87–132). The IC50
values of [125I]AGRP(87–132) displacement by NDPMSH are as follows: at the hMC3R 5 1.9 6 0.15 nM;
hMC4R 5 3.75 6 0.1 nM; and hMC5R 5 11.2 6 2.1 nM.
The [125I]NDP-MSH and [125I]AGRP(87–132) displacement data and the cAMP data reveal a hierarchy of
melanocortin receptor subtype sensitivity to AGRP such
that hMC3R 5 hMC4R . hMC5R.
DISCUSSION
The identification of AGRP has added additional complexity to our attempts to understand weight ho-
meostasis. Recent pharmacological and anatomical
data have further strengthened the link between melanocortins and weight control and indicate that melanocortins act downstream of the fat hormone leptin
(16–19). Levels of AGRP mRNA exhibit up to 10-fold
alterations in different obesity models (1, 2) and, therefore, as a naturally occurring orexigenic agent that
antagonizes melanocortins, AGRP may represent a
unique target for antiobesity drug development.
While our previous studies of the action of baculovirus-produced AGRP allowed us to determine some
aspects of this regulatory protein’s action, our inability
to produce highly purified product led us to consider
an alternative approach. Chemical protein synthesis
uses native chemical ligation of unprotected synthetic
peptide segments in aqueous solution, followed by
folding/disulfide formation to give the functional protein molecule (14). Our present experiments demonstrate that the techniques of chemical protein synthesis can be used to rapidly produce highly purified,
biologically active AGRP molecules in amounts of tens
of milligrams in a convenient and straightforward fashion. In the present correspondence we capitalized on
AGRP Binding
the use of these synthetic techniques to build upon our
previous observations of AGRP. The availability of
highly purified AGRP protein variants allowed us not
only to readily develop an AGRP radioligand with
which the site of AGRP action could be explored, but
also enabled us to perform more detailed pharmacological analysis of this molecule.
Both chemically synthesized mouse [Leu127Pro]
AGRP and AGRP(87–132) have similar inhibitory potency
and efficacy as baculovirus-produced human AGRP
Form A 1 B. In cAMP assays [Leu127Pro] AGRP,
AGRP(87–132), and recombinant human AGRP Form A
1 B are essentially equipotent at inhibiting the hMC4R.
Both chemically synthesized variant AGRP molecules
were also found to display a similar nanomolar range of
activity as previously observed for human recombinant
Form A 1 B at the hMC3R (2). Like recombinant human
Form A 1 B, both chemically synthesized AGRP variants
had only minimal activity at the hMC5R, and neither
displayed any inhibitory activity at the hMC1R or the
hMC2R. AGRP(87–132) was only slightly more potent
than either longer synthetic or recombinant forms of
AGRP in displacing [125I]NDP-MSH from the hMC4R.
Having demonstrated the biological activity of the
chemically synthesized AGRP variants, we used
AGRP(87–132) to further study the actions of this protein. Our ability to radiolabel AGRP(87–132) with 125I
allowed us to directly study AGRP(87–132) binding.
[125I]AGRP(87–132) bound only to those heterologous
cell lines expressing melanocortin receptor subtypes
susceptible to AGRP inhibition in cAMP assays and at
which [125I]NDP-MSH was displaced by mouse
[Leu127Pro]AGRP (Fig. 3). Typical displacement
curves appear to indicate that the iodination process
did not alter the biological activity of AGRP(87–132).
The finding that the displacement of [125I]AGRP(87–
132) from the hMC5R was shifted to the right is consistent with the decreased potency of AGRP at this
receptor subtype observed in cAMP assays.
A persistent controversy that has existed regarding
the action of Agouti protein is whether it has effects
independent of its antagonism of a-MSH (11–13).
Much of this speculation is based on the sequence
similarity between agouti protein, and cone snail
(conotoxins) and spider (plectoxins) toxins. These toxins, which affect calcium channels, contain a cysteinerich motif that can be closely aligned against 10 cysteine residues present in the C terminus of both Agouti
and AGRP (Fig. 1). While some of the effects of Agouti
in the absence of a-MSH may be explained by its
ability to act as inverse agonist, it has been suggested
that a separate agouti receptor may exist (20, 21). This
controversy has been approached by examining the
action of Agouti on melanoma cell lines lacking the
MC1R (13). More recently, epitope-tagged Agouti has
been used (22). However, this matter has been somewhat difficult to study since a radiolabeled Agouti has
not been developed. Because of this controversy we
used the novel radioligand [125I]AGRP(87–132) to examine the binding sites of AGRP. Both conventional
153
binding studies and photoemulsion studies indicate
that [125I]AGRP(87–132) only binds to melanocortin
receptors demonstrated to be susceptible to AGRP
inhibition in cAMP assays. This does not, however,
exclude the possibility that an endogenous cell type
that expresses a native hMC3R, hMC4R, or hMC5R
may also possess additional binding sites.
The competitive pattern of AGRP inhibition of melanocortins binding to the MC3R and MC4R observed in
the present studies does not necessarily imply that
AGRP and melanocortin agonist occupy the same site
on the receptor. It is possible that the two ligands
simply influence each other’s binding through an allosteric mechanism. In fact, there is no significant sequence similarity between melanocortins and AGRP,
although this does not exclude some similarity on the
basis of three-dimensional structure. Future receptor
mutagenesis studies using our novel radioligand
[125I]AGRP(87–132) and the radioligand [125I]NDPMSH should be helpful in this respect.
Although AGRP(87–132) was approximately 3-fold
more potent than mouse [Lue127Pro] AGRP in its ability
to displace [125I]MSH-MSH from hMC4R-expressing
cells, the antagonists were equipotent in their ability to
inhibit a-MSH-induced cAMP accumulation mediated by
the hMC4R. Regardless of the reasons for this apparent
difference, these results indicate that the structural determinants for both MCR binding and melanocortin antagonism are located within the cysteine-rich C-terminal
domain. Furthermore, AGRP(87–132) retains the pattern
of melanocortin receptor selectivity displayed by the fulllength molecule. Further truncation and other manipulations of human AGRP(87–132) will help identify its minimally active form, and modification of residues within this
fragment should provide insight into the determinants of
receptor subtype selectivity.
In summary, these studies demonstrate the ability to
chemically synthesize biologically active AGRP variants. These studies also demonstrate that AGRP(87–
132), a variant lacking the N terminus of AGRP and
consisting of only the C-terminal cysteine-rich AGRP
module, retains the biological activity of full-length
AGRP. Finally, these studies describe the AGRP radioligand, [125I]AGRP(87–132), and demonstrate the
binding of this radioligand directly to melanocortin
receptor protein. [125I]AGRP(87–132) should be a helpful tool for anatomical studies of the natural sites of
AGRP binding, development of an AGRP RIA, and
identification of small molecule antagonists of AGRP
interaction with the melanocortin receptors. The latter
compounds could have potential applications as regulators of human feeding behavior.
MATERIALS AND METHODS
Mouse [Leu127Pro]AGRP and AGRP(87–132) Synthesis
Peptides were synthesized by Boc chemistry using manual
stepwise solid-phase peptide synthesis as previously de-
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scribed (23). The 46-amino acid polypeptide corresponding
to the C-terminal module, human AGRP(87–132), was assembled on Thr-OCH2-Pam-resin (Perkin-Elmer Applied Biosystems, Foster City, CA). The N-terminal basic segment,
mouse AGRP 21–85, was assembled on a thioester resin.
Peptides were cleaved from the resin with hydrogen fluoride
containing 5–10% p-cresol (Fluka, Buchs, Switzerland) for 1 h
at 0 C, lyophilized, and then purified using reversed phase
HPLC on C4 columns (Vydac, Murrieta, CA) with water (0.1%
trifluoroacetic acid)/acetonitrile (0.1% trifluoroacetic acid)
gradients. The molecular weights of these peptides were
confirmed by electrospray ionization mass spectrometry
(Perkin-Elmer SCIEX, Foster City, CA). To generate the fulllength construct, purified mouse AGRP(21–85) thioester and
human AGRP(87–132) were dissolved in 6 M guanidine hydrochloride and 200 mM phosphate (pH 7.0) containing 1%
thiophenol at a concentration of 2–4 mM and stirred overnight. Under these conditions native chemical ligation
joined the two peptides to form full-length mouse
[Leu127Pro]AGRP(21–131), appearing as a new peak on analytical HPLC with mol wt indicative of segment condensation by peptide bond formation [observed: 12,397.4 6 1.50;
calculated: 12,394.5 (average isotopes)]. Ligated peptides
were then fully reduced by incubating 1 h with 20% b-mercaptoethanol, purified by HPLC, and lyophylized [24 mg
mouse AGRP(21–85)] thioester 1 16.9 mg human AGRP(87–
132) yielded 14.3 mg mouse [Leu127Pro]AGRP. Protein folding of human AGRP(87–132) and mouse [Leu127Pro]AGRP
was initiated by dissolving the lyophilized peptide in a solution of 2 M guanidine hydrochloride and 100 mM Tris (pH 8.0)
containing 8 mM cysteine and 1 mM cystine (Fluka), and
stirring overnight. The folded proteins were then
purified by HPLC and lyophilized. Human AGRP(87–132)
(138.2 mg) (reduced) yielded 52.5 mg AGRP(87–132) (oxidized); 14.3 mg [Leu127Pro]AGRP (reduced) yielded 4.7 mg
[Leu127Pro]AGRP (oxidized). Two-dimensional nuclear magnetic resonance studies of AGRP(87–132) confirmed the existence of a single homogeneous folded state (K. Bolin, J.
Trulson, and G. L. Millhauser, unpublished results). This observation was supported by the formation of a sharp peak on
analytical reverse phase HPLC eluting earlier than the reduced state, and the loss of 10 mass units by electrospray
ionization mass spectrometry, which is consistent with the
formation of five disulfides in the oxidized form (AGRP(87–
132) observed: 5,191.1 6 1.05; calculated: 5,191.2 (average
isotopes); mouse [Leu127Pro]AGRP observed: 12,384.9 6
1.11; calculated: 12,383.5 (average isotopes).
Baculovirus-produced recombinant human AGRP Form A
1 B was produced and partially purified as previously described (2, 24). Form A 1 B refers to nonhomogeneous
fractions of recombinant AGRP that run closely together on
Western blot (2). Form A consists of mature AGRP minus its
signal sequence of 20 amino acids, and Form B contains
several fragments cleaved after residues 46, 48, or 50.
cAMP Assays
cAMP generation was measured using a competitive binding
assay kit (TRK 432, Amersham, Arlington Heights, IL) according to a standardized protocol (6). Heterologous cell lines
stably expressing the human (h) melanocortin receptors that
have been previously described were used in these assays
(6). For assays, culture media were removed and cells were
incubated with 0.5 ml Earle’s balanced salt solution that
contained AGRP and melanocortin agonist for 30 min at 37 C
in the presence of 1023 M isobutylmethylxanthine. The reaction was stopped by adding ice-cold 100% ethanol (500
ml/well). The cells in each well were scraped and transferred
to a 1.5-ml tube and centrifuged for 10 min at 1900 3 g, and
the supernatant was evaporated in a 55 C water bath with
prepurified nitrogen gas. cAMP content was measured according to instructions accompanying the assay kit. a-MSH
and human ACTH 1–39 were obtained from Peninsula Lab-
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oratories, Inc. (Belmont, CA). Each experiment was performed a minimum of three times with duplicate wells. The
mean value of the dose-response data were fit to a sigmoid
curve with a variable slope factor using the nonlinear squares
regression in Graphpad Prism (Graphpad Software, San
Diego, CA). EC50 values determined from these fits were used
for plotting Schild analysis linear regressions. pA2 values
were derived from the y 5 0 intercept of the Schild plot of the
log of dose ratio minus one (log DR-1) as previously described (6). Ki values were determined as the negative log of
the pA2. All statistical analyses represent the mean of the
data 6 SE.
Radioiodination
NDP-MSH, a long acting superpotent melanocortin agonist,
was obtained from Peninsula Laboratories, Inc. (Belmont, CA)
(25). [125I]NDP-MSH and [125I]AGRP(87–132) were prepared
by a modification of a chloramine-T method previously described (26). 125I-labeled Na (0.5 mCi) (Amersham) was added
to 20 mg of either NDP-MSH or AGRP(87–132) in 5 ml of 50
mM sodium phosphate buffer (pH 7.4). Ten microliters of a 2.4
mg/ml solution of chloramine T (Sigma Chemical Co., St.
Louis, MO) in 50 mM sodium phosphate (pH 7.4) were added
for 15 sec, and the reaction was stopped with 50 ml of a 4.8
mg/ml solution of sodium metabisulfite (Sigma). The reaction
mixture was then diluted in 800 ml of 50 mM ammonium
acetate (pH 5.8) and purified by reverse phase chromatography. BSA (100 ml of a 2% solution) was added to all fractions containing radioactivity.
Binding Experiments
After removal of media the cells were washed twice with
MEM and then preincubated with AGRP in 0.5 ml MEM (Life
Technologies, Gaithersburg, MD) containing 0.2% BSA for 30
min before incubation with radioligand. Binding experiments
were performed using conditions previously described (6).
[125I]NDP-MSH (3 3 105 cpm; ;61 fmol) or 3 3 105 cpm
125
I-AGRP(87–132) (;55 fmol) were used. Binding reactions
were terminated by removing the media and washing the
cells twice with MEM containing 0.2% BSA. The cells were
lysed with 0.1 N NaOH 1% Triton X-100, and the radioactivity
in the lysate was quantified in an analytical g-counter. Nonspecific binding was determined by measuring the amount of
125
I-label remaining bound in the presence of 1025 M unlabeled ligand, and specific binding was calculated by subtracting nonspecifically bound radioactivity from total bound
radioactivity. Typically, total binding of [125I]AGRP(87–132)
was about 13.5 6 1.3 3 104 cpm, and nonspecific binding
was 3.0 6 0.4 3 103 cpm. For photoemulsion studies, the
binding assays were performed directly on a chambered
microscope slides (SlideFlask, NUNC, Roskilde, Denmark).
Approximately 105 cells were placed on each slide and allowed to grow for 12 h. After binding experiments were
performed, slides were fixed with gluteraldehyde and dried.
Slides were then dipped in Kodak NTB2 photoemulsion
(Eastman Kodak Co., New Haven, CT) and exposed for 3
days before being developed, examined, and photographed
using a Leica DMRB microscope (Leica, Inc., Deerfield, IL).
Acknowledgments
Received July 7, 1998. Revision received September 15,
1998. Accepted September 18, 1998.
Address requests for reprints to: Ira Gantz, M.D., 6504
MSRB I, 1150 West Medical Center Drive, Ann Arbor,
Michigan 48109-0682. E-mail: [email protected].
This work was supported by a Veterans Administration
Merit Review Award (I.G.), funds from the University of Michigan Gastrointestinal Peptide Research Center (NIH Grant
AGRP Binding
P30 DK-34933), RO1 DK-47398 (C.J.D.), and RO1 DK-28506
(G.S.B). G.S.B is an Associate Investigator of the Howard
Hughes Medical Institute.
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