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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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-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,
-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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Ubiquitous expression of Agouti, which occurs in mice that carry
mutations in the 5'-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
).
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-MSH and ACTH at specific melanocortin
receptor subtypes. Agouti potently antagonizes the action of
melanocortins at the melanocyte melanocortin receptor (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 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-MSH-Stimulated cAMP Generation in Cell Lines Transfected with
Melanocortin Receptors-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 -MSH at the hMC3R and hMC4R. With
increasing concentrations of mouse [Leu127Pro]AGRP, a progressive
rightward shift of the -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 -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 -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 ± 0.6 nM at the hMC3R and
a Ki of 2.5 ± 0.25 nM at the hMC4R (Table 1).
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-MSH at the hMC3R and hMC4R of 3.3 ± 0.28 nM
and 2.6 ± 0.21 nM, respectively. We also examined
the effect of recombinant human AGRP Form A + B on -MSH-stimulated
cAMP generation at the hMC4R (Fig. 2F
). Although the dose-response
curves for recombinant human AGRP Form A + B were not parallel, a
linear regression of the data revealed a slope of 0.94 and
Ki of 1.2 ± 0.17 nM (Fig. 2F, inset). In contrast to chemically synthesized mouse
[Leu127Pro]AGRP, the Emax of -MSH in the presence of
recombinant human AGRP Form A + 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
hMCR-expressing 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 with the actions of mouse
[Leu127Pro]AGRP and AGRP(87–132) in the cAMP assays.
IC50 values for [125I]NDP-MSH displacement
are hMC1R>10-6 M, hMC3R = 17.4 ±
3.7 nM, hMC4R = 15.7 ± 4.1 nM,
hMC5R = 310.6 ± 18.7 nM. Figure 3B compares the ability of mouse
[Leu127Pro]AGRP, AGRP(87–132), and recombinant human AGRP Form A + B
to displace [125I]NDP-MSH from the hMC4R. The
displacement curves of baculovirus-produced human AGRP Form A + 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 + B was 13.4 ± 2.9
nM.
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2.5 x 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 = hMC3R >
hMC5R).
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). 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 equipotent at the two receptor subtypes. The
IC50 values of [125I]AGRP(87–132)
displacement by AGRP(87–132) at the hMC3R = 11.2 ± 3.1
nM, hMC4R = 9.0 ± 1.7 nM, and
hMC5R = 25.6 ± 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 NDP-MSH are as
follows: at the hMC3R = 1.9 ± 0.15 nM;
hMC4R = 3.75 ± 0.1 nM; and hMC5R =
11.2 ± 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 = hMC4R > hMC5R.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 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 + B. In cAMP assays [Leu127Pro] AGRP, AGRP(87–132), and recombinant human AGRP Form A + 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 + B at the hMC3R (2). Like recombinant human Form A + 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 -MSH (11, 12, 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 cysteine-rich 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 -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 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]NDP-MSH 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 -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 full-length 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Baculovirus-produced recombinant human AGRP Form A + B was produced and partially purified as previously described (2, 24). Form A + 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 10-3 M
isobutylmethylxanthine. The reaction was stopped by adding ice-cold
100% ethanol (500 µl/well). The cells in each well were scraped and
transferred to a 1.5-ml tube and centrifuged for 10 min at 1900 x
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. -MSH and human ACTH 1–39
were obtained from Peninsula Laboratories, 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 = 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 ±
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 µg of either NDP-MSH or AGRP(87–132) in 5 µl 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 µl of a 4.8 mg/ml solution of
sodium metabisulfite (Sigma). The reaction mixture was then diluted in
800 µl of 50 mM ammonium acetate (pH 5.8) and purified by
reverse phase chromatography. BSA (100 µl 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 x
105 cpm; 61 fmol) or 3 x 105 cpm
125I-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 
-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 calculated by subtracting
nonspecifically bound radioactivity from total bound radioactivity.
Typically, total binding of [125I]AGRP(87–132) was about
13.5 ± 1.3 x 104 cpm, and nonspecific binding
was 3.0 ± 0.4 x 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).
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FOOTNOTES |
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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 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.
Received for publication July 7, 1998. Revision received September 15, 1998. Accepted for publication September 18, 1998.
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REFERENCES |
|---|
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-melanocyte stimulating hormone
receptor antagonist. Biochemistry 34:12341–12346[CrossRef][Medline]
-melanotropin and agouti on B16 melanoma
cells: evidence for inverse agonism of agouti. J Recept Signal
Transduct Res 17:75–98[Medline]
-melanotropin. Science 213:1025–1027This article has been cited by other articles:
 |
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
 |
|
 M. A. Smith, K. Hisadome, H. Al-Qassab, H. Heffron, D. J. Withers, and M. L. J. Ashford Melanocortins and agouti-related protein modulate the excitability of two arcuate nucleus neuron populations by alteration of resting potassium conductances J. Physiol., January 15, 2007; 578(2): 425 - 438. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Aja, S. Bi, S. B. Knipp, J. M. McFadden, G. V. Ronnett, F. P. Kuhajda, and T. H. Moran Intracerebroventricular C75 decreases meal frequency and reduces AgRP gene expression in rats Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2006; 291(1): R148 - R154. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. W. M. Creemers, L. E. Pritchard, A. Gyte, P. Le Rouzic, S. Meulemans, S. L. Wardlaw, X. Zhu, D. F. Steiner, N. Davies, D. Armstrong, et al. Agouti-Related Protein Is Posttranslationally Cleaved by Proprotein Convertase 1 to Generate Agouti-Related Protein (AGRP)83-132: Interaction between AGRP83-132 and Melanocortin Receptors Cannot Be Influenced by Syndecan-3 Endocrinology, April 1, 2006; 147(4): 1621 - 1631. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Pan, A. J. Kastin, Y. Yu, C. M. Cain, T. Fairburn, A. M. Stutz, C. Morrison, and G. Argyropoulos Selective Tissue Uptake of Agouti-Related Protein(82-131) and Its Modulation by Fasting Endocrinology, December 1, 2005; 146(12): 5533 - 5539. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. S. Tallam, J. J. Kuo, A. A. da Silva, and J. E. Hall Cardiovascular, Renal, and Metabolic Responses to Chronic Central Administration of Agouti-Related Peptide Hypertension, December 1, 2004; 44(6): 853 - 858. [Abstract] [Full Text] [PDF]
|
|
|
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|
|
L. Huo, H. Munzberg, E. A. Nillni, and C. Bjorbaek Role of Signal Transducer and Activator of Transcription 3 in Regulation of Hypothalamic trh Gene Expression by Leptin Endocrinology, May 1, 2004; 145(5): 2516 - 2523. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Doghman, P. Delagrange, A. Blondet, M.-C. Berthelon, P. Durand, D. Naville, and M. Begeot Agouti-Related Protein Antagonizes Glucocorticoid Production Induced through Melanocortin 4 Receptor Activation in Bovine Adrenal Cells: A Possible Autocrine Control Endocrinology, February 1, 2004; 145(2): 541 - 547. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Yang, M. Chen, Y. Lai, I. Gantz, A. Yagmurlu, K. E. Georgeson, and C. M. Harmon Molecular Determination of Agouti-Related Protein Binding to Human Melanocortin-4 Receptor Mol. Pharmacol., July 1, 2003; 64(1): 94 - 103. [Abstract] [Full Text] [PDF]
|
|
|
|
|
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W. A. J. Nijenhuis, K. M. Garner, R. J. van Rozen, and R. A. H. Adan Poor Cell Surface Expression of Human Melanocortin-4 Receptor Mutations Associated with Obesity J. Biol. Chem., June 13, 2003; 278(25): 22939 - 22945. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. S.H. Yeo, E. J. Lank, I. S. Farooqi, J. Keogh, B. G. Challis, and S. O'Rahilly Mutations in the human melanocortin-4 receptor gene associated with severe familial obesity disrupts receptor function through multiple molecular mechanisms Hum. Mol. Genet., March 1, 2003; 12(5): 561 - 574. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Nickolls, M. I. Cismowski, X. Wang, M. Wolff, P. J. Conlon, and R. A. Maki Molecular Determinants of Melanocortin 4 Receptor Ligand Binding and MC4/MC3 Receptor Selectivity J. Pharmacol. Exp. Ther., March 1, 2003; 304(3): 1217 - 1227. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Zheng, M. M. Corkern, S. M. Crousillac, L. M. Patterson, C. B. Phifer, and H.-R. Berthoud Neurochemical phenotype of hypothalamic neurons showing Fos expression 23 h after intracranial AgRP Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2002; 282(6): R1773 - R1781. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-K. Yang, C. Dickinson, Y.-M. Lai, J.-Y. Li, and I. Gantz Functional properties of an agouti signaling protein variant and characteristics of its cognate radioligand Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2001; 281(6): R1877 - R1886. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Lechan and J. B. Tatro Editorial: Hypothalamic Melanocortin Signaling in Cachexia Endocrinology, August 1, 2001; 142(8): 3288 - 3291. [Full Text] [PDF]
|
|
|
|
|
|
J. J. Hwa, L. Ghibaudi, J. Gao, and E. M. Parker Central melanocortin system modulates energy intake and expenditure of obese and lean Zucker rats Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2001; 281(2): R444 - R451. [Abstract] [Full Text] [PDF]
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T. M. Gunn, T. Inui, K. Kitada, S. Ito, K. Wakamatsu, L. He, D. M. Bouley, T. Serikawa, and G. S. Barsh Molecular and Phenotypic Analysis of Attractin Mutant Mice Genetics, August 1, 2001; 158(4): 1683 - 1695. [Abstract] [Full Text] [PDF]
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 E. H. Hani, S. Dupont, E. Durand, C. Dina, S. Gallina, I. Gantz, and P. Froguel Naturally Occurring Mutations in the Melanocortin Receptor 3 Gene Are Not Associated with Type 2 Diabetes Mellitus in French Caucasians J. Clin. Endocrinol. Metab., June 1, 2001; 86(6): 2895 - 2898. [Abstract] [Full Text] [PDF]
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A. Katsuki, Y. Sumida, E. C. Gabazza, S. Murashima, T. Tanaka, M. Furuta, R. Araki-Sasaki, Y. Hori, K. Nakatani, Y. Yano, et al. Plasma Levels of Agouti-Related Protein Are Increased in Obese Men J. Clin. Endocrinol. Metab., May 1, 2001; 86(5): 1921 - 1924. [Abstract] [Full Text]
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 W. A. J. Nijenhuis, J. Oosterom, and R. A. H. Adan AgRP(83-132) Acts as an Inverse Agonist on the Human-Melanocortin-4 Receptor Mol. Endocrinol., January 1, 2001; 15(1): 164 - 171. [Abstract] [Full Text]
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 A. Slominski, J. Wortsman, T. Luger, R. Paus, and S. Solomon Corticotropin Releasing Hormone and Proopiomelanocortin Involvement in the Cutaneous Response to Stress Physiol Rev, July 1, 2000; 80(3): 979 - 1020. [Abstract] [Full Text] [PDF]
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M. M. Hagan, P. A. Rushing, L. M. Pritchard, M. W. Schwartz, A. M. Strack, L. H. T. Van der Ploeg, S. C. Woods, and R. J. Seeley Long-term orexigenic effects of AgRP-(83---132) involve mechanisms other than melanocortin receptor blockade Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2000; 279(1): R47 - R52. [Abstract] [Full Text] [PDF]
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J.-Y. Li, S. Finniss, Y.-K. Yang, Q. Zeng, S.-Y. Qu, G. Barsh, C. Dickinson, and I. Gantz Agouti-Related Protein-Like Immunoreactivity: Characterization of Release from Hypothalamic Tissue and Presence in Serum Endocrinology, June 1, 2000; 141(6): 1942 - 1950. [Abstract] [Full Text] [PDF]
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D. M. Dinulescu and R. D. Cone Agouti and Agouti-related Protein: Analogies and Contrasts J. Biol. Chem., March 15, 2000; 275(10): 6695 - 6698. [Abstract] [Full Text] [PDF]
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 A. Inui Transgenic Approach to the Study of Body Weight Regulation Pharmacol. Rev., March 1, 2000; 52(1): 35 - 62. [Abstract] [Full Text] [PDF]
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Y.-k. Yang, C. J. Dickinson, Q. Zeng, J.-Y. Li, D. A. Thompson, and I. Gantz Contribution of Melanocortin Receptor Exoloops to Agouti-related Protein Binding J. Biol. Chem., May 14, 1999; 274(20): 14100 - 14106. [Abstract] [Full Text] [PDF]
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J. Oosterom, K. M. Garner, W. K. den Dekker, W. A. J. Nijenhuis, W. H. Gispen, J. P. H. Burbach, G. S. Barsh, and R. A. H. Adan Common Requirements for Melanocortin-4 Receptor Selectivity of Structurally Unrelated Melanocortin Agonist and Endogenous Antagonist, Agouti Protein J. Biol. Chem., January 5, 2001; 276(2): 931 - 936. [Abstract] [Full Text] [PDF]
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, which also denotes the N terminus of mouse
[Leu127Pro]AGRP. 
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.
, No AGRP; 
, AGRP 10-9
M; 
, AGRP
10-7 M; •, AGRP 10-6
M.
