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Departments of Internal Medicine (Y-K.Y., T.Y.), Pediatrics (C.D.),
Physiology (T.Y.), and Surgery (I.G.) University of Michigan
Ann Arbor, Michigan 48109-0682
Howard Hughes Medical
Institute and The Departments of Pediatrics and Genetics Stanford
University (M.M.O., B.D.W., G.S.B.) Stanford, California
94305-5428
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-MSH or ACTH at the five known human melanocortin receptor subtypes
(hMCR 1–5). When stably expressed in L cells (hMC1R, hMC3R, hMC4R,
hMC5R) or in the adrenocortical cell line OS3 (hMC1R, hMC2R, hMC4R),
purified recombinant ASIP inhibits the generation of cAMP stimulated by
-MSH (hMC1R, hMC3R, hMC4R, hMC5R) or by ACTH (hMC2R). However,
dose-response and Schild analysis indicated that the degree of ASIP
inhibition varied significantly among the receptor subtypes; ASIP is a
potent inhibitor of the hMC1R, hMC2R, and hMC4R, but has relatively
weak effects at the hMC3R and hMC5R. These analyses also indicated that
the apparent mechanism of ASIP antagonism varied among receptor
subtypes, with characteristics consistent with competitive antagonism
observed only at the hMC1R, and more complex behavior observed at the
other receptors. ASIP inhibition at these latter receptors,
nonetheless, can be classified as surmountable (hMC3R, hMC4R and hMC5R)
or nonsurmountable (hMC2R). Recombinant ASIP also inhibited binding of
radiolabeled melanocortins,
[125I-Nle4,
D-Phe7]-MSH and
[125I-Phe2,
Nle4]ACTH 1–24, to the hMCR 1–5 receptors,
with a relative efficacy that paralleled the ability of ASIP to inhibit
cAMP accumulation at the hMC1R, hMC2R, hMC3R, and hMC4R. These results
provide new insight into the biochemical mechanism of ASIP action and
suggest that ASIP may play an important role in modulating melanocortin
signaling in humans. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The effects of agouti on fur color require the presence of a functional
melanocortin 1 receptor (MC1R), a G protein-coupled receptor in which
activating or loss-of-function mutations result in constitutive
synthesis of eumelanin or pheomelanin, respectively. The MC1R can be
activated by several peptide hormones derived from POMC, including
-MSH and ACTH. These peptides are produced at very high levels in
the brain and pituitary gland, but the role of melanocortins in the
normal control of pigmentation is uncertain. By contrast, activation of
the adrenal-specific MC2R by circulating ACTH is the major stimulus for
cortisol production, and activation of the MC3R and MC4R in the central
nervous system is thought to account for effects of melanocortins on
learning, behavior, and cardiovascular regulation.
Insight into the mechanism by which agouti exerts its control over coat
color has come from studies of Lu et al. (5), in which
recombinant mouse agouti protein was shown to antagonize the effects of
-MSH on heterologous cells that expressed the melanocortin-1
receptor (MC1R). Lu et al. found that mouse agouti protein
had no effect on the MC3R or MC5R but was a potent antagonist of the
MC4R, a brain-specific receptor that responds to ACTH as well as to
-MSH (6). Additional studies by Blanchard et al. (7) have
suggested that mouse agouti protein is a competitive antagonist of the
endogenous MC1R expressed on B16F10 mouse melanoma cells.
Potential pathophysiological action of agouti protein at one or more of the extrapigmentary melanocortin receptors has been suggested by the phenotype of mutant mice in which agouti is expressed ubiquitously, such as viable yellow (Avy) or lethal yellow (Ay), which have pleiotropic effects including obesity, diabetes, and increased tumor susceptibility (8, 9, 10). These findings have generated additional interest in the possible role of agouti, melanocortins, and their receptors in weight homeostasis, but the relevance of ASIP in human physiological or pathophysiogical function remains to be elucidated. Here we report the biochemical properties of ASIP relative to the full spectrum of human melanocortin receptors including the adrenal-specific MC2R. Our findings indicate that ASIP is a potent inhibitor of MC1R, MC2R, and MC4R signaling, but that its mechanism of action is complex and unlikely to be explained by simple competitive antagonism.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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90%
to >99%. Cell lines that stably expressed each of the human
melanocortin receptor subtypes, hMCR 1–5, were exposed to various
concentrations of ASIP for 30 min before the addition of melanocortin
agonist, and cAMP accumulation was then measured after an additional 30
min incubation. Control experiments indicated that ASIP had no effect
on basal cAMP levels in cell lines transfected with the MCRs and also
had no effect on histamine-stimulated cAMP levels in L cells that
stably express the histamine H2 receptor (data not shown). As shown in
Fig. 1
, ASIP is an antagonist of melanocortin action at
all the human MCRs, but the extent of this inhibition is variable.
Furthermore, the apparent mechanism of ASIP inhibition at the various
receptor subtypes is also different.
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-MSH-stimulated cAMP
generation and has no significant effect on maximal stimulation (Fig. 1A
). To gain additional insight into the mechanism of ASIP action, we
plotted the EC50 of -MSH (dose ratio) as a function of
antagonist concentration (inset, Fig. 1A). For a competitive
antagonist, the agonist dose ratio is proportionate to the change in
antagonist concentration, and the slope of this plot should approximate
unity (Schild analysis). Our results with the hMC1R are consistent with
competitive antagonism, since the slope of the Schild regression is
0.81 ± 0.19 (Fig. 1A), with an inhibitory constant
(Ki) of 0.47 ± 0.06 nM (Table 1). Similar results were obtained when we expressed the
hMC1R in an adrenocortical cell line, OS3 (see below), with a Schild
regression slope of 0.84 ± 0.24 and a Ki of 0.53
± 0.05 nM (Fig. 2A and Table 1).
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), in contrast to
hMC2R-transfected L cells or 293 cells, in which there is no response
to ACTH (data not shown). The magnitude of this response is especially
striking compared with other MCRs expressed in L cells, which generally
exhibit a maximal level of ligand-stimulated cAMP accumulation less
than 10-fold (Fig. 1), and is not due to differences in levels of
receptor expression, because similar results were observed using hMC2R
expressed in two different vectors (data not shown), and competitive
binding assays indicated similar levels of expression for all five
receptor subtypes (see legend to Fig. 1).
When OS3 cells that expressed the hMC2R were exposed to different
concentrations of ACTH and ASIP, we observed an unexpected pattern of
antagonism (Fig. 1B). Similar to its effects at the hMC1R, ASIP is a
potent antagonist of the hMC2R; however, the pattern of dose-response
inhibition is very different in that the maximum stimulatory responses
elicited by ACTH are markedly diminished and plateau well below the
levels attained in the absence of ASIP. This characteristic is a
hallmark of noncompetitive antagonism, but the effects we observed are
not completely consistent with the textbook definition of
noncompetitive antagonism because the EC50 values for cAMP
generation at different ASIP concentrations do not remain constant. In
the discussion that follows, we describe the effects of ASIP on the
hMC2R as nonsurmountable antagonism.
The effects of ASIP on the hMC3R, hMC4R, and hMC5R were examined in L
cells (Fig. 1, C-E); for the hMC4R, the effects of ASIP were also
examined in OS3 cells (Fig. 2B). At all three receptors, the effects of
ASIP are surmountable at high agonist concentrations, similar to the
pattern observed at the hMC1R (Fig. 1A, C-E). The Schild regression
slopes for the hMC3R, hMC4R, and hMC5R are significantly less than
unity; therefore, the empirical Ki values for these
receptors can only be used as an estimate of the dissociation constant
(Table 1). Nonetheless, comparison of Ki values suggest a relative
order of sensitivity to ASIP antagonism of MC4R > hMC1R >
hMC5R > hMC3R (Table I). Analysis of the hMC4R expressed in OS3
cells gave nearly identical results to those obtained in L cells, with
Schild regression slopes of 0.73 ± 0.01 and 0.71 ± 0.1 and
Ki values of 0.16 ± 0.03 nM and 0.14
± 0.02 nM, respectively (Fig. 2B and Table 1).
To investigate whether the effects of ASIP on cAMP generation could be
explained by effects on melanocortin binding, we used the radiolabeled
melanocortin agonist [I125-Nle4,
D-Phe7]-MSH (NDP-MSH) (12) to study the
hMC1R, hMC3R, hMC4R, and hMC5R (Fig. 3A). With the
exception of the hMC5R, the ability of ASIP to inhibit
I125-NDP-MSH to these receptors parallels the ability of
ASIP to inhibit cAMP generation, with MC4R hMC1R >
hMC3R > hMC5R. I125-NDP-MSH did not bind to the hMC2R
(data not shown), and therefore we used
[I125-Phe2, Nle7]ACTH1–24 (Fig. 3B). The effects of ASIP on binding of this melanocortin to the hMC2R
are approximately equivalent to that seen at the hMC1R and hMC4R and
indicate that while ASIP interacts specifically with all human MCRs, it
is a potent antagonist of the hMC1R, hMC2R, and hMC4R, and a relatively
weak antagonist of the hMC3R and hMC5R.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Our conclusions regarding ASIP action at the hMC1R and hMC4R are
consistent with previous analyses of the mouse agouti protein (5, 7).
However, we found that ASIP could antagonize melanocortin signaling at
the hMC3R and hMC5R, which differs from the results of Lu et
al. (5), who found that mouse agouti protein had no effect on the
rat MC3R or the mouse MC5R. These differences are unlikely to be
explained by variation between the sequences for mouse agouti and ASIP
or by impurities in the preparations used, because we have observed
similar results with a preparation of mouse agouti protein that is
99% pure (data not shown). Instead, the absence of rat MC3R
inhibition by mouse agouti protein, as reported by Lu et al.
(5), may reflect a quantitative rather than qualitative difference from
our observations with the hMC3R, because we observed significant
inhibition of the hMC3R only at ASIP or mouse agouti
concentrations 10-7 M. With the mouse
MC5R, however, Lu et al. (5) found no inhibition of
10-7 M agouti protein; therefore, our
observations that the hMC5R is sensitive to relatively low
concentrations of ASIP or mouse agouti may reflect differences between
the MC5R of rodents and humans.
In examining the effects of ASIP on the MC2R we made the surprising observation that the ACTH response of OS3 cells transfected with the hMC2R is extremely robust, with a nearly 100-fold increase in levels of cAMP accumulation. Expression of a functional MC2R has not been possible in L cells or 293 cells, but recently, Cammas et al. (13) reported that a functional mouse MC2R could be expressed in HeLa cells. The basis of the difference between OS3 cells or HeLa cells and other cell types is not known, but is likely to involve both receptor-specific and cell-specific components, because several attempts at expressing the hMC1R and hMC4R receptor in OS3 cells gave results similar to those observed when these receptors were expressed in L cells, with no more than a 6-fold increase in levels of cAMP accumulation.
The nonsurmountable nature of the hMC2R response to ASIP inhibition in
OS3 cells is also surprising. This characteristic is receptor-specific
and not cell-specific, since the effects of ASIP on the hMC1R or the
hMC4R were nearly identical in L cells compared with OS3 cells. At the
hMC3R, hMC4R, and hMC5R, -MSH can surmount the effects of ASIP, but
inhibition of these receptors is not consistent with competitive
antagonism, since changes in the EC50 of -MSH (dose
ratio) are not proportionate to changes in ASIP concentration. In fact,
it is only the hMC1R at which the effects of ASIP conform to those
predicted for a competitive antagonist; in this regard, our findings
are consistent with those studies by Blanchard et al. (7) of
the endogenous MC1R of mouse B16F10 melanoma cells (7). Nonetheless,
our conclusions, and those of others, are not based on direct
measurement of ASIP binding, and further studies that utilize a
radiolabeled or tagged form of ASIP may help determine whether or not
the molecular nature of ASIP action exhibits fundamental differences
among melanocortin receptor subtypes.
Our data underscore the potential for ASIP or a closely related molecule to act as a physiological or pathophysiological antagonist of the hMC1R, hMC2R, or hMC4R. By analogy to the phenotypes displayed by mice that carry various agouti alleles, three hypotheses might be considered. First, inhibition of MC1R signaling in mice produces a switch from eumelanin to phaeomelanin, a cysteine-rich yellow pigment. Very high levels of phaeomelanin are found in bright red human hair, and it is possible that dominant inheritance of this phenotype through pedigrees represents normal action of ASIP in humans. If so, ASIP structure and/or expression is likely to be highly polymorphic in humans, a hypothesis that is easily testable. Second, if ASIP or a closely related molecule normally functions to inhibit hMC2R activation, loss-of-function mutations might result in adrenocortical hyperplasia with hypercortisolism and secondary suppression of ACTH production, while gain-of-function mutations would produce a relative deficiency of corticosteroids and increased serum ACTH levels. Closer study of ASIP expression in normal and abnormal adrenal samples may shed light on this possibility. Finally, the pleiotropic effects observed in rodents with ectopic expression of agouti protein (obesity, diabetes, predisposition to tumorigenesis) suggests that somatic or germline mutations leading to ectopic ASIP expression in humans would have pleiotropic effects similar to those observed in mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and 2 have
been obtained from single clones; qualitatively similar results have
been obtained with independent clones in replicate experiments.
Receptor number determined by competitive binding assay with the
Graphpad Prism program (Graphpad Software, San Diego, CA) was found to
be similar for the different cell lines depicted in Fig. 1 and is given
in the figure legend.
cAMP Assays
For our studies, we measured intracellular cAMP using an assay kit
(TRK 432, Amersham, Arlington Heights, IL). Cells transfected with the
coding regions of the human melanocortin receptor genes were grown to
confluence in 12-well (2.4 x 1.7 cm) tissue culture plates. L
cells were maintained in DMEM ( Life Technologies; Gaithersburg, MD)
containing 4.5 g/100 ml glucose, 10% fetal calf serum, 1
mM sodium pyruvate. OS3 cells were grown on Ham’s F-10
nutrient mixture containing L-glutamine (Life
Technologies). Media for both cell lines contained 100 U/ml penicillin
and streptomycin and, in the case of transfected cells, 1 mg/ml of
geneticin (G418). For assays, the media was removed and cells were
washed twice with Earle’s balanced salt solution (EBSS, Life
Technologies) containing 10 mM HEPES (pH 7.4), 1
mM glutamine, 26.5 mM sodium bicarbonate, and 1
mg/ml BSA. Cells were preincubated for 30 min with human recombinant
ASIP in 0.5 ml EBSS before the addition of melanocortin agonist and 5
µl of 2 x 10-2 M
isobutylmethylxanthine. Cells were then incubated for another 30 min at
37 C at which time 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 Eppendorf tube and centrifuged for 10 min at
1900 x g. The supernatant was evaporated in a 55 C
water bath with prepurified nitrogen gas. cAMP content was measured by
competitive binding assay according to the assay instructions. Human
ACTH (1–39), -MSH, [Phe2, Nle7]ACTH
(1–24), and [Nle4,
D-Phe7]-MSH were obtained from Peninsula
Laboratories, Inc. (Belmont, CA). Recombinant mouse agouti protein and
human ASIP were prepared according to the method of Ollmann et
al. (unpublished results). Each experiment was performed a minimum
of three times with duplicate wells. The mean value of the
dose-response data was fit to a sigmoid curve with a variable slope
factor using the nonlinear squares regression in Graphpad Prism
(Graphpad Software). Values determined from these fits were used for
calculating the Schild analysis linear regression plot. 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 (20). Ki values (the negative log of the
pA2) presented in Table 1 were determined for relative
comparison of ASIP potency. Since the slopes of the linear regression
analysis of hMCR 3, 4, and 5 are not unity, by strict definition, true
Ki values cannot be determined.
Binding Assays
After removal of media the cells were washed twice with EBSS and
preincubated with ASIP in 0.5 ml MEM (Life Technologies) containing
0.2% BSA for 30 min before incubation with 106 cpm of
radioligand. [125-Nle4,
D-Phe7]-MSH was prepared by the
chloramine-T method according to the protocol modified from Tatro and
Reichlin (21), and binding experiments were performed using conditions
previously described (15, 16). [I125-Phe2,
Nle7]ACTH(1–24) was prepared as described by Hofmann
et al. (22), and binding experiments using this radioligand
were performed using conditions described by Rainey et al.
(23). Binding reactions were terminated by removing the media and
washing the cells twice with MEM containing 0.2% BSA. The cells were
lysed with 1% Triton X-100, and the radioactivity in the lysate was
quantified in a model 1285 Tracor Analytic 
-counter. Nonspecific
binding was determined by measuring the amount of
125I-labeled melanocortin remaining bound in the presence
of 10-5 M unlabeled melanocortin, and specific
binding was calculated by subtracting nonspecifically bound
radioactivity from total bound radioactivity.
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ACKNOWLEDGMENTS |
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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 P30DK-34933), and a grant from the NIH to G.S.B. (DK-28506). M.M.O. and B.D.W. are supported by Graduate (EY07106) and Medical Scientist Trainee (GM07365) grants. G.S.B. is an Assistant Investigator of the Howard Hughes Medical Institute.
Received for publication May 15, 1996. Revision received December 13, 1996. Accepted for publication December 18, 1996.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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