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-Melanocyte-Stimulating Hormone (MSH) than with -MSH
Research Centre for Developmental Medicine and Biology (K.G.M.), Department of Paediatrics, University of Auckland, Auckland 1, New Zealand; and Glaxo Wellcome Inc. Research Institute (W.O.W., D.H.W.), Research Triangle Park, North Carolina 27709
Address all correspondence and requests for reprints to: Kathleen G. Mountjoy, Research Centre for Developmental Medicine and Biology, Department of Paediatrics, University of Auckland, Auckland 1, New Zealand. E-mail: kmountjoy{at}auckland.ac.nz
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-MSH is more abundant than -MSH in the brain, the
fetus, human blood, and amniotic fluid, but there is little information
on its ability to interact with melanocortin receptors. The aim of this
study is to compare and contrast the ability of desacetyl--MSH and
-MSH to couple melanocortin receptors stably expressed in HEK293
cells, to the protein kinase A (PKA) signaling pathway.
Desacetyl--MSH activated mouse MC1, MC3, MC4 and MC5 receptors with
EC50s = 0.13, 0.96, 0.53, and 0.84 nM, and
-MSH activated these receptors with EC50s = 0.17,
0.88, 1.05, and 1.34 nM, respectively. Mouse agouti protein
competitively antagonized -MSH and desacetyl--MSH coupling to the
MC1-R similarly. In contrast, mouse agouti protein antagonized
desacetyl--MSH much more effectively and potently than -MSH
coupling the MC4-R to the PKA signaling pathway. Furthermore, mouse
agouti protein (10 nM) significantly reduced (1.4-fold) the
maximum response of mMC4-R to desacetyl--MSH and 100 nM
mouse agouti significantly increased (4.8-fold) the EC50.
Minimal antagonism of -MSH coupling mMC4-R to the PKA signaling
pathway was observed with 10 nM mouse agouti, whereas both
50 and 100 nM mouse agouti appeared to reduce the maximum
reponse (1.1- and 1.3-fold, respectively) and increase the
EC50 (2.5- and 3.4-fold respectively). Mouse agouti protein
did not significantly antagonize either -MSH or desacetyl--MSH
coupling mouse MC3 and MC5 receptors. Understanding the similarities
and differences in activation of melanocortin receptors by
desacetyl--MSH and -MSH will contribute to delineating the
functional roles for these endogenous melanocortin peptides. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-MSH
produced by pituitary melanotrophs, desacetyl--MSH is the
predominant form of ACTH1–13 NH2 produced in
the brain (1, 2), the fetus (3, 4), human blood (5), and amniotic fluid
(6). Desacetyl--MSH may also be more abundant than -MSH in the
pituitary of the Avy yellow mouse (7). The biological
activity of many peptide hormones and neuropeptides is altered by
N-terminal acetylation; for example, ß-endorphin completely loses its
opiate activity, whereas N-terminal acetylation of desacetyl--MSH to
form -MSH enhances some activities of ACTH1–13 and
virtually eliminates others. -MSH injected daily to rats is 10- to
100-fold more effective than desacetyl--MSH at increasing
pigmentation (8), arousal, memory, attention, and excessive grooming
(1). Desacetyl--MSH, however, is more effective than -MSH at
blocking opiate analgesia and opiate receptor binding in
vivo (1). -MSH and desacetyl--MSH also differentially affect
feeding and weight gain. Weight gain of agouti obese mice is
increased by sc administered desacetyl--MSH, as is food intake and
fat pad weight, but -MSH injections do not significantly increase
food intake or body weight (9). Similarly, both desacetyl--MSH and
-MSH given sc positively stimulate neonatal rat growth during the
first 2 weeks of life, but desacetyl--MSH is much more effective and
potent than -MSH (10). Both peptides increased muscle and brain
growth, whereas desacetyl--MSH also induced a rise in fat deposits.
Intracerebroventricular (ICV) administered -MSH and
ACTH1–24 inhibit food intake in food deprived (11) and fed
(12) rats, whereas ICV injections of desacetyl--MSH had no
effect.
Much interest has focused on the dominant agouti locus
mutations, lethal yellow (Ay) and viable yellow
(Avy), which give rise to mice with yellow coat
color as well as obesity and noninsulin dependent diabetes (13). This
phenotype is now known to be a result of ectopic expression of agouti
protein (14). However, the precise mechanism by which ectopic
agouti expression causes obesity and noninsulin-dependent diabetes
mellitus is yet to be determined. The mouse agouti gene
encodes a secreted protein primarily produced in the hair follicle (14, 15). Mouse agouti protein is normally involved in regulating the
production of two pigments, phaeomelanin and eumelanin, which produce
red/yellow and brown/black coloration, respectively. Agouti
is expressed during the mid-portion of hair growth, and this results in
hairs with a subapical yellow band on an otherwise black or brown
background, commonly known as agouti banding. Another genetic locus,
extension, that controls the relative amounts of
phaeomelanin or eumelanin, encodes the MSH (MSH) receptor (16). -MSH
binding to the MSH receptor, now known as the melanocortin-1 receptor
(MC1-R), leads to increases in cAMP and dark pigmentation. Recently the
mechanism of action of agouti on pigmentation was delineated when mouse
agouti protein was discovered to be a competitive antagonist of -MSH
coupling the mouse MC1-R to the protein kinase A (PKA) intracellular
signaling pathway (17, 18).
Following the cloning of the MC1-R, a family of five melanocortin
receptors have been identified. Two of these, MC3-R (19, 20) and MC4-R
(21, 22), are expressed in the ventromedial nucleus of the hypothalamus
(VMH). This region of the brain is considered important in the
regulation of feeding behavior and lesions to the VMH are associated
with an increase in body weight (23). It was therefore of considerable
interest when agouti protein was discovered to be an antagonist of
-MSH at the MC4-R in addition to the MC1-R (17). One hypothesis to
explain the Ay and Avy obesity phenotypes is
that agouti protein is antagonizing the MC4-R in the VMH. Targeted
deletion of the MC4-R gene recently identified this receptor to be
involved in appetite control and weight regulation, as the animals
lacking MC4-R are obese and hyperphagic (24). Further evidence
supporting this hypothesis is in vivo antagonism of
hypothalamic MC3 and MC4 receptors by 1) a synthetic peptide results in
attenuation of the cyclic melanocortin analog, MTII, induced decrease
in feeding (25) and 2) transgenic mice ubiquitously expressing
agouti-related protein develop obesity (26).
Despite the recent surge of interest in exploring the roles of
melanocortin peptides and their receptors in feeding and weight
homeostasis, there is still very little known about the functions of
desacetyl--MSH and nothing is known about agouti protein antagonism
of this peptide. In this study, we have extensively characterized
-MSH and desacetyl--MSH coupling mouse MC1, MC3, MC4, and MC5
receptors to the PKA intracellular signaling pathway and compared mouse
agouti protein antagonism of these melanocortin peptides and their
receptors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of mouse and human agouti protein
The production of recombinant human and mouse agouti has been
previously described (29). A 614bp XbaI/PstI
fragment of the full-length human agouti complementary DNA (cDNA) (30)
or mouse agouti cDNA (14, 15) was subcloned into the baculovirus
expression vector pAcMP3 (PharMingen, San Diego, CA).
Virus incorporating this vector were produced by standard methods (17).
Spodoptera frugiperda cells (Sf-9) were propagated in Grace’s
supplemented medium containing 10% CS and 0.1% pluronic F-68 and used
to produce high titer viral stocks. Fifteen-liter scale production runs
of human and mouse agouti were produced using Trichoplusia
ni (T. ni) cells adapted to suspension (JRH Biosciences, Woodland, CA). T. ni. cells were
infected 24 h postseeding at a density of 1 x
106 cells/ml and conditioned media from the infected cells
harvested 48 h later and purified using cation exchange columns
(approximately 60% and 90% purity for mouse and human respectively)
or reverse phase HPLC (
99% purity for both mouse and human). The
purity was estimated from 4–20% Tris/Glycine SDS-PAGE, ISS Pro Blue
staining.
Adenylyl cyclase assays
Adenylyl cyclase activity was determined directly by measuring
the ability of cells to convert [3H]adenine to
[3H]cAMP (31) following exposure of the cells to
increasing doses of either -MSH or desacetyl--MSH in the presence
and absence of different doses of human or mouse agouti protein.
Duplicate wells containing approximately 1 x 106
cells were incubated for 2 h with 2.5 µCi of
[3H]adenine in DMEM containing 10% NCS. After this the
medium was aspirated and the cells washed once with PBS warmed to 37 C.
The cells were then exposed for 1 h to increasing concentrations
of melanocortin peptides in the presence of DMEM containing 0.1% BSA
and 0.5 mM IBMX with or without mouse agouti protein. The
medium with peptides plus or minus agouti protein was aspirated and the
cells solubilized with 1 ml of 2.5% perchloric acid, 0.1
mM cAMP. Lysate (0.8 ml) was removed, neutralized with 80
µl of 4.2 N KOH, and 0.42 ml of H2O. The
samples were mixed and then the sediment was allowed to settle. A total
of 0.1 ml of each lysate was counted in a ß counter to
determine the total amount of [3H]adenine incorporated
into cells. cAMP was separated from the lysate following sequential
chromatography over Dowex and alumina columns. cAMP was eluted from the
alumina columns in 4 ml of Tris (pH 7.4) and counted in a ß counter.
Relative cyclase activity was calculated by determining the percentage
of [3H] adenine converted into [3H]cAMP.
The Kaleidagraph software package (Synergy Software,
Reading, PA) was used for fitting curves to the data and calculating
EC50 and maximum response values.
Statisitics
Comparisons between groups were made using Sigmastat (Jandel
Scientific) and one- or two-way ANOVA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-MSH and desacetyl--MSH increase adenylyl cyclase activity
via mouse MC1, MC3, MC4, and MC5 receptors-MSH and desacetyl--MSH,
stimulated dose dependent increases in adenylyl cyclase activity with
similar potencies on mouse MC1, MC3, MC4, and MC5 receptors. The
EC50 values for these peptides are shown in Table 1
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-MSH than -MSH coupling the mouse MC4-R (mMC4-R) to the
PKA signaling pathway-MSH
coupling of the mMC4-R to the PKA intracellular signaling pathway (Fig. 1). As shown in Fig. 1, mouse agouti
protein shifted the EC50 from 5.22 x
10-10 M to 3.51 x 10-9
M in an apparent dose dependent manner. However, this shift
was less pronounced when -MSH was used as a ligand (Fig. 2), shifting from 1.61 x
10-9 M to 6.12 x 10-9
M. Mouse agouti protein (100
nM) significantly increased the EC50 (4.8-fold)
for desacetyl--MSH (Fig. 3), whereas as little as 10 nM
protein was sufficient to significantly reduce the maximum response
(1.4-fold) of mMC4-R to desacetyl--MSH (Fig. 4). Mouse agouti protein (50 and 100
nM) appeared to increase the EC50 (2.5- and
3.4-fold, respectively) and decrease maximum response (1.1- and
1.3-fold) of -MSH coupling mMC4-R to the PKA pathway, but these
changes were not significant. Agouti protein antagonism of the maximum
response was significantly greater for desacetyl--MSH than with
-MSH. The pA2 values are not provided since agouti antagonism of the
MC4-R is not competitive.
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-MSH and desacetyl--MSH
coupling the mMC1-R to the PKA signaling pathway-MSH
and -MSH coupling of the mMC1-R to the PKA intracellular signaling
pathway (Figs. 1
and 2). As shown in Fig. 3, mouse agouti protein
shifted the EC50 in a dose-dependent manner from 1.74
x 10-10 M to 1.02 x 10-9
M (5.9-fold) and from 1.26 x 10-10
M to 8.67 x 10-10 M
(5.4-fold) for -MSH and desacetyl--MSH, respectively. The maximum
response appeared to be decreased by 1.2- and 1.3-fold for -MSH and
desacetyl--MSH respectively, but these changes were not significant
(Fig. 4). The pA2 values for agouti antagonism of -MSH and
desacetyl--MSH were 0.98 M and 0.25 M,
respectively.
Mouse agouti protein has little effect on -MSH or
desacetyl--MSH coupling mMC3-R and mMC5-R to the PKA intracellular
signaling pathway
Mouse agouti protein (10–100 nM) had minimal effect
on either -MSH or desacetyl--MSH coupling the mouse MC3 and MC5
receptors to the PKA intracellular signaling pathway (
Figs. 1–4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-MSH than -MSH coupling mMC4-R to the PKA signaling
pathway. Mouse agouti protein significantly decreased both the
sensitivity and maximum responsiveness of the mMC4-R to
desacetyl--MSH and appeared to decrease the sensitivity and
responsiveness of the mMC4-R to -MSH but these differences were not
significant. This is the first time antagonism of desacetyl--MSH
activating melanocortin receptors has been studied.
Desacetyl--MSH exists but very little is known about its function(s)
or how it activates the cloned melanocortin receptors. It has
previously been proposed that a defect in -MSH acetylation could
contribute to obesity and diabetic phenotypes, based on
characterization of a variant "yellow" mouse. Desacetyl--MSH is
more potent than -MSH at increasing feeding and weight gain in
yellow obese (Avy/a) mice (9) and Avy/a mice
may have a higher ratio of desacetyl--MSH to -MSH in the
pituitary than their lean (a/a) littermates (7).
The potent antagonism of desacetyl--MSH at the MC4-R by mouse agouti
protein suggests that desacetyl--MSH induced increases in feeding
and weight gain in obese yellow agouti mice (9), and also in neonatal
rats (10), may not be through agonism of the MC4-R.
To explain the effects of desacetyl--MSH on feeding and weight gain
we propose that desacetyl--MSH activates the MC3-R, which in
contrast to the MC4-R, is expressed not only in the VMH but also in the
arcuate nucleus where the blood brain barrier is leaky, and is not
significantly antagonized by agouti protein. We also propose that
desacetyl--MSH activates the MC5 receptor that is expressed in
adipose tissue and muscle (32). The MC5-R couples to the PKA signaling
pathway in response to desacetyl--MSH and mouse agouti protein is
not an antagonist of either -MSH or desacetyl--MSH coupling this
receptor to the PKA signaling pathway. Hence, increased activity of the
MC3 and MC5 receptors in response to desacetyl--MSH in the yellow
obese mouse may contribute to the mechanism for agouti-driven obesity.
Although targeted deletion of the MC5-R in mice did not lead to an
obese phenotype (33), the MC5-R has recently been linked with human
obesity (34).
Mouse agouti protein antagonism of desacetyl--MSH induced cAMP
increases via the mMC4-R is not consistent with competitive antagonism.
This is in contrast with the competitive antagonism of -MSH coupling
the mMC1-R via the PKA signaling pathway (17, 18). Levels of mouse
agouti protein (10 nM), which are without effect on -MSH
coupling the mMC4-R, significantly reduce the maximum response
resulting from desacetyl--MSH activation of this receptor.
Increasing agouti concentrations fail to further decrease the maximum
response for the ligand. Recently antagonism of -MSH coupling the
hMC4-R to the PKA signaling pathway by agouti-related protein (26) was
shown to be inconsistent with competitive antagonism. The role this
effect plays in energy homeostasis is not clear. Interestingly,
administration of 5 µg/day desacetyl--MSH, but not -MSH, to
obese yellow mice (Avy) increases feeding and weight gain
and 15 µg/day -MSH, but not desacetyl--MSH, induces eumelanin
synthesis (9). Furthermore, NDP--MSH, a superpotent analog of
-MSH, when sc administered to obese yellow mice, reverses fur
pigmentation but not obesity and insulin resistance (35). It appears
therefore that -MSH can compete with agouti at the MC1-R, but
neither -MSH nor desacetyl--MSH when administered sc can compete
with agouti antagonism at the MC4-R. Furthermore, administration of
desacetyl--MSH actually potentiates the agouti-driven obesity
phenotype. We offer two possible explanations for the decrease in
maximum response induced by mouse agouti protein. First, agouti protein
may down-regulate the MC4-R independent of melanocortin peptides or
potentiate melanocortin peptide induced down-regulation. This issue
will be resolved when antibodies to the MC4-R become available and
regulation of receptor protein can be studied. Second, agouti protein
may bind irreversibly to the MC4-R.
In summary, mouse MC1, MC3, MC4, and MC5 receptors are similarly
activated by both -MSH and desacetyl--MSH. Mouse agouti protein
antagonizes both -MSH and desacetyl--MSH coupling MC1-R and MC4-R
to the PKA signaling pathway; a greater effect is seen with
desacetyl--MSH than -MSH for the MC4-R only. Mouse agouti protein
antagonizes desacetyl--MSH on the MC4-R by decreasing both the
sensitivity and maximum response. Understanding the similarities and
differences in activation of melanocortin receptors by
desacetyl--MSH and -MSH will contribute to delineating the
functional roles for these endogenous melanocortin peptides.
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Footnotes |
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2 Present address: Zen-Bio, P.O. Box 12503, Research Triangle Park,
North Carolina 27709.
Received July 10, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-melanotropin in a multineurotransmitter neuron. Science 215:1125–1127-melanocyte stimulating
hormone throughout development. Biol Neonate 54:86–92[Medline]
-MSH and not -MSH is the major form of -MSH in
amniotic fluid. J Endocrinol Invest 11:345–349[Medline]
-melanocyte stimulating hormone
regulation in rat ageing. Neurosci Lett 201:65–68[CrossRef][Medline]
-MSH antagonize feeding behavior stimulated by
kappaopiate agonists. Peptides 7:843–848[CrossRef][Medline]
melanotropin and other
proopiomelanocortin peptides in the hypothalamus and limbic system.
Proc Natl Acad Sci USA 90:8856–8860-melanocyte stimulating hormone receptor antagonist. Biochemistry 34:12341–12346[CrossRef][Medline]
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