INTERACTION OF MELANIN-CONCENTRATINGHORMONE (MCH), NEUROPEPTIDE E-I (NEI),

NEUROPEPTIDE G-E (NGE), AND a-MSHWITH MELANOCORTIN AND MCH RECEPTORS

ON MOUSE B16 MELANOMA CELLS

Edith Hintermann, Heidi Tanner, Christiane Talke-Messerer,

Sophie Schlumberger, Urs Zumsteg, and Alex N. Eberle*

Laboratory of Endocrinology, Department of Research (ZLF),

University Hospital and University Children's Hospital, CH-4031 Basel,

Switzerland

ABSTRACT

Melanin-concentrating hormone (MCH) and a-melanocyte-stimulating

hormone (a-MSH) are known to exhibit mostly functionally antagonistic,

but in some cases agonistic activities, e.g., in pigment cells and in the brain.

Neuropeptide E-I (NEI) displays functional MCH-antagonist and MSH-

agonist activity in different behavioral paradigms; the role of neuropeptide

G-E (NGE) is not known. This study addressed the question of possible

molecular interactions between a-MSH, MCH and the MCH-precursor-

derived peptides NEI and NGE at the level of the pigment cell MCH receptor

subtype (MCH-Rpc) and the different melanocortin (MC) receptors. Radio-

receptor assays using [125I]MCH, [125I]a-MSH and [125I]NEI as radioligands

and bioassays were performed with MC1-R-positive and MC1-R-negative

mouse B16 melanoma cells and with COS cells expressing the different MC

receptors. The IC50s of a-MSH and NEI or NGE for [125I]MCH displacement

from mouse MCH-Rpc were 80-fold and, respectively, >300-fold higher than

that of MCH, and the IC50s for MCH and NEI or NGE for [125I]a-MSH

displacement from mouse MC1-R were 50,000-fold and >200,000-fold higher

than that of a-MSH. No high-af®nity binding sites for NEI were detected on

J. OF RECEPTOR & SIGNAL TRANSDUCTION RESEARCH, 21(1), 93±116 (2001)

93

Copyright # 2001 by Marcel Dekker, Inc. www.dekker.com

*Corresponding author. E-mail: Alex-N.Eberle@unibas.ch

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B16 melanoma cells and there was no signi®cant displacement of [125I]a-MSH

by MCH, NEI or NGE with MC3-R, MC4-R and MC5-R expressed in COS

cells. At concentrations of 100 nM to 10 mM, however, MCH, NEI and NGE

induced cAMP formation and melanin synthesis which could be blocked by

agouti protein or inhibitors of adenylate cyclase or protein kinase A. This shows

that mammalian MCH-precursor-derived peptides may mimic MSH signalling

via MC1-R activation at relatively high, but physiologically still relevant

concentrations, as e.g. found in autocrine/paracrine signalling mechanisms.

INTRODUCTION

Melanin-concentrating hormone (MCH) is a vertebrate hypothalamic peptide

which was discovered by its pigment aggregation activity in teleost melanophores

(1,2). The widespread projections of MCH neurons in the mammalian brain (3±5)

suggests that MCH together with neuropeptide E-I (NEI: H-Glu-Ile-Gly-Asp-Glu-

Glu-Asn-Ser-Ala-Lys-Phe-Pro-Ile-NH2) and neuropeptide G-E (NGE: H-Gly-Ser-

Val-Ala-Phe-Pro-Ala-Glu-Asn-Gly-Val-Gln-Asn-Thr-Glu-Ser-Thr-Gln-Glu-OH)Ð

two additional peptides originating from the MCH precursor (6,7)Ðin¯uence

various activities in the brain. These include processes associated with emotional

responses, cognition and general arousal (8,9), memory retention (10), differentia-

tion effects in brain neurons (11), reduction of dopamine in the ventromedial

nucleus (12), increased grooming behaviour and locomotor activity (13) as well as

antagonism by MCH on stress-induced release of ACTH in the rat (14). Interest-

ingly, the stimulation of grooming behavior by NEI is inhibited by simultaneous

application of MCH (13). Other studies in rat and sheep showed an involvement of

MCH and NEI in the control of water and electrolyte balance (15±17). Of

particular interest at present is the role of MCH in regulating feeding behaviour:

although initially controversially discussed (18±20), it is now generally accepted

that MCH exerts a strong orexigenic response in mice (21±23). Animals lacking

the MCH precursor gene are hypophagic and lean (24), whereas MCH over-

expression in transgenic mice leads to obesity and insulin resistance (25). Other

central effects of MCH concern the regulation of luteinizing hormone release in

rats (26±30) and in primates (31), and the anxiolytic response of MCH in rats (32,

33). In humans, a variant gene for MCH has been isolated (34,35) from which a

variant MCH peptide (MCHv) is derived; the variant gene appears to be expressed

during brain development (36).

To study the sites of action of MCH, a receptor binding assay was established

using biologically active tritiated (37) or monoiodinated (38) MCH radioligand.

With these, MCH binding sites were discovered on rat brain synaptosomes (37)

and on plasma membranes of various cell types, including melanoma cells (39),

neuroblastoma cells (39), or keratinocytes (40). Crosslinking experiments with

photoreactive MCH revealed a labelled band on melanoma cells of approximately

45±50 kDa (40,41), indicating a G-protein-coupled receptor. In 1999, the 353-

amino acid orphan somatostatin-like receptor 1 (SLC-1), a G-protein-coupled

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receptor signalling via Ca2� mobilization, was found to represent a functional

receptor for MCH, named MCH-R1 (42±46). MCH-R1 is widely distributed

throughout the rat brain (47,48) and the human brain (49) and it is also found on

porcine and human ciliary epithelial cells (50). Various human tumours such as

phaeochromocytomas, glangioneuroblastomas and neuroblastomas contain MCH-

R1 mRNA (51). In the brain, MCH-R1 is now known to constitute a central target

of leptin action (52). MCH-R1 is also discovered in the periphery, e.g., in rat

adipocytes where MCH triggers leptin release (53), or in insulin-producing cells

(RINm5F and CRI-G1 cell lines) which respond with insulin release when exposed

to MCH (54). Stimulation of MCH-R1 expressed in CHO or HEK-293 cells by

MCH not only leads to the mobilization of intracellular free Ca2� but also to a

reduction of forskolin-stimulated cAMP production (42) in certain cells. It appears

that MCH-R1 couples to multiple G proteins and activates diverse intracellu-

lar signalling pathways (55). Recently, a second MCH-R subtype was cloned, the

340-amino acid SLT or MCH-R2 with a distribution pattern similar to MCH-R1

(56±60). MCH-R2 signals through the Gq protein (58). Neither MCH-R1 nor

MCH-R2 seem to be produced by B16 mouse melanoma cells whose MCH

binding sites most likely correspond to a separate pigment-cell type MCH-Rpc. No

information is currently available on receptors for NEI and NGE.

MCH and a-MSH and their receptors are now known to antagonistically

regulate energy homeostasis (61). For example, stimulation of central melano-

cortin receptors leads to an increase of TRH, TSH and circulating thyroid

hormones (62), whereas icv injected MCH signi®cantly reduces plasma TSH

(63). The antagonism of the orexigenic activity of MCH by the tonic inhibition

of feeding by a-MSH (64±66) also represents an important switch in the

mediation of satiety signals through leptin (67). A physiological antagonism

between MCH and a-MSH was observed in several other regulatory functions of

these peptides in the brain, e.g., in a CNS auditory gating paradigm (68), in

grooming and locomotor activities (13), and in the modulation of hypothalamic

monoaminergic levels (12). The best studied example of antagonism between

MCH and a-MSH, however, is the control of skin colour of teleost ®shes which

was the basis for the molecular identi®cation of MCH. In teleost melanophores,

MCH stimulates melanosome aggregation (2) and a-MSH induces melanosome

dispersion (69). On the other hand, in tetrapod melanophores and in mammalian

melanoma cells, MCH was shown to exert weak MSH agonist activity (70),

possibly by interaction with melanocortin MC1 receptors (69), which are the

pigmentary subtype of the ®ve known MC receptors (71,72). A similar MSH-

like activity of MCH was found after application of the peptide into the medial

preoptic area of the female rat which stimulated sexual behavior in the same way

as a-MSH but antagonized a-MSH-induced aggression and exploration (73).

Whereas the molecular mechanism of action of MCH and a-MSH in the

nanomolar concentration range through their speci®c receptors is documented,

the different forms of interaction between MCH, NEI, NGE and a-MSH at a

higher concentration range is less clear.

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The aim of the present study was to analyze interactions of MCH, NEI, NGE

and a-MSH with the MCH-Rpc and the different melanocortin receptor subtypes.

The ®rst target were mouse B16 melanoma cells which either express MCH-Rpc or

both MCH-Rpc and MC1-R and with which receptor binding analysis and

bioassays can be performed (!determination of agonist vs. antagonist activity).

The interaction with the other subtypes of melanocortin receptors was then studied

with MC-Rs transiently expressed in COS cells. Also, potential receptors for NEI

were sought using a novel [Tyr2]-NEI analogue as radioligand.

MATERIALS AND METHODS

Peptides and Chemicals

The peptides NGE, NEI, [Tyr2]-NEI, MCHv and [D-Phe13, L-Tyr19]-MCH

were synthesized on a Milligen 9050 automated peptide synthesizer using the

continuous ¯ow technology with TentaGel-based (Rapp, Tubingen, Germany)

NovaSyn TGA resin (Calbiochem-Novabiochem, LaÈufel®ngen, Switzerland)

containing 4-hydroxymethylphenoxyacetyl linker as well as Fmoc-protection of

a-amino groups, in virtually the same way as described by Drozdz and Eberle (38).

The following protecting groups were used for o-protection: Trt for Cys; Boc for

Lys and Trp; tBu for Asp, Glu, Ser, Thr and Tyr; Pbf for Arg, and Trt for Asn and

Gln. Cleavage from the resin was carried out in 87% TFA=5% anisol=2.5%

thioanisol=0.5% ethane dithiol=5% water. Cyclization of the linear MCH peptides

was performed by iodine oxidation using a solution of iodine in glacial acetic acid

(30-fold to 40-fold excess). The peptides were puri®ed by conventional chromato-

graphy and semipreparative HPLC and characterized by analytical HPLC and

MALDI or electrospray mass spectrometry. Figure 1 shows the analytical data for

[Tyr2]-NEI as an example for all peptides described here. It should be noted for

this particular sequence that the quality of the peptide after cleavage from the resin

and puri®cation on Sephadex G-25 yielded a peptide of high purity. After HPLC,

the peptide was completely homogenous.

MCH, [Nle4, D-Phe7]-a-MSH and a-MSH were obtained from Bachem AG

(Bubendorf, Switzerland). The protein kinase A inhibitor H89 and the adenylate

cyclase inhibitor SQ22536 were supplied by Calbiochem-Novabiochem Interna-

tional, San Diego, CA. Recombinant mouse agouti protein was a gift of Glaxo Inc.,

Research Triangle Park, NC. All other reagents were of highest grade available.

Cell Culture

B16-F1 and B16-G4F mouse melanoma cells were cultivated in modi®ed

Eagle's medium (MEM) with Earle's salts (Gibco, Paisley, UK) supplemented with

1% MEM non-essential amino acids (100x; Gibco), 1.5% MEM vitamin solution

(100x; Gibco), 10% heat-inactivated fetal calf serum (Amimed, Basel, Switzerland),

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Figure 1. A: HPLC elution pro®le of [125I]-[Tyr2]-NEI after radioiodination and prepuri®cation on a C18 reversed-phase

minicolumn. The HPLC system used corresponds to that described in (75). B: HPLC elution pro®le of synthetic [Tyr2]-NEI

after cleavage from the resin and prepuri®cation on Sephadex G-25. The HPLC system used corresponds to that described in

(38). C: Electrospray mass spectrometry of [Tyr2]-NEI.

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2 mM L-glutamine (Gibco), penicillin (50 units/ml; Gibco), and streptomycin

(50mg/ml; Gibco) at 37�C in a humidi®ed atmosphere and 5% CO2. SKMel28

human melanoma cells were grown in RPMI 1640 medium containing folic acid

(40 mg/l) and NaHCO3 (2 g/l; Seromed, Berlin, Germany) which was supplemented

with 10% fetal calf serum, 2 mM L-glutamine, penicillin (50 units/ml), and

streptomycin (50 mg/ml). COS-7 cells were cultured in Dulbecco's modi®ed

Eagel's medium (DMEM; Gibco) containing 4.5 g/l glucose, supplemented with

10% heat-inactivated fetal calf serum, 2 mM L-glutamine, penicillin (50 units/ml)

and streptomycin (50mg/ml).

Receptor Overexpression in COS-7 Cells

The cDNAs for the mouse MC1 and MC5 receptor (gift of Dr. R. Cone,

Vollum Institute, Portland, OR) were inserted into the pcDNAINeo vector (Invitro-

gen), and the human MC3 and MC4 receptor cDNAs (gift of Dr. R. Cone) were

ligated into the pcDNA3 vector (Invitrogen). Plasmid DNA was introduced into

COS-7 cells by DEAE-dextran-mediated transfection (74). Three days after

transfection, the cells were detached with trypsin and washed in ice-cold

phosphate-buffered saline (137 mM NaCl, 8 mM Na2HPO4, 2.7 mM KCl,

1.47 mM KH2PO4, pH 7.2). Cell numbers were determined with a hemocytometer.

Radioligand Preparation

Monoiodinated [Nle4,D-Phe7]-a-MSH and [D-Phe13,L-Tyr19]-MCH radio-

ligands were obtained by enzymatic iodination using solid phase bound glucose

oxidase=lactoperoxidase, as described previously for the a-MSH analogue by

Eberle et al. (75) and the MCH analogue by Hintermann et al. (76). Monoiodin-

ated [Tyr2]-NEI was prepared by the `equimolar' chloramine T method (75) at

4�C; the HPLC pro®le of this radiotracer after prepuri®cation on a C18 reversed-

phase minicolumn is shown in Fig. 1. In this text, the three radioligands are

named [125I]MCH, [125I]a-MSH and [125I]NEI.

Receptor Binding Assay

The binding medium consisted of MEM medium with Earle's salts (Gibco)

containing 25 mM HEPES, 0.2% gelatin, 0.3 mM 1,10-phenanthroline and

0.16 mM phenylmethylsulfonyl¯uoride (PMSF). The binding reaction was started

by adding 50ml (0.05 pmol) of either [125I]MCH, [125I]a-MSH or [125I]NEI to

prelubricated 1.5 ml polypropylene tubes (Corning Costar Corporation,

Cambridge, MA) containing 0.5 ml of cell suspension (2� 106 cells per ml of

binding medium) and 50ml of competitor peptide solution. Cells were incubated

for 2 h at 10�C. Triplicate aliquots (150ml) were layered onto 150ml ice-cold

silicon oil of a density of 1013 kg/cm3 which was prepared by mixing equal

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volumes of AR-20 and AR-200 oil (Wacker Chemie, Munich, Germany).

Unbound radioactivity was removed by centrifugation at 4�C for 2 min at

12,000 g. Radioactivity was counted in a Packard Riastar g-counter and binding

data were analyzed with Prism (GraphPad Software, San Diego, CA).

Melanin Assay

Melanin assays with B16-F1 melanoma cells and determination of total

protein content were performed as described in (77). Brie¯y, 2500 cells per well

were seeded in 96-well plates in medium enriched with 0.3 mM L-tyrosine. After

24 h, peptide or vehicle was added to a ®nal volume of 250ml, the cells were

further incubated for 3 days and absorbance was measured at 405 nm. In the

experiments with SQ22536 and H89, the inhibitors were added 24 h after seeding

and the peptides 48 h after seeding. Again, absorbance was measured three days

after addition of peptide. Then, the culture medium was removed by a ¯ick of the

wrist and the cells were ®xed with 100 ml of a solution containing 10% (wt=vol)

formaldehyde and 0.9% NaCl for at least 30 min at room temperature. After

removing the ®xative, the cells were stained with 50ml of ®ltered 1% (wt=vol)

methylene blue in 0.01 M borate buffer (pH 8.5) for 30 min. The wells were then

washed with 4� 200 ml borate buffer and the dye was eluted with 100 ml of 1 : 1

(vol=vol) ethanol and 0.1 M HCl overnight at 4�C. Absorbance of a 1 : 30 dilution

of the dye in borate buffer was measured at 650 nm.

Cyclic AMP Determination

B16-F1 cells were seeded into 12-well plates and grown for two days to

con¯uency. Cells were then washed twice with prewarmed culture medium

containing 0.2% gelatin and stimulated for 30 min at 37�C with peptide or vehicle.

The medium was aspirated and cAMP extracted with 0.5 ml ice-cold absolute

ethanol. Plates were maintained at room temperature for 10 min, swirled, the

extract transferred to microcentrifuge tubes and dried in a vacuum concentrator for

30 min. The dried residue was dissolved in 1 ml sodium acetate buffer provided in

the EIA kit, further dilutions in the same buffer were prepared and 100ml aliquots

were used for the cAMP assay (Amersham International, Little Chalfont, UK). The

protein content of the cells was determined by dissolving the residue of each dry

tissue culture well in 1 ml 1 N NaOH. After 4 h of gentle rotary swirling, aliquots

were assayed by the Bradford method, using BSA as standard.

Detection of MCH-R1 and MCH-R2 mRNA by RT-PCR

Total RNA was extracted from B16 melanoma cells with the RNeasy kit

(Qiagen) including DNase I digestion according to the instructions of the

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manufacturer. First strand cDNA was produced by M-MLV reverse transcriptase

(Promega) using 1 mg of RNA and 0.2 mg oligo (dT)15. Each RNA preparation was

controlled for the presence of genomic DNA and subjected to an RNase ONE

digestion (Promega) to show speci®c ampli®cation from cDNA. PCR was

performed with cDNA corresponding to 50 ng of total RNA, 200mM dNTP,

1mM of each primer, 1.5 mM MgCl2 and 2 U Taq-DNA-polymerase (Promega) in

a total volume of 40ml with 40 cycles of 94�C (45 s), 59�C (50 s), 72�C (45 s).

Primer sequences were (i) for MCH-R1 50-ACCATGTGCACCCTCATCAC-30

(sense primer) and 50-TCTCACACAGAGCACTATGTAC-30 (antisense primer),

and (ii) MCH-R2 50-CTGCCAGTGTGGTGGATACAG-30 (sense primer) and

50-AACGTGTCAGTCGAAATGGTTG-30 (antisense primer), respectively. PCR

products were resolved on a 1.5% agarose gel and visualized by ethidium bromide

staining.

RESULTS

Competition Binding Studies with MCH, NEI, NGE

and a-MSH at the MCH-Rpc

Competition binding experiments were performed with B16-G4F mouse

melanoma cells which express MCH-Rpc but are de®cient from MC1-R (78).

Using a concentration range of unlabelled ligand from 1.7� 10ÿ6 M to

2.6� 10ÿ10 M, the peptides MCH and MCHv inhibited binding of [125I]MCH

in a dose-dependent manner (Fig. 2) with an IC50 of 7.7� 0.5� 10ÿ8 M for MCH

and 3.0� 0.7� 10ÿ7 M for MCHv (Table 1). This is in contrast to NEI and NGE

which showed almost no displacement activity (Fig. 2), even when 10-fold higher

Figure 2. Log dose±response curve of MCH (j), MCHv (m), NEI (u), NGE ( ) and a-MSH (s)

in a competition binding assay with B16-G4F cells and [125I]MCH as radioligand. For each

competitor one representative curve out of three independent experiments is shown. IC50 values

are listed in Table 1.

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concentrations were used (Table 1). a-MSH displayed weak displacement of

[125I]MCH from the MCH-Rpc at micromolar concentration.

Competition binding experiments with human SKMel28 melanoma cells

which express both MCH-Rpc and MC1-R produced similar IC50 values for MCH

and MCHv. This means that independent of the presence or absence of MC1-R on

melanoma cells or the origin of the cell lines used, the binding characteristics of

[125I]MCH remain about the same (Table 1).

Binding of MCH to MC1 Receptor

To investigate the interaction of MCH, NEI and NGE with MC1-R, binding

studies with [125I]a-MSH were performed using B16-F1 mouse melanoma cells

which express both MCH-Rpc and MC1-R (Table 1). As positive control,

competition binding studies with unlabelled a-MSH were done. Binding of a-

MSH radioligand to these cells was competitively inhibited by MCH at high

concentrations (IC50� 2.2� 0.5� 10ÿ5 M), whereas NEI and NGE showed no

competition (IC50 >> 2� 10ÿ5 M). This shows that at pharmacological peptide

concentrations MCH can bind to the MC1-R.

Stimulation of Melanin Synthesis by MCH, NEI and NGE

Maximal melanin synthesis in B16-F1 cells induced by a-MSH is observed

over a broad concentration range (mM to pM) of the peptide (Fig. 3A). It should be

noted that during the time of experiment, the B16-F1 cells used were particularly

sensitive to a-MSH, reacting with a full melanogenic response at MSH

Table 1. Competition of [125I]a-MSH and [125I]MCH Binding to Mouse and Human Melanoma

Cells by a-MSH, MCH, MCHv, NEI and NGE

Cell line Radioligand Competitor IC50

B16-F1 [125I]a-MSH a-MSH 3.9� 0.1� 10ÿ10 MB16-F1 [125I]a-MSH MCH 2.2� 0.5� 10ÿ5 MB16-F1 [125I]a-MSH NEI >> 2� 10ÿ5 MB16-F1 [125I]a-MSH NGE >> 2� 10ÿ5 MB16-G4F [125I]MCH MCH 7.8� 0.5� 10ÿ8 MB16-G4F [125I]MCH MCHv 3.0� 0.7� 10ÿ7 MB16-G4F [125I]MCH a-MSH 6.1� 0.0� 10ÿ6 MB16-G4F [125I]MCH NEI >> 2� 10ÿ5 MB16-G4F [125I]MCH NGE >> 2� 10ÿ5 MSKMel 28 [125I]MCH MCH 5.2� 1.4� 10ÿ8 MSKMel 28 [125I]MCH MCHv 1.2� 0.6� 10ÿ7 M

Values are means� SEM (n� 3). Mouse B16-F1 and human SKMel 28 melanoma cells express both

MCH-Rpc and MC1-R whereas B16-G4F cells only express MCH-Rpc. IC50 values were obtained

from competition binding curves and represent ligand concentration required for half-maximal

inhibition of radioligand binding.

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Figure 3. Stimulation of melanin production by MCH prohormone peptides and a-MSH. B16-F1 cells were

incubated with a-MSH (A), MCH (B), NEI (C) or NGE (D) at different concentrations for three days and relative

melanin production was determined by measuring the absorbance of the wells at 405 nm. The melanin values were

then normalized to the total protein content in the wells. Data points represent the mean� SEM of quadruplicate

measurements of one out of four independent experiments.

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concentrations as low as 1 pM. Incubation of the cells in the presence of high

concentrations of either MCH, NEI or NGE also resulted in a concentration-

dependent yet only partial or submaximal melanin secretion (Fig. 3B±D). MCH

and NGE induced a small but distinct melanogenesis at 1� 10ÿ8 M or higher

concentrations, whereas NEI was only effective at 1� 10ÿ6 M. The melanogenic

response to MCH and NEI was clearly smaller than that to NGE. In order to

exclude any contamination with tiny amounts of a-MSH, a series of experiments

with MCH and NGE was run in the presence of a-MSH antibody known to

suppress stimulatory activity by MSH (69): no difference in the melanogenic

activity of MCH and NGE was seen with or without MSH antibody.

Signalling Pathway for MCH-, NEI- and NGE-Induced

Melanin Production

Activation of MC1-R by a-MSH has been shown to lead to an increase in

cAMP and stimulation of protein kinase A (PKA) (69). To investigate whether

stimulation of melanin synthesis by the MCH-prohormone-derived peptides is also

mediated by this signalling pathway, cAMP concentrations in B16-F1 cells were

determined after a 30-min stimulation of the cells with peptide. Although the

relative melanin content was similar when the cells were incubated with

1� 10ÿ6 M or 1� 10ÿ12 M a-MSH (Fig. 3A), the cAMP concentration was

approximately 26-fold lower when stimulated with the lower peptide concentration

(Fig. 4), con®rming earlier observations that a partial cAMP response is suf®cient

to induce maximal melanin production. Incubation of the cells with MCH, NEI or

Figure 4. Cyclic AMP production of B16-F1 cells after stimulation with a-MSH or the MCH

prohormone peptides. Cells were incubated with vehicle or peptide for 30 min, cAMP was extracted

and measured by enzyme immunoassay. Results are the mean� SEM of triplicate values of three

determinations.

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NGE at 1� 10ÿ6 M led to a cAMP content which was only slightly above the

basal level (Fig. 4), although at this very same concentration, the peptides clearly

induced melanin synthesis (Fig. 3B±D). In order to check whether PKA activation

was involved, melanin assays were done in the presence of the adenylate cyclase

inhibitor SQ22536 (Fig. 5A) and the PKA inhibitor H89 (Fig. 5B). In both types

of experiment, melanin production was substantially blocked by the corresponding

inhibitor, demonstrating the importance of the cAMP/PKA pathway in MCH-,

NEI- and NGE-induced melanin synthesis.

MCH-, NEI- and NGE-Induced Melanin Synthesis Proceeds

via MC1-R Activation

To investigate whether MCH, NEI and NGE induce melanin synthesis by

activation of MC1-R, the melanin assays were performed in the presence of

Figure 5. Effects of inhibitors of the cAMP/protein kinase A pathway on melanin synthesis

induced by a-MSH, MCH, NEI or NGE. B16-F1 cells were stimulated with the appropriate peptide

concentration in the absence (light bars) or presence (dark bars) of (A) 2 mM SQ22536 (adenylate

cyclase inhibitor) or (B) 10mM H89 (PKA inhibitor). Absorbance was measured at 405 nm and

relative melanin synthesis calculated by normalizing the melanin values to total protein content. Two

representative experiments measured in quadruplicates (� SEM) are shown.

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1� 10ÿ7 M agouti protein which is an inverse agonist of a-MSH and it is bound

by MC1-R with the same af®nity as a-MSH (79). Agouti does not compete for

[125I]MCH binding to the MCH-Rpc (data not shown). As reported previously (79),

a 10-fold excess of agouti as compared to a-MSH did not inhibit melanin synthesis

whereas a 100-fold excess of agouti blocked a-MSH-induced melanin production

completely (Fig. 6A). When the cells were treated with MCH, NEI and NGE,

agouti also caused complete or almost complete inhibition of melanin synthesis

(Fig. 6B±D), indicating that stimulation of melanogenesis by the MCH peptide

family is mediated via MC1-R.

Figure 6. Effect of agouti protein on a-MSH-, MCH-, NEI- and NGE-induced melanin synthesis.

Melanin assays were performed with B16-F1 cells stimulated with a-MSH (A), MCH (B), NEI (C) or

NGE (D) at the indicated concentrations in the absence (light bars) or the presence (dark bars) of

100 nM agouti protein. Absorbance was measured at 405 nm and relative melanin synthesis was

calculated after normalizing the melanin values to total protein content. Each value represents the

mean� SEM of a single experiment performed in triplicates.

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MCH, NEI and NGE Do Not Antagonize a-MSH

in Costimulation Experiments

B16-F1 mouse melanoma cells were costimulated with a-MSH and the

MCH-prohormone-derived peptides in order to study possible antagonism of

MCH, NEI or NGE. The cells were exposed to a constant concentration of

1� 10ÿ12 M a-MSH and varying concentrations of MCH, NEI and NGE. As

shown in Table 2, a-MSH-stimulated melanin production was not changed

signi®cantly by any peptide concentration added. This is also true if other

concentrations of a-MSH used (data not shown). Thus under these conditions,

neither MCH nor NEI or NGE displayed antagonistic or synergistic activity on the

melanogenic response of these cells to a-MSH.

Expression of Receptors for MCH and NEI on Melanoma Cells

The primer pairs for MCH-R1 and MCH-R2 (see Materials and Methods),

which had been shown to work well for the detection of mRNA of these receptors

by RT-PCR in other cells, did not reveal a PCR product in B16 melanoma cells,

indicating that neither MCH-R1 nor MCH-R2 are expressed by these cells. This is

further evidence that the MCH-Rpc is an MCH-R subtype different from MCH-R1

or R2. Binding studies using [125I]-NEI did not reveal speci®c binding sites on

B16 melanoma cells which means that this cell line does not express receptors

for NEI.

Interaction of MCH, NEI and NGE with Overexpressed

Melanocortin Receptors

Following the demonstration of activation of MC1-R by MCH, NEI and

NGE, we investigated a possible interaction of these peptides with some of the

Table 2. Melanin Synthesis of B16-F1 Mouse Melanoma Cells Following Costimulation with

1� 10ÿ12 M a-MSH and Various Concentrations of MCH, NEI and NGE

Concentrationof peptide added

Percent of relative melanin production after stimulation with

MCH NEI NGE

0 100% 100% 100%1� 10ÿ9 M 110� 10% 112� 7% 98� 5%1� 10ÿ8 M 98� 7% 110� 10% 110� 1%1� 10ÿ7 M 106� 5% 113� 1% 92� 2%1� 10ÿ6 M 110� 5% 114� 9% 101� 1%1� 10ÿ5 M 103� 8% 100� 1% 108� 1%

Relative melanin production induced by stimulation with a-MSH alone was normalized to 100%.

Values represent the mean� SEM of two experiments, each done in triplicates.

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other melanocortin receptors. Binding of [125I]a-MSH to transiently overexpressed

MC3-R, MC4-R and MC5-R was compared with that of MC1-R in the presence of

MCH, MCHv, NEI or NGE as well as of a-MSH or agouti protein as controls.

Each peptide was applied at a ®nal concentration of 1.7� 10ÿ6 M. As expected,

a-MSH almost completely inhibited radioligand binding to MC1-R to an extent of

97.4� 0.4% (mean� SEM, n� 3) and agouti was only slightly less potent (Fig. 7).

Similar results for a-MSH and agouti were obtained with MC3-R, MC4-R and

MC5-R, except that agouti was a weak displacer in the latter. MCH, NEI and NGE

had a very slight inhibitory activity on [125I]a-MSH binding to MC1-R whereas

with the other melanocortin receptors, MCH, NEI or NGE did not show any

competition with [125I]a-MSH binding.

DISCUSSION

The functional antagonism of MCH and a-MSH in teleost melanophores has

been unequivocally demonstrated and is thought to result from different receptor-

effector activation pathways in these cells (2,8). In vitro, the melanin-concentrating

effect of MCH is not dependent on extracellular Ca2� (80), in contrast to the

melanosome-dispersing effect of a-MSH that requires Ca2� for ligand-receptor

binding and for receptor signaling (81,82). Whereas a-MSH is well known to

signal via adenylate cyclase and protein kinase A (69), MCH was thought to

Figure 7. Displacement of [125I]a-MSH from overexpressed MC1-R, MC3-R, MC4-R and MC5-R

by a-MSH, agouti, MCH, MCHv , NEI or NGE. COS-7 cells were transiently transfected with

plasmids encoding the corresponding melanocortin receptors and incubated with [125I]a-MSH alone

(for total binding) or in the presence of 1.7 mM competitor. Each value represents the mean� SEM of

three determinations.

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activate protein kinase C in teleost melanophores (83). However, a2-adrenoceptor

activation synergizes with MCH-Rpc signalling in teleost melanophores (84) but

antagonizes MC1-R signalling in most lower vertebrate pigment cells (69). The

molecular basis of these interactions is insuf®ciently understood and becomes

more complicated because of the weak MSH-agonist activity of MCH reported for

tetrapod melanophores (70,85) and melanoma cell tyrosinase (70). This latter

effect of MCH was explained by either direct interaction of MCH with melano-

cortin receptors or by indirect activation of the intracellular signalling cascade via

stimulation of MCH-Rpc and its effector(s). Earlier studies with structural analogs

of salmon MCH (86,87) and different assay conditions (80) had suggested that the

N-terminal part of MCH was responsible for eliciting the Ca2�-dependent MSH-

agonist activity of MCH whereas the central and C-terminal part of MCH was

responsible for the Ca2�-independent MCH activity. From these studies, it was

concluded that MCH and MSH are structurally and evolutionarily related (88).

This notion, however, is no longer maintained, particularly in view of more recent

MCH structure-activity studies (89).

The current interest in the orexigenic activity of MCH (see Introduction), its

involvement in the leptin-NPY signalling cascade (90) as well as its reported

functional antagonism with a-MSH at the level of the central nervous system made

it necessary to clarify a possible interactions of mammalian MCH, a-MSH and the

MCH-prohormone-derived neuropeptides NEI and NGE at the MCH-Rpc and MC

receptors. A natural cell line expressing suf®cient numbers of either MCH-R1 or

MCH-R2 and simultaneously MC4-R would have been the ideal tissue for these

studies, but at present there are no (natural) cell lines meeting these criteria.

Therefore we chose cells from peripheral tissues known to express both melano-

cortin and MCH receptors, such as different subclones of B16 mouse melanoma

cells, in order to investigate a possible correlation between MCH-Rpc and MC1-R

binding and signalling. The interaction of MCH and its related peptides with

central melanocortin receptors was then studied with MC1 and MC3±5 receptors

expressed in COS cells.

Like salmon MCH, the mammalian form does not share structural elements

with a-MSH or with agouti protein and therefore MCH was not expected to bind to

melanocortin receptors. However, the present study now shows that at high

concentrations, MCH, NEI and NGE can elicit a marked melanogenic response

in melanoma cells. Since this effect was blocked in the presence of inhibitors for

adenylate cyclase or protein kinase A or by agouti protein, the MSH-like activity

of MCH, NEI and NGE is thought to be mediated via MC1-R. In costimulation

experiments aimed at testing for a possible a-MSH-antagonizing activity of MCH

at concentrations with marginal MSH-like activity, no inhibition of a-MSH by

MCH was observed (data not shown). This ®nding and the fact that MCH induced

only weak cAMP formation indicates that MCH-Rpc is not coupled or only weakly

coupled to Gi or to Gs proteins. It is well possible that only the overexpression of

the MCH-Rpc will lead to a receptor number high enough to study the signalling

pathway(s) for MCH in pigment cells in vitro.

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To investigate whether MCH interacts with other MC receptors, radioligand

binding studies were performed with transiently overexpressed MC1-, MC3-,

MC4- and MC5-R. Since MCH at mmolar concentration did not displace a-MSH

from MC3-R, MC4-R or MC5-R, it can be assumed that the in vivo antagonistic

effect of MCH seen in physiological experiments is not based on blockage at MC

receptors and subsequent inhibition of cAMP formation. Hence, stimulation of

food intake by administration of MCH is likely to proceed by a mechanism

different from the effect elicited by agouti which inhibits binding of [125I]a-MSH

to MC4-R in the nmolar range (91). Even when peptide concentrations were

increased, e.g., for the determination of IC50 of MCH competing with [125I]a-

MSH, no further inhibition of radioligand binding to MC4-R was measurable (data

not shown). The weak competition of radioligand binding to MC5-R by agouti

correlates with published data (91). The fact that agouti protein competed with

[125I]a-MSH binding to MC3-R (this study) but did not antagonize a-MSH

activation of MC3-R (91) is an interesting observation which needs further

clari®cation.

The existence of a variant gene for MCH in man (34,35) and the identi®ca-

tion of transcripts of this gene in the human brain may mean that other forms of

MCH exist in the CNS. Although the corresponding peptide MCHv could not yet

be isolated, we included synthetic MCHv in our experiments studying a possible

antagonism of a-MSH. However, as found for authentic MCH, no inhibition of

speci®c [125I]a-MSH binding by MCHv was detected when the peptide was tested

at mmolar concentration with the different MC receptor subtypes. At the MCH-R

of mouse or human melanoma cells, the IC50 for MCHv was higher than that of

MCH, indicating that if the variant peptide plays a physiological function, it

may bind to a different MCH-R subtype or is only active during development

phases (36).

Since it had been demonstrated that MCH and NEI are secreted from

cultured rat hypothalamic cells (92) and that NEI antagonizes behavioral effects

elicited by MCH, the question arose whether NEI and/or NGE represent natural

ligands for MCH-R. At B16-G4F mouse melanoma cells, which express MCH-Rpc

but not MC1-R, [125I]MCH could not be displaced by NEI or NGE which means

that these two neuropeptides most likely interact with receptors different from

MCH-Rpc. Interestingly, NEI was shown to bear epitopes which are recognized by

antisera against a-MSH (9) and therefore, it had been assumed that NEI might be a

speci®c ligand recognized by one of the melanocortin receptors. In fact, binding

experiments demonstrated that NEI and NGE did not exhibit speci®c af®nity for

MC3-R, MC4-R or MC5-R at mmolar concentration. However, both NEI and NGE

stimulated melanin synthesis in mouse melanoma cells by activation of MC1-R. In

spite of only weak binding of MCH, NEI and NGE to MC1-R, activation of the

cAMP/PKA signalling cascade and melanogensis is induced by peptides derived

from the MCH prohormone.

In summary, our results demonstrate that crossreaction between the MCH

and POMC hormonal systems and their receptors is possible and thus may explain

HORMONES AND NEUROPEPTIDES 109

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the MSH-like effect of MCH found in melanophores and melanoma cells as well

as as at the level of the CNS. This is of particular interest in those situations where

high local MCH concentrations may occur, such as in autocrine=paracrine

regulatory mechanism, possibly leading to a dual effect of MCH, i.e., an

interaction with MCH receptors at low concentrations and with MC receptors at

high concentration. In the periphery MCH-Rpc and MC1-R as well as POMC have

been shown to be expressed not only in melanocytes or melanoma cells but also in

keratinocytes or other cells of the skin. It is possible therefore that the POMC and

MCH system may form a peripheral autocrine/paracrine regulatory network

similar to that found in the brain and therefore may represent a useful model to

study the molecular mechanism of the MCH/NEI/NGE/MSH agonism and

antagonism. Yet, cloning of MCH-Rpc, NEI-R and NGE-R (if existing) and

analysis of the corresponding signalling pathways in cells with overexpressed

MCH-R will be the basis for elucidating these interactions in more detail.

ACKNOWLEDGMENTS

We thank Dr. Roger C. Cone, Vollum Institute for Advanced Biomedical

Research, Oregon Health Sciences University, Portland, OR, for providing us with

the cDNAs for the melanocortin receptors, and Dr. D. Willard, Glaxo Research

Institute, Glaxo Inc., Research Triangle Park, NC, for recombinant mouse agouti

protein and Dr. C. Guenat, Novartis Pharma Basel, Switzerland, for the mass

spectra. Financial support was obtained from the Swiss National Science Founda-

tion and Millenium Pharmaceuticals Inc., Cambridge, MA.

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