JOURNAL OF RECEPTOR RESEARCH, l l ( 1 - 4 ) , 379-390 (1991)

HETEROGENEITY OF THE MSH RECEPTOR AMONG B16 MURINE MELANOMA SUBCLONES

Flavio F.A. Solcal , Yoram Salomon2 and Alex N. Eberlel*

Department of Research, University Hospital and University Children's Hospital,

CH-4031 Basel, Switzerland 2 Department of Hormone Research, The Weizmann Institute of Science,

Rehovot 761 00, Israel

ABSTRACT

The heterogeneity of melanotropin receptors on B16 sublines was tested by using photoaffinity crosslinking techniques and the superpotent a- MSH derivative [Nle4, D-Phe7, 1'-(2-nitro-4-azido-phenylsulfenyl)-Trp9]- a-MSH (NAPS-MSH). Specific crosslinking of this compound to B16-F1, 81 6-F10, B16-M2R or B16-W4 cells revealed three different subtypes of MSH receptor based on SDS-PAGE analysis. Binding of monoiodinated a-MSH to these different subclones is saturable and characteristic for a single class of complexes (0.9 nM CKD < 1.6 nM). In this article the nature of the different MSH receptor subtypes as well as their possible correlation to the melanogenic potential of a particular cell line is discussed.

]NTRODUCTlON

Melanocyte-stimulating hormones (MSH) such as a-MSH and 6-MSH have been shown to bind to a specific membrane receptor on B16

379

C'opyripht 0 199 1 by Marcel Dckker, I n L

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3 80 S O L C A , SALOMON, AND E B E R L E

murine melanoma cells (1, 2), to increase the intracellular CAMP level (2, 3), and to stimulate tyrosinase (EC 1.14.18.1) activity (4, 5) and melanogenesis (6). More recently, the MSH receptor for a-MSH on B16- F1 cells was identified by photoaffinity labeling (7, 8 ) and shown to consist of single broad band of 45 kDa as identified by SDS-PAGE analysis using [Nles, D-Phe7, 1’-(2-nitr0-4-azido-phenylsulfenyl)-Trp9]- a-MSH (NAPS-MSH) as photoactivatable probe (8). Very similar results were obtained by Gerst et al. (9) using a radioiodinated azidophenyl derivative of porcine O-MSH as a specific photoprobe for B16-M2R cells. In these cells two specific bands of apparent molecular weight of 43 kDa and 46 kDa were labeled upon photoactivation. In the present report we describe the use of 1251-NAPS-MSH for the identification of the different subtypes of MSH receptors on various B16 murine melanoma subclones. On the basis of competition experiments we suggest that these various MSH receptor subtypes represent very similar proteins.

MATERIALS AND METHODS

Peptides

[Nle4, D-Phe7, Trp(NAPS)g]-a-MSH (NAPS-MSH) was synthesized in our laboratory and radioiodinated to its monoiodinated form, 1251-NAPS- MSH, as previously described (8). a-MSH was a gift from Ciba-Geigy AG, Basel, Switzerland and was iodinated to 1251-a-MSH according to Siegrist et at. (10). 1251-a-MSH and 1251-NAPS-MSH were purified to homogeneity by reversed-phase high pressure liquid chromatography before each experiment.

Tissue Culture

All media and additives were purchased from GIBCO (Paisley, U.K.). Cells were maintained at 37OC in a humidified atmosphere of 95% air and 5% carbon dioxide. B16-F1 and B16-W4 subclones were obtained from Professor M.M. Burger, Friedrich Miescher Institute, Basel, and subcultured in complete minimal essential medium (MEM) with Earle’s salts supplemented with 10% heat-inactivated fetal calf serum, 2mM glutamine, 1.5% non-essential vitamin solution, 50 U/ml penicillin and

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HETEROGENEITY OF THE MSH RECEPTOR 381

5Opg/ml streptomycin (cMEM). B16-M2R cells were subcultured in Ham's F12 and Dulbecco's modified Eagle's medium mixed 1 :I (DMEM/Fl2) and supplemented with 10% heat-inactivated horse serum.

Photoaffinity Labeling of B16 Cells

5x1 0 6 cells were incubated in 600pl binding buffer (MEM supplemented with 25 mM HEPES, 0.2% bovine serum albumin and 0.3 mM 1, lO- phenanthroline) containing 0.33 nM (600,000 cprn/600pl) [(1251)Tyr2, Nle4, D-Phe', Trp(NAPS)g]-a-MSH in the presence or absence of cold peptide (3000-fold molar excess) in bovine serum albumin coated tubes. After incubation at 25% for 90 minutes the cells were UV-irradiated for 5 min on ice, using the 310-550 nm spectrum (180 rnW/crn2). Photolysed cells were washed with cold EDTA solution (0.02% EDTA in PBS pH 7.4), resuspended in 5 mM Tris/HCI, pH 7.4, containing 1 mM 1, lO- phenanthroline, and maintained for 15 min on ice. The cells were lysed by sonication and the total membrane fraction was recovered by centrifugation at 16,000 g in a microcentrifuge. The pellets were then equilibrated in PBS (pH 7.4) before DNAse I treatment (1500 U DNAse I / ml cell pellet, 10 min at 30OC). After a second DNAse I digestion step the membranes were solubilized in 150 pl SDS-PAGE sample buffer (2% SDS, 62.5 rnM Tris/HCI pH 6.8, containing 0.001% bromophenol blue, 5% O-mercaptoethanol and glycerol) and treated with 20 mM N- ethylmaleinimide for 20 min at 30% prior to electrophoresis on 10% polyacrylamide gels.

Radioreceptor Assay

For competition experiments the cells were harvested from roller bottles by EDTA treatment. The binding reaction was started by adding 5x106 cells in MEM medium supplemented with 25 mM HEPES, 0.2% bovine serum albumin and 0.3 mM 1,l O-phenanthroline to polystyrene tubes containing 50 PI of 1251-~-MSH and 50 pI of unlabeled ligand. The final concentration of radioligand was set to 200,000 cpm/600 pi. Equilibrium binding was reached after 3 hours at 15OC. The cells were occasionally resuspended by gentle mixing. Triplicate aliquots of 150 pl were

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382 SOLCA, SALOMON, AND EBERLE

centrifuged over silicon oil (density: 1013 kgm-3) in 400 pl polyethylene microtubes. After centrifugation the radioactivity associated with the cell pellets was determined in a LKB ycounter.

Bioassay

Melanogenesis was assessed by the in situ melanin assay described by Siegrist and Eberle (6). Briefly, B16 subclones were seeded in a 96- well rnicrotiter tissue culture plate (Falcon) at a density of 2500 cells/well containing 200 ~l of melanin assay medium. Two different media were used in this assay: (i) cMEM or (ii) DMEM/F12, both adjusted to a final L-tyrosine concentration of 500 pM. After 24 hours of preincubation serial dilutions of a-MSH (50pl) were added to the adherent cells. Each concentration was assayed in sextuplicate. Melanin formation was assessed 72 hours later by measuring the absorbance at 405 nm in a Dynatech MR 600 microplate reader.

RESULTS

Photoaffinity Labeling Reveals Different Forms of the MSH Receptor

These experiments were performed with 1251-NAPS-MSH, a radioiodinated photoactivatable MSH derivative. This compound was previously shown to bind specifically to B16-F1 cells and to membranes derived thereof and displays full biological activity. It further allowed the identification and the characterization of the B16-F1 MSH receptor which was shown to be a 45 kDa acidic glycoprotein (8 ) (see also Fig. 1). A similar MSH-binding protein can be labeled on F10 cells as illustrated in Fig. 1. W4 cells express a low molecular weight form of the MSH receptor which migrates as a 42 kDa protein. Using 1251-NAPS- MSH for photoaffinity labeling of M2R cells we identified two labeled bands of 46 kDa and 43 kDa respectively by SDS-PAGE analysis of whole cell lysates. These two proteins incorporate the MSH derivative specifically since a 3000-fold excess fully displaces labeling (Fig. 1 ). Taken together our data show that B16 subclones express different subtypes of the MSH receptor.

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HETEROGENEITY OF THE MSH RECEPTOR 383

FIG. 1. Photoaffinity labeling of B16 subclones with 1251-NAPS-MSH. Covalent labeling was achieved by incubating 5x1 06 cells with 0.2 pmoles of 1251-NAPS-MSH in the absence (1) or presence (2) of a 3000- fold excess of a-MSH. The labeled cellular extracts were analyzed by SDS-PAGE on 10% polyacrylamide gels. The B16 subclones used in these experiments were maintained in culture for more than 3 months except for the W4 cell line which was used after 1 month of subculture.

Binding of 1251- a -MSH to F1, M2R and W4 B16 Subclones

The affinity of a-MSH for its receptor and the number of binding siteskell were determined by LIGAND (Il), an iterative non linear regression analysis using the data obtained from competition curves (Table 1). As shown in Fig. 2, a-MSH binds to a single class of receptors displaying dissociation constants (KD) ranging between 0.9 and 1.6 nM. The M2R cell line expresses about twice as many MSH receptors as the F1 clone. In attempting to identify whether the different culture conditions used for M2R and F1 cells could play an active role in the level of expression of MSH receptors, we maintained the F1 clone in DMEM/F12 and the M2R cells in cMEM. This switch in culture conditions was apparently withou? dramatic effect on the level of expression of MSH receptors based on competition experiments even after 2 months of subculture. These data suggest that the various subclones tested express a very similar MSH receptor although at different levels.

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384 SOLCA, S A L O M O N , A N D EBERLE

TABLE 1 Characteristics of '251-a-MSH Binding to Different B16 Subclones

Cell line Culture Medium KD (nMIa Si tes/Cell

B16-F1 cMEM

DMEM/Fl2

B16-M2R cMEM

DMEM/F12

81 6-W4 cMEM

0.9 6694

1 .o 9929

1.6 13720

1.3 9996

1.4 13683

a Competition binding data were obtained by incubating the cells at 15OC for 3 hours in the presence of a constant concentration of 1251-a- MSH and increasing amounts of cold a-MSH. Dissociation constants were calculated from 2-3 independent experiments using LIGAND (1 1).

Bioassa ys

In a preliminary attempt to correlate the potential of melanization of the B16 variants in response to a-MSH with the expression of different receptor subtypes we assessed melanin formation with the in situ melanin assay (6). Typical dose-response curves obtained with B16 cells are shown in Fig. 3. B16-F1 cells are responsive to a-MSH when the cells are assayed in cMEM supplemented with tyrosine (a melanin precursor) to a final concentration of 500 pM. Interestingly, if cMEM is replaced by DMEM/Fl2 medium in the assay the F1 cells lose their responsiveness to the hormone as illustrated in Fig. 3B. M2R cells are not able to secrete melanin in this assay after a 72 h exposure to a-MSH independent of the assay medium used (Fig. 3A, 3B). Another surprising

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HETEROGENEITY OF THE MSH RECEPTOR

0.14-

0.12-

0.10-

0.08 -

0.06 -

0.04 -

0.02 -

0.00 1

385

b

c9 .

Fig. 2. Log dose-response curves of a-MSH in competition binding assays (3h, 15OC). The following cell lines were tested for their affinity to MSH and for the numbers of binding sites expressed: F1 cells grown in cMEM ( 0 ) or in DMEM/F12 ( W ) , M2R cells grown either in cMEM (0) or in DMEM/F12 (0). The W4 cells (A) used in this experiment were 1 month old and cultured in cMEM medium. The experimental procedure is described in "Materials and Methods".

observation was that W4 cells recover the ability to produce a-MSH- induced melanin after 3 months of continuous subculturing (Fig. 3C).

Hormone binding to the MSH receptor is thought to be the initial step in the regulation of melanin synthesis in mammalian systems. In cultured B16-F1 murine melanoma cells 1251-NAPS-MSH interacts with a 45 kDa membrane glycoprotein (8) (Fig. 1). Ligand binding induces an elevation of the intracellular CAMP level which in turn is responsible for the activation of tyrosinase. This enzyme is located in melanosomes and carries out the rate limiting step in melanin biosynthesis.

Photoaffinity labeling with '*%NAPS-MSH of the M2R cell line reveals two different proteins of molecular weight 46 kDa and 43 kDa which are

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386 SOLCA, SALOMON, AND EBERLE

5 v, 0 d

1.51

0.3:

0.0:

5 v, 0 P

0

c m ; n c

1.5 1.2 1 0.6 B

0.0 0.3LJ--- - - 1

- 0 . 3 4 . 8 . 7 . ' 7 . - I

- 1 1 - 1 0 - 9 - 8 - 7 - 6 - 5 - 4 - 1 1 - 1 0 - 9 - 8 - 7 - 6 - 5 - 4 LOG (Molarity) LOG (Molarity)

0.6 - E

0

13 2 0.2-

p 0.1- 2 0.0-

-0.1 - - 1 2 - 1 1 - 1 0 - 9 - 8 - 7

LOG (Molarity)

Fig. 3. Log dose-response curves of a-MSH using the in situ melanin assay. The B16 cell lines used in this assay were grown either in cMEM (A, C) ( F1 (M), M2R (U), W4-1 month old cultures (0) and W4-3 months old cultures ( 0 ) ) or in DMEM/F12 (B) (F1 (M), M2R (0)) and assayed in their respective media supplemented with L-tyrosine to a final concentration of 500 pM. Each point corresponds to the average of six determinations. The absorbance of the wells was measured at 405 nm after 72 h of stimulation with a-MSH.

able to bind melanotropins. These two bands had been previously identified by us using a radioiodinated azidophenyl derivative of porcine O-MSH. At present, it is difficult to speculate whether these two bands represent subunits of the MSH receptor or two different types of MSH receptor. Analyses of the data obtained from competition experiments show that the receptors in all three cell lines behave kinetically like a single class. Thus the most likely explanation for the two bands observed in M2R cells is that they correspond either to differentially spliced melanotropin receptors or to a single MSH receptor showing different glycosylation states. Furthermore, a multisubunit structure of the MSH

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HETEROGENEITY OF THE MSH RECEPTOR 387

receptor appears unlikely because all G-coupled receptors cloned so far have been shown to consist of a single polypeptide chain with molecular weights ranging between 43 and 75 kilodaltons (12, 13, 14). These considerations fit the hypothesis of differential splicing or glycosylation of the MSH receptor. Nevertheless, we can not exclude the possibility that the 43 kDa band represents a degradation product of the upper labeled protein band (46 kDa).

We also assessed the melanogenic potential of the three subclones by an in sifu melanin assay. These experiments were performed in order to determine whether the expression of various subtypes of the MSH receptor could be correlated with melanogenesis. In contrast to the F1 cell line, M2R cells are unable to secrete melanin in response to a-MSH when assayed under the same conditions. Nevertheless, they have been shown to respond to 0-MSH (2) and to [Nle4, D-Phe71-a-MSH by an enhanced adenylate cyclase activity, and to bind the latter iodinated derivative (Salomon unpublished). This discrepancy might be explained in two ways: (i) The in situ melanin assay is predominantly based on the release of melanin into the medium and it is possible that the secretion step is impaired in M2R cells; (ii) tyrosinase plays a pivotal role in melanin synthesis and its activation by melanotropins in the M2R cell line has not so far been reported. Thus, M2R tyrosinase might be present either in an inactive or in a constitutively active form but incorrectly located within the cell. Both of these phenomena could be explained by altered glycosylation pathways. Indeed, lmokawa and Mishima (1 5) have good evidence that glycosylation plays an important role in the intracellular trafficking of tyrosinase.

The increase of tyrosinase activity may vary according to the culture conditions used (16). We therefore performed melanin assays in the two culture media supplemented with tyrosine to a final concentration of 500 pM (Fig. 3). B16-F1 cells lost their ability to produce MSH-induced melanin when incubated in DMEM/F12 suggesting the presence of essential melanogenic factors in fetal calf serum or inhibitory factors in horse sera. The same cells were fully active when the assay was performed in cMEM. This phenotype does not require long term culture of F1 cells in DMEM/F12 since identical observations were made when F1 cells cultured in cMEM were assayed in DMEM/F12, containing 500 pM L-tyrosine.

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3 aa SOLCA, SALOMON, AND EBERLE

If W4 cells are kept in culture for more than 3 months they regain the ability to rnelanize as shown in Fig. 3. This is probably due to a loss of the specific W4 phenotype. Indeed, W4 cells have been selected for their resistance to toxic concentrations of wheat-germ agglutinin (1 7). This resistance has been attributed to a change in the glycosylation pathway of these cells (18, 19). Since tyrosinase is a glycoprotein, an alteration in the glycosylation state of the enzyme will probably impair its activity. Further support for this hypothesis is that W4 cells show elevated cAMP levels (even to a greater extent than F1 cells) in response to a-MSH despite their failure to rnelanize. Since the intracellular cAMP level is raised in response to MSH these receptors seem to be functional and the default must be situated either in the activation step of tyrosinase or in its intracellular location.

The data presented in this article suggest that the F1, M2R and W4 B1 6 subclones express similar MSH receptors (0.9 nM c KD < 1.6 nM) although at slightly varying levels and in different forms. Nevertheless, further investigations such as crosslinking experiments performed with affinity labeled cells or membranes are required to clarify the subunit structure of the MSH receptor.

ACKNOWLEDGEMENTS

We are grateful to Mrs.Caroline Kuenlin and to Mrs. Sibylla Stutz for their expert technical assistance. Special thanks are due to Dr. J. Baumann for the critical reading of this manuscript. This work was supported by grant 31-25653.88 from the Swiss National Science Foundation. Y.S. is the Charles and Tillie Lubin Professor of Hormone Research.

REFERENCES

1. Siegrist, W.; Oestereicher, M.; Stutz, S., Girard, J.; and Eberle, A.N. Radioreceptor assay for a-MSH using mouse B16 melanoma cells. J. Receptor Res. 8,323-343, 1988.

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HETEROGENEITY OF THE MSH RECEPTOR 389

2.

3.

4.

5.

6.

7.

8.

9.

Gerst, J.E.; Sole, J.; Mather, J.P.; and Salomon, Y. Regulation of adenylate cyclase by O-melanotropin in the M2R melanoma cell line. Mol. Cell. Endocrinol. 46, 137-1 47, 1986.

Niles, R.M.; and Makarski, J.S. Hormonal activation of the adenylate cyclase in mouse melanoma metastatic variants. J. Cell. Physiol. 96, 355-360, 1978.

Jimenez, M.; Kameyama, K.; Maloy, W.; and Tomita, Y. Mammalian tyrosinase: Biosynthesis, processing, and modu lat i o n by me Ian ocyt e -st i mu lat i ng hormone . Proc. Natl. Acad.

Lee, T.H.; Lee, M.S.; and Lu, M. Effects of a-MSH on melanogenesis and tyrosinase of B16 melanoma. Endocrinology 91 , 11 80- 11 88, 1972.

Siegrist, W.; and Eberle, A.N. In situ melanin assay for MSH using mouse B16 melanoma cells in culture. Anal. Biochem. 159,

Scimonelli, T.; and Eberle, A.N. Photoaffinity labeling of melanoma cell MSH receptors. FEBS Letters 226, 134-137, 1987.

Solca, F.; Siegrist, W.; Drozdz, R.; Girard, J.; and Eberle, A.N. The receptor for a-melanotropin of mouse and human melanoma cells. J. Bid. Chem. 264, 14277-1 4281, 1989.

sci. USA 85, 3830-3834, 1988.

191-197, 1986.

Gerst, J.E.; Sole, J.; Hazum, E.; and Salomon, Y. Identification and characterization of melanotropin binding proteins from M2R melanoma cells by covalent photoaffinity labeling. Endocrinology. 123, 1792-1797, 1988.

10. Siegrist, W.; Solca, F.; Stutz, S.; Giuffre, L.; Carrel, S.; Girard, J.; and Eberle, A.N. Characterization of receptors for a- melanocyte-stimulating hormone on human melanoma cells. Cancer Res. 49, 6352-6358, 1989.

11. Munson, P.J.; and Rodbard, D. Ligand: a versatile computerized approach for the characterization of ligand-binding systems. AnaLBiochem. 107, 220-239, 1988.

12. Loosfelt, H.; Misrahi, M.; Atger, M.; Salesse, R.; Thi, M.; Jolivet, A.; Guiochon-Mantel, A.; Sar, S.; Jallal, B.; Garnier, J.; and Milgrom, E. Cloning and sequencing of porcine LH- hCG receptor cDNA: Variants lacking transmembrane domain. Science 245, 525-528, 1989.

Jour

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and

Sig

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rans

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from

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Gie

ssen

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use

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y.

390 S O L C A , SALOMON, AND EBERLE

13. Masu, Y.; Nakayama, K.; Tamaki, H.; Harada, Y.; Kuno, M.; and Nakanishi, S. cDNA cloning of bovine substance-K receptor through oocyte expression system. Nature 329, 836-838. 1987.

14. Dohlman, H.G.; Caron, M.G.; and Lefkowitz, R.J. A family of receptors coupled to guanine nucleotide regulatory proteins. Biochemistry. 26, 2657-2664, 1987.

15. Imokawa, G.; and Mishima, Y. Analysis of tyrosinases as asparagin linked oligosaccharides by concanavalin A lectin chromatography: appearance of new segments of tyrosinases in melanoma cells following interrupted melanogenesis induced by glycosylation inhibitors. J. Invest. Derm. 85, 165-1 68, 1985.

16. Saeki, H.; and Oikawa, A. Effects of pH and type of sugar in the medium on tyrosinase activity in cultured melanoma cells. J. Cell. PhySiOl. 94, 139-1 46, 1978.

17. Tao, T.; and Burger, M.M. Non-metastasising variants selected from metastasising melanoma cells. Nature 270, 437-438, 1977.

18 Finne, J.; Tao, T.; and Burger, M.M. Carbohydrate changes in glycoproteins of a poorly metastasizing wheat germ agglutinin- resistant clone. Cancer

lectin resistant phenotype: Increase in a fucosyltransferase in mouse melanoma ce1ls.J. Cell. Biol. 92, 277-282, 1982.

Res. 40, 2580-2587, 1980.

19. Finne, J.; Burger, M.M.; and Prieels, J.P. Enzymatic basis for a

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