JOURNAL OF MOLECULAR RECOGNITION, VOL. 8, 167-168 (1995)

EXTENDED ABSTRACT Regulation of Melanotropin Receptors in Melanoma Cells

Walter Siegrist,* Sibylla Stutz and Alex N. Eberle Lahoratory of Endocrinology, Department of Research (ZLF), University Hospital and University Children's Hospital, CH-40.31 Basel. Switzerland

Keywords: MSH; melanoma; receptor; MC 1; up-regulation; down-regulation

Human and mouse melanoma cells have been shown to express high-affinity binding sites for a-melanocyte- stimulating hormone (a-MSH) (Eberle, 1988; Ghanem et al. , 1988; Siegrist et al . , 1989) Photo-affinity labeling revealed a molecular weight of approximately 45 kDa ( S o b et af., 1989). This subtype of the G protein- coupled melanocortin receptors was designated MC1 after its molecular cloning (Chhajlani et al. , 1992; Mountjoy et ul., 1992). It was found that a-MSH up- regulates its receptors in three human melanoma cell lines. The extent of up-regulation was at about two-fold above control values. In contrast, homologous down- regulation was observed in six human and two mouse melanoma lines, and in two human melanoma lines no change of receptor numbers was observed. Down-regulation was particularly conspicuous in mur- ine cells where about 80% of specific MSH binding disappeared after incubation with a-MSH. A less pro- nounced down-regulation was observed in some human cells where MSH reduced its receptor number by only 20 to 30% as compared to untreated cells. Up- and down-regulation of MSH receptors was confirmed by Scatchard analysis which proved that receptor affinities did not change after incubation with a-MSH. The hormone up-regulated MC1 at a half maximal effective concentration (EC,,,) of 1.6 n M in human cells. This effect was only marginal in the first 5 h of incubation but reached a maximum after 20 to 30 h. The peptide analogs ACTH,-,7 and [Nle', ~ - P h e ~ ] - a - M s H had a higher potency, whereas ACTH,_,,, desacetyl-a-MSH, and [Nle']-c(-MSH were less effective when compared to a-MSH. I n mouse B16-F1 cells, a-MSH down- regulated its receptors with an EC5,, of 0.23 n M . The time-course of down-regulation was maximal after 15 h and thus faster than up-regulation. In contrast to up- regulation, down-regulation could be mimicked by G,-protein activation with cholera toxin (Table 1). Elevation of CAMP by treatment with forskolin or isobutylmethylxanthine (IX) alone induced only a par- tial down-regulation in B16 cells. Furthermore, the MSH-induced down regulation was diminished in the presence of forskolin, suggesting the existence of a CAMP-dependent mechanism counter-regulating the

Thi\ work was supported by the Swiss National Science Foundation. Author t o whom correspondence should be addressed.

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Table 1. Modulation of MSH receptor (MCl) numbers in three melanoma cell lines

B16-Fl HBL D10 Treatment mean* ( f S D ) mean* ( t S D ) mean* (?SDI Control 100 100 100

a-MSH 10 nM 19 ( k 7 ) 76 ( 2 4 ) 2 3 8 ( + 2 3 )

u-MSH +forskolin Forskolin 3 p~

8Br-CAMP 1 mM

8Br-CAMP + IX Cholera toxin 0.1 mM Cholera toxin 10 nM

TPA 30 nM

Ix 0.2 mM

54 ( f 1 0 ) 57 7 9 ( f 1 6 ) 72 81 ( k 9 ) 58 50 ( + 1 ) 71

28 ( 5 1 ) 52 15 ( 2 3 ) 96

72 ( 2 9 ) 108

90 ( f 4 ) 101

(28 ) 165 (+14) 5 1 6 ) 105 ( f 4 ) (21 ) 131 ( 2 5 ) 512) 81 (510) +13) n.d. ( + 5 ) 8 6 ( + 1 0 ) +19) 90 ( + l o )

213) 54 ( k 5 )

Retinoic acid 2 VM 6 5 ( + 1 9 ) 1 0 2 ( + 1 3 ) 49 ( f 8 )

* MSH binding of untreated cells was assigned t o 100 per cent . SD = Standard deviation. n.d. = Not determined.

effect of MSH. Down-regulation was partially abo- lished in the presence of PKA inhibitors, providing further evidence that MSH receptor down-regulation in B16 cells proceeds through a cAMP/PKA-dependent and an independent process.

In mouse B16 cells both types of treatment, a-MSH receptor stimulation and PKC activation by TPA, induced down-regulation of MC1. By contrast, in human D10 cells TPA down-regulated MSH receptors whereas a-MSH had an up-regulating effect. Treatment with retinoic acid had a similar effect as TPA. MSH receptors on the human HBL cell line, however, were insensitive to both TPA and retinoic acid.

Taken together, evidence is presented for both a positive and a negative pathway regulating melanotro- pin receptors in melanoma cells after incubation with the homologous ligand. In addition MSH receptor down-regulation could be induced by PKC activation and after treatment with retinoic acid. Furthermore, it has been shown that the number of MSH receptors expressed on the surface of melanoma cells is the result of different regulatory mechanisms. The contribution of each of these pathways seems to vary markedly between different melanoma cell lineages.

Received I 0 Lkcember 199.3 Acceprrd 28 December 199.1

CCC 0952-3499195/0 1 0 167-02 0 I995 hy John Wiley & Sons. Ltd.

168 W. SlEGRlST E T A L .

References

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Eberle, A. N. (1988). The Melanotropins: Chemistry, Physiology and Mechanism of Action. Karger, Basel.

Ghanem, G. E., Comunale, G., Libert, A., Vercammen, G. A. and Lejeune, F. J. (1988). Evidence for alpha-melanocyte- stimulating hormone (alpha-MSH) receptors on human malignant melanoma cells. Int. J. Cancer 41, 248-255.

Mountjoy, K. G., Robbins, L. S., Mortrud, M. T. and Cone, R. D. (1992). The cloning of a family of genes that encode the melanocortin receptors. Science 257, 1248-1251.

Siegrist, W., Solca, F., Stutz, S., Giuffre, L., Carrel, S., Girard, J. and Eberle, A. N. (1989). Characterization of receptors for alpha-melanocyte-stimulating hormone on human mela- noma cells. Cancer Res. 49, 6352-6358.

Solca, F., Siegrist, W., Drozdz, R., Girard, J. and Eberle, A. N. (1989). The receptor for a-melanotropin of mouse and human melanoma cells. J. Biol. Chem. 264, 14277-14281.