L -Dopa binding sites in rodent melanoma cells

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Biochimau ct Biophy~i('a t~ ta. 113'i t i~t)2132J, 32', ~: 1992 Elsevicr Science Publishers I~;.V. All rights rcserx,cd nq25 413~ ,t)2 $tP~ (t~!

Rapid Report

c-Dopa binding sites in rodent melanoma cells Andrzej Slominski and Daniel Pruski Department of Microbiology, Immunology and Molecular Genetics, Albany Medical College, Albany, NY (USA) (Received 12 June 1992)

Key words: L-Dopa;Melanoma cells; Membrane; Nucleis Rapid, saturable, specific and stereoselective binding of L-dopa to crude membranes and purified nuclei from rodcnt amelanotic melanoma cells is reported. Cross-linking of [3H]dopa to melanoma cell surface emphasized proteins of approx. 55, 30, 25 and < 20 kDa. It is suggested that these binding sites may regulate melanocyte activity.

In melanocytes, L-dopa, a product of enzymatic hydroxylation of L-tyrosine, is oxidized by tyrosinase to dopaquinone and transformed further through a series of oxidation and reduction reactions called melanogenesis into melanin [1,2]. In the vertebrate pigmentary system, evidence is accumulating that L-dopa and its derivatives can act as inducers and regulators of the entire melanogenic apparatus and regulate the expression and activity of MSH receptors, melanocyte proliferation and intermediary metabolism [3]. Interestingly, direct bioregulatory effects by L-dopa were documented for some non-pigmentary systems, such as embryonal epithelial cells, lymphocytes and neuronal cells

[3]. Recently, we have documented that the induction and regulation of melanogenesis in hamster amelanotic melanoma cells by L-tyrosine and L-dopa is specific for those amino acids and follows pathways different from those linked to the activation of dopaminergic and adrenergic receptors [4,5] and L-dopa had no effect on intracellular levels of cAMP, cGMP and IP3 (6). Others have documented that L-dopa can stimulate proliferation of murine amelanotic melanoma cells and phosphorylated isomers of L-dopa can regulate MSH receptor activity [7,8]. Previously, we proposed that L-dopa may act as a hormone-like bioregulator of mammalian pigmentation [3] and others have suggested a neurotransmitter role for L-dopa [9-11]. Such functions would require a presence of receptors for this amino acid on neuronal cells and on melanocytes. It has been shown that

Correspondence to: A. Slominski, D e p a r t m e n t of Microbiology, Immunology and Molecular Genetics, Albany Medical College, Albany, NY 12208, USA.

u-dopa binds to membranes from bovine brain [12], interacts with glycine receptors on spinal cord neurons of lamprey [13] and binds to membrane preparations isolated from coelenterates [12]. To elucidate the mechanism of dopa bioregulation in malignant melanocyte, we started a characterization of binding sites for [3H]dopa in crude membrane preparations and nuclei purified from hamster and mouse amelanotic melanoma cells. Bomirski hamster AbC1 and Cloudman (clone #6) amelanotic melanoma ceils were cultured in Ham's F10 medium plus 10% horse serum [4]. Cells were harvested, centrifuged and pellets frozen at -70°C. The protocols to isolate crude membranes and purify nuclei were modifications of those described by Hulme, [14] and Van der Klis et al. [15]. Briefly, frozen pellets were thawed and homogenized in 20 mM Tris-HCl, 2 mM CaCI2, 1 mM MgCI 2, 5% glycerol, 0.25 M sucrose, 0.01% aprotinin, 1 mM PMSF (phenylmethylsulfonyl fluoride) (pH 7.6) and centrifuged at 1000 Xg. The supernatants were centrifuged at 16 000 x g for 30 rain and resulting pellets, defined as crude membrane preparations, were washed in the above buffer and stored at - 7 0 ° C until use. For isolation of nuclei, the 1000 × g pellet was washed four times by suspension in the above buffer and centrifugation at 1000 X g. The pellet was purified further by centrifugation (45 rain, 45000 x g) through 2.4 M sucrose in homogenization buffer. After two further washed with homogenization buffer, the isolated nuclei were suspended in 20 mM Tris-HCl, 0.25 M sucrose, 1 mM EDTA, 50 mM NaC1, 5% glycerol (pH 7.6) plus proteinase inhibitors and stored at -70°C. For electron microscopic examination, the nuclei were fixed in a mixture of glutaraldehyde (2.5%) plus formaldehyde (2%), buffered to pH 7.2 with sodium cacodylate buffer (0.1 M) and pro-

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Fig. 1. Time-course of [3H]L-dopa binding. The concentration of [3H]L-dopa was 0.1 izM. Values represent the means from duplicate determinations of specificbinding to membranes (A) or nuclei (B). Specificbinding was defined as total binding minus nonspecificbinding in the presence of 1 mM of L-dopa. Ordinate: cpm of [3H]L-dopabound/mg protein. Abscissa: time of binding. Abe1 amelanotic melanoma cells (©); $91 amelanoticmelanoma cells (e).

cessed further as described previously [4]. Electron microscopy was done by Mrs E. Kuklinski (Dept. of Dermatology Yale University). Binding experiments were performed with L-dihydroxyphenylaline, L-3,4-[ring-2,5,6,-3H]dopa (spec. act. 32.3 Ci/mmol, NEN, DuPont), L-tyrosine, L-[ring-3,53H]tyrosine (spec. act. 50 C i / m m o l , NEN, Dupont) and [3H]L-leucine (spec. act. 5 Ci/mmol, NEN, DuPont) as ligands. The incubations were done at room temperature in (total volume) 100/zl of PBS (pH 6.5), containing 0.3225 p~Ci (0.1 izM) or 0.645/.~Ci (0.2 ~ M ) of [3H]dopa, 1 mM CaCI 2 1 mM MgC12, 1 mM PMSF and 0.01% aprotinin per well in 96-well plates with continuous shaking. Following incubations, the assay mixtures were rapidly filtered under vacuum through Gelman A / E glass-fiber filters. Filters were washed 5 times with excess cold PBS and radioactivity

retained on filters was measured by liquid scintillation spectroscopy. Cross-linking experiments followed protocols described for cross-linking of glucagon receptors by UVradiation [16]. Binding of [3H]L-dopa to intact cells followed protocols described previously for MSH binding to melanoma cells [17]. Cells were detached from the culture flasks in Ca 2+- and Mg2+-free Tyrode's solution containing 1 mM EDTA, collected by centrifugation, washed and suspended at concentration 2 . 1 0 6 cells per 50/xl of binding buffer (Ca 2+- and MgZ+-free PBS (pH 7.0) plus 0.1% glucose) and transferred into 96-well plates. 50 ~1 of above binding buffer plus 1.29 p~Ci of [3H]e-dopa, to reach final concentration 0.4 ~ M and different additions listed were added immediately to 50/xl of cell suspension. The plates were incubated for 1 h at 10°C with continuous shaking. Plates were

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Fig. 2. Competition curves of [3H]L-dopa binding to membranes. The concentration of [3H]L-dopa was 0.2 p.M. Values represent the m e a n s from duplicate determinations of total counts minus binding to filters. Ordinate: % of [3H]L-dopa bound. Abscissa: L-dopa ( ~ []) and o-dopa (m n ) were added at various concentrations (0, 1, 10, 100 and 1000 p,M); L-tyrosine ( + - - - - - - + ) and L-leucine ( o - - - - - - o ) were added (1000 ~tM). (A), s91 amelanotic m e l a n o m a cells; (B), abC1 amelanotic m e l a n o m a cells. The binding studies were performed for 1 h at room temperature.

326 then placed on ice and exposed to UV emission from minlarlight lamp UVSL13 from the distance of 1 cm for 10 min [16]. After removing from incubation wells, cells were centrifuged, washed in PBS and lysed with ice-cold PBS (pH 6.8) 1% Triton X-100, 0.01% aprotinin and 2 mM PMSF. The homogenates were centrifuged at 1 6 0 0 0 × g for 30 min and supernatants were diluted in the 4 × strength Laemmli buffer at ratio 1:4 (supernatant/buffer). An equal amount of protein was separated by 0.1% S D S - P A G E (8% acrylamide) and gels, after fixing and drying, were processed for fluorography [18]. Protein content was estimated with the help of a Bio-Rad kit and bovine albumin as a standard. In the present paper, we demonstrated a rapid binding of [3H]L-dopa to crude m e m b r a n e preparations and purified nuclei from hamster and murine amelanotic melanoma cells that reached a plateau after 1 h of incubation (Fig. 1). The binding to crude membranes was stereoselective, with L-dopa demonstrating greater competition than D-dopa (Fig. 2). Binding a p p e a r e d also to be specific for dopa, since L-tyrosine and L-leucine were less efficient competitors than D- or L-dopa (Fig. 2). The m e m b r a n e binding sites for L-dopa were most likely heterogeneous, i.e., D-dopa and L-leucine partially competed binding of [3H]L-dopa. To further characterize cell surface dopa-binding proteins [3H]k-dopa was cross-linked to intact hamster m e l a n o m a cells by UV radiation. Proteins of approx. 55, 30, 25 and < 20 kDa were labeled (Fig. 3). This pattern was similar to that reported for [~4C]L-tyrosine A

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29-Fig. 3. Fluorography of t,-dopa binding proteins derived from the surface of AbCl hamster amelanotic melanoma cells. Cells (2.106 cells/100 t*l) were incubated for 60 min at 10°C in binding buffer containing 0.1 mM cycloheximide,0.1 mM PMSF, 0.001% aprotinin, 0.4 ~M [3H]L-dopa, and the additions listed below. The radioligand was crosslinked to binding proteins with irradiation by UV light using mineralight lamp model UVSL13 as recomended for glucagon receptors (16). Proteins were extracted with 1% triton X-100, then suspended in Laemmli buffer and separated by SDS-PAGE. Gels, after fixing and drying were processed for fluorography (18). Left, molecular weight markers (kDa). A: no addition; B: 1 mM L-dopa; C: 1 mM D-dopa; D: 1 mM L-tyrosine; E: 1 mM L-arginine; F: 1 mM L-alanine; G: 1 mM dopaminc.

cross-linked to hamster melanoma cells [1~1. Furthcrmore, the cross-linking of [3H]l.-dopa was compctcd out by nonradioactive l.-dopa and l-tyrosine but not by D-dopa, dopamine, ~:arginine and t:alaninc, suggested a specificity for the precursors of melanogenesis. In this context, the moderate competition of i -tyrosinc for dopa binding sites and the ability of L-Icucinc to compete with dopa seen in binding to crude membranc preparation require explanation (Fig. 2). In cross-linking experiments, radioligand was incubated with intact cells at low temperature, e.g., 10°C. This most likely eliminated binding to intracellular membrane structures (present in crude membrane preparation), reduced sensitivity, and apparently reduced binding to amino acids transporters, e.g., l.-arginine and t:alaninc did not compete with L-dopa (Fig. 3). Although this suggests presence on melanoma cell surface of proteins with high affinity for l,-dopa, defining their role will require their purification with subsequent molecular characterization and comparative studies with nonmelanocytic cells. Some authors have reported that L-dopa stimulated R N A synthesis in isolated nuclei from the rat brain [20]. Since in melanocytes L-dopa is produced intracellularily (in melanosomes) and can accumulate in the cytoplasm [21], we have undertaken an effort to detect nuclear binding sites for L-dopa. The nuclei used in our assays were free from membraneous contamination (not shown). The binding of [~H]L-dopa to nuclei of hamster and murine amelanotic melanoma cells was rapid (Fig. 1), saturable (not shown), stereoselective and specific, i,e., L-dopa had greater competing potency than D-dopa and L-tyrosine and L-leucine had no competing potency (Fig. 4A and B). No specific binding of radiolabelled L-leucine and very low binding of t:tyrosine were detected in nuclei (not shown). The binding of [~H]dopa to nuclei was reversed by the addition of nonradioactive L-dopa (Fig. 4C). The fact that l:dopa, at alkaline pH, can autooxidize to dopaquinone, which will bind exposed sulfhydryl groups, may question the specificity of the observed effect. Here, we argue that the binding was specific for L-dopa. The binding was done under slightly acidic conditions (pH 6.5) and for a relatively short time of incubation (i h), which should inhibit dopa autoxidation. Furthermore, the binding was stereoselective and reversible (Fig. 4). Therefore, it is suggested that the nuclear dopa binding sites are specific for i,-dopa and may be involved in the regulation of melanocyte activity. In summary, the presence of specific binding sites for L-dopa in crude membranes and purified nuclei of rodent malignant melanocytes was demonstrated. Thus, the first step toward identification of putative cell surface dopa ' r e c e p t o r s ' / c a r r i e r s and putative nuclear dopa 'receptors' has been accomplished. The next step

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Fig. 4. Binding of [3H]L-dopa to nuclei. (A) and (B): Competition curves of [3H]L-dopa binding to nuclei. The concentration of [3H]L-dopa was 0.2/zM. Values represent the means from duplicate determinations of total counts minus binding to filters. Ordinate: % of [3H]L-dopa bound. Abscissa: L-dopa ( [ ~ - - - - - - D ) and x-dopa (11 II) were added at various concentrations (0, 1, 10, 100 and 1000 /zM); L-tyrosine (z~ z~) and L-leucine ( • - - - • ) were added (1000/~M). (A), $91 amelanotic melanoma cells; (B), abC1 amelanotic melanoma cells. The binding studies were performed for 1 h at room temperature. (C): Reversibility of [3H]L-dopa binding to nucleis of $91 mouse melanoma cells. The concentration of [3H]L-dopa was 0.1 /zM. Values represent means from triplicate determinations of total counts minus binding to filters. Ordinate: cpm of [3H]L-dopa bound/mg protein. Abscissa: time of binding. (e e), time-course of [3H]L-dopa binding; ( © - - - - - - © ) , time-course of reversion of [3H]L-dopa binding by addition of 1 mM of unlabelled L-dopa after 1 h of incubation (arrowhead).

is a comparative study with nonmelanocytic cells and further molecular characterization of the binding proteins. We thank Mrs. E. Kuklinski from the Dept. of Dermatology, Yale School of Medicine for examination of purified nuclei by electron microscope, and Drs. G. Moellmann, R. Paus, R. Ambros, R. Marois and T. Friedrich for critical reading of the manuscript and valuable comments. The work was supported by Lawrence M. Gelb 'Foundation (Clairol), and BRSG grant SO7RR05395-30 to AS. References 1 Pawelek, J. and Korner, A. (1982) Am. Sci. 70, 136-145. 2 Moellmann, G., Slominski, A., Kuklinska, E. and Lerner, A.B. (1988) Pigment Cell Res. Suppl 1, 79-87.

3 Slominski, A. and Paus, R.J. (1990) Theor. Biol. 143, 123-138. 4 Slominski, A., Moellmann, G., Kuklinska, E., Bomirski, A. and Pawelek, J. (1988) J. Cell Sci. 89, 287-296. 5 Howe, J., Costantino, R. and Slominski, A. (1991) Acta Dermatovenereol. 71, 150-152. 6 Slominski, A., Moellmann, G. and Kuklinska, E. (1989) J. Cell Sci. 92, 551-559. 7 Pawelek, J., Bolognia, J., McLane, J., Murray, M., Osber, M. and Slominski, A. (1988) Prog. Clin. Biol. Res. 256, 143-154. 8 McLane, J., Osber, M. and Pawelek, J. (1987) Biochem. Biophys. Res. Comm. 145, 719-725. 9 Goshima, Y., Nakamura, S., Ohna, K. and Misu, Y. (1991) Neurosci. Lett. 129, 214-216. 10 Misu, Y., Goshima, Y., Nakamura, S. and Kubo, T. Brain Res. (1990) 520, 334-337. l l Komori, K., Fuiji, T. and Nagatsu, 1. (1991) Neurosci. Lett. 133, 203-206. 12 Carlberg, M. (1990)J. Neural Transm. Genet. Sect. 81, 111-119. 13 Rusin, K.I., Bayev, K.V., Batueva, I.V., Safronov, B.V. and Suderevskaya, E.I. (1988) Neirofiziologia 20, 706-708.

328 14 Hulme, E.C. (ed.) (1990) Receptor Biochemistry. A Practical Approach. Oxford University Press, Oxford. 15 Van Der Klis, F.R.M., Wiersinga, W.M. and Vijloder, J.M. (1989) FEBS Lett. 246, 6-12. 16 Iwanij, V. and Hur, K.C. (1985) Proc. Natl. Acad. Sci. USA 82, 325-329. 17 Slominski, A., Jastreboff, P. and Pawelek, J. (1989) Biosci. Rep. 9, 579-587.

18 Laemmli, U.K. (1970) Nature 227, 68U-,~85. 19 Slominski, A. (1991) In Vitro Cell. Dev. Biol. 27A, 735 738. 20 Arkhipova, L.V., Tretyak, T.M. and O~;~din, O.N 119881 Biokhimiya 53, 1078-1(181. 21 Agrup, G., Hansson, C,, Rorsman, H., Rosengren, A.M. and Rosengren, E. (1979) Acta Dermatovener. 59. 355 356.