Volume 129, number 1 FEBS LETTERS June 1981 PHOTOAFFINITY LABELLING OF MSH RECEPTORS REVEALS A DUAL ROLE OF CALCIUM IN MELANOPHORE STIMULATION Pierre N. E. DE GRAAN, Alex N. EBERLE* and Frans C. G. VAN DE VEERDONK Zoological Laboratory, State University of Utrecht, 3508 TB Utrecht, The Netherlands and *Institute of Molecular Biology and Biophysics, Swiss Federal Institute of Technology (ETH), 8093 Ziirich, Switzerland Received 7 May 1981 1. Introduction UV irradiation of Xenopus laevis melanophores in the presence of photoreactive [Pap13]-ol-MSH induces irreversible pigment dispersion [ 13. It appears that specific covalent labelling of MSH receptors results in the generation of a continuous signallasting for several hours. This longlasting effect makes it possible to study the involvement of various factors, such as Ca*“, at the different steps (see [2]) of hormonal signal transmission. Ca*+ is well known to be indispensable for medi- ating the effect of (u-MSH on melanophores of several species [3,4]. From earlier studies with melanophores of Rana pipiens [S] and Xenopus laevis [6] it was concluded that the interaction of c+MSH with its receptor leads to the activation of an adenylate cyclase system, as was shown with mouse melanoma cells [ 71. The subsequent pigment dispersion is controlled by cAMPand does not depend on extracellular Ca*+, since the dispersion can be brought about by dbcAMP in the absence of Ca*+ [3,8]. Thus the effect of Ca*” seems to precede the action of CAMP; however, the precise site of Ca*+ action remained unclear. Photoaf- fmity labelling of MSH receptors enabled us to distin- guish between receptor-associated and post-receptor Ca*+ requirement. Here we show that Ca*+ is essential for hormone-receptor binding, for coupling of the hormone receptor to adenylate cyclase and/or for activation of the enzyme. Abbreviations: dbcAMP, dibutyryl CAMP; EGTA, ethylene glycol bis(paminoethy1 ether) N,N’-tetracetic acid; MI, mela- nophore index; MSH, melanotropin (melanocyte-stimulating hormone); Pap, pazidophenylalanine * Present address: MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England ElsevierfNorth-Holland Biomedical Press 2. Materials and methods (w-MSH and [Pap13]a-MSH were prepared by a frag- ment condensation approach using the strategy in [ 91. Racemlzation of a-MSH was performed in 0.1 N NaOH at 60°C for 30 min. Methoxyverapamil (D 600) was a gift of Krioll AG (Ludwigshafen). Photolysis experiments were done as in [ 11, except for variations in the composition of the preincubation and wash media. Briefly, ventral tail-fin pieces (2 X 2 mm) of Xenopus laevis tadpoles were preincubated at 20°C for 20 min in assaymedia with or without Ca*+ (2X 10-3M)orwithD600(8X lo-‘M),andwere then incubated in the peptide solutions for 20 min under normal lamp light. During the next 20 min the tail-fin pieces were exposed to UV irradiation (controls were kept under normal lamp light). The pieces were then washed continuously at 15°C in media with or without Ca*+ or with D 600. Ca*+-free medium was prepared by adding 2 X 10e3 M Mg*+ to Ca*+- and Mg*+-free medium containing 1O-“ M EGTA. Melano- some dispersion was quantified microscopically every 20 min using the melanophore index of [lo]. 3. Results and discussion Pigment dispersion in ventral tail-fin melanophdres of Xenopus tadpoles, induced in vitro by (Y-MSH or [Pap13]+MSH, depends on the presence of extracel- lular Ca2+ (fig.1). In Ca*+-free medium melanosome dispersion is completely abolished or readily reversed; readdition of Ca*+ to medium containing the hormone leads to normal dispersion. These results confirm that stimulation of tadpole melanophores by (Y-MSH requires Ca*+, as was shown with melanophores of 113