Photoaffinity Labelling of Msh Receptors on Anolis Melanophores: Irradiation Technique and Msh Photolabels for Irreversible Stimulation

Abstract

Excised dorsal skin of Anolis carolinensis was exposed to high intensity UV-irradiation in the presence of different photoreactive α-MSH derivatives. The resulting covalent binding of the hormone to its receptor induced irreversible pigment dispersion. The duration of the longlasting response depended on the type and length of irradiation; it was maximal after two 5 min irradiation phases with a light intensity of ∼180 mW/cm² and a spectrum from 310 to 550 nm, fresh hormone being added after the first phase. [Nα-(4-Azidophenyl-acetyl-serine¹]-α-MSH (I), [2′-(2-nitro-4-azidophenylsulphenyl)-tryptophan⁹]-α-MSH (II) and [p-azidophenylalanine¹³]-α-MSH (III) all inserted into the receptor to about the same extent, as judged from the persistence of the longlasting signal. In contrast, [D-alanine¹, p-azidophenylalanine², norvaline⁴]-α-MSH (IV) and [Nα-(4-azidophenylacetyl)-serine¹, leucine⁹]-α-MSH (V) gave much less insertion and [leucine⁹, p-azidophenylalanine¹³]-α-MSH (VI) hardly any insertion when applied in the same relative excess (5-fold the concentration inducing a maximal response). Covalent attachment of the cleavable photolabel [Nα-(4-azidophenyl)-1, 3′-dithio-propionyl-serine¹]-α-MSH (VII) and subsequent washing of the skin in buffer containing 1% β-mercaptoethanol released the peptide from the receptor. Insertion of the C-terminal photolabel [p-azidophenylalanine¹³]-α-MSH was reduced by the weak antagonist H-Phe-Ala-Trp-Gly-Gly-Pro-Val-NH₂. These experiments prove that hormone receptors can be covalently labelled in tissue with very limited light transparency.