Involvement of multiple sulfhydryl groups in melatonin signal transduction in chick brain

To gain insight into the molecular mechanism underlying melatonin binding and signal transduction in the chick brain, we have investigated the role of -SH groups, using a sulfhydryl alkylating reagent N-ethylmaleimide (NEM). At least two -SH groups are involved in the formation of the receptor-G protein complex: one is sensitive to and the other relatively insensitive to NEM. Alkylation of the sensitive group selectively abolishes high affinity binding of 2-[125I]iodomelatonin ([125I]MEL), similar to the effect induced by GTP, thus leading to a complete loss of sensitivity to nucleotides. Modification of both groups causes a marked reduction in binding capacity. Agonists with high affinity, but not other compounds with low affinity for the melatonin receptor, protect against alkylation by NEM. GTP gamma s does not significantly alter the reactivity of -SH groups towards NEM, but agonist-protected receptors remain sensitive to this nucleotide. Moreover, NEM pretreatment blocks the inhibitory effect of melatonin on forskolin-stimulated adenylate cyclase activity in chick brain. These data suggest that the -SH group modulating agonist affinity may lie within the coupling domain between the receptor and G protein but outside of the GTP binding site. In addition, sulfhydryl groups are essential for melatonin binding and signal transduction in chick brain.