Inhibition by melittin and fluphenazine of melanotropin receptor function and adenylate cyclase in M2R melanoma cell membranes.

Abstract

Melanotropin (MSH) receptor activity in the M2R mouse melanoma cell line is tightly controlled by calcium by an unknown mechanism. The possibility that calcium regulation is mediated by calmodulin or a calmodulin-related calcium binding protein has been addressed in this report by studying the effects of two known calmodulin antagonists, fluphenazine and melittin, on MSH receptor function. Stimulation of adenylate cyclase (AC) in M2R plasma membranes by beta MSH was strongly inhibited by both antagonists. The concentrations of fluphenazine and melittin yielding half-maximal inhibition (IC50) of AC were 16 microM and 2.4 microM, respectively. Both fluphenazine and melittin also inhibit prostaglandin E1-, GTP gamma S, and forskolin-stimulated AC activity, as well as that of unstimulated enzyme, although inhibition is shown to occur at significantly higher concentrations of antagonist. We have shown that the calcium-dependent rate-limiting step in MSH stimulation of adenylate cyclase, that of hormone binding, is strongly inhibited by these antagonists at concentrations identical to, if not lower than, those required for the inhibition of AC activity (fluphenazine-IC50, 14 microM; melittin-IC50, 0.7 microM). The actions of these antagonists, furthermore, appear to be calcium insensitive, as melittin affects the stability of both the high affinity (calcium containing) and low affinity (calcium depleted) receptor-MSH complexes. The sensitivity of the MSH receptor to inhibition by calmodulin antagonists resembles that described for purified calmodulin-sensitive enzyme systems, which suggests a possible role for calmodulin in MSH receptor function. Among peptide hormone receptors, this effect by calmodulin antagonists appears to be unique for the MSH receptor.