CALMODULIN-BINDING PEPTIDES INTERFERE WITH MELANOCYTE-STIMULATING HORMONE-RECEPTOR ACTIVITY AND STIMULATE ADENOSINE 3,5-MONOPHOSPHAT PRODUCTION IN M2R MOUSE MELANOMA-CELLS

The MSH receptor belongs to a unique class of G-protein-coupled recept ors, in which calcium ions control the binding affinity of MSH by a ye t unknown mechanism. Possible involvement of a calcium-binding protein [e.g. calmodulin (CaM)] in the regulation of MSH receptor activity ha s been studied in the M2R mouse melanoma cell line. In this study, we tested the inhibitory effects of a group of calmodulin-binding peptide s (CBPs) on MSH receptor activities in intact M2R cells and membrane p reparations derived from them. We also report here on stimulatory effe cts of CBPs on cAMP production in M2R cells that could not be produced in other cell lines lacking MSH receptors. This group of CBPs include s synthetic peptides comprising the CaM-binding domains of Ca2+/CaM-de pendent enzymes, cytotoxic venom peptides, and peptide hormones that h ave been reported to directly interact with CaM. The results show that CBPs, at micromolar concentrations, inhibit MSH binding and consequen t adenylate cyclase stimulation in a specific and concentration-depend ent manner, but have no effect on adenylate cyclase stimulation by pro staglandin E(1). On the other hand, when MSH was omitted and forskolin (0.5-1 mu M) was added instead, CBPs had the opposite effect on cAMP production, stimulating it in M2R cells, but not in other cell types t ested. Thus, these peptides can be considered as antagonists of MSH re ceptor and partial agonists of M2R adenylate cyclase. In contrast to M SH, the stimulatory effects of CBPs were unaffected by EGTA, suggestin g a Ca2+-independent action of these peptides. Using phospholipid vesi cles and M2R cells, we recently showed that CBP activity in M2R cells may include direct partition into the lipid bilayer of the cell membra ne, permitting interaction with hydrophobic lipid-inserted domains of components of the signal transducing machinery. Based on these finding s, we suggest that the mechanism of action of CBPs in the M2R cells in cludes two major components: 1) interaction with the cell surface memb rane and penetration into the lipid milieu, and 2) interaction with ex posed or lipid-embedded protein epitopes intrinsically associated with the MSH-receptor system, thereby affecting the MSH receptor cascade.