Induction of constitutive melanogenesis in amelanotic mouse melanoma cells by transfection of the human melanocortin-1 receptor gene

Abstract

The human melanocortin-1 (MC1) receptor was stably expressed in the amelanotic mouse melanoma cell clone B16-G4F which does not express its own (mouse) MC1 receptor and hence is unresponsive to alpha melanocyte stimulating hormone (alpha MSH). From several stable transfectant cell lines expressing the human MC1 receptor in relatively high numbers, three melanin producing clones (G4F-12, 14, and 15) and one amelanotic clone (G4F-7) were further analyzed in competition binding experiments and in cAMP and melanin assays. The dissociation constants (KD) for [Nle4, D-Phe7]-alpha MSH in all four clones ranged from 0.187 to 0.705 nmol/l, thus corresponding to the KD observed with the different human melanoma cell lines so far studied. Intracellular cAMP content was 3- to 5-fold higher than that of control cells, and alpha MSH induced an additional 1.5- to 1.7-fold increase. G4F-15 cells secreted melanin into the medium whereas the other clones did not secrete melanin. The extent of melanin secretion was similar to that of fully alpha MSH-stimulated B16-F1 mouse melanoma cells but the onset of secretion was delayed. alpha MSH induced an additional dose-related increase (up to 1.3-fold) in melanin production which could be suppressed by the addition of specific alpha MSH antibodies without altering the constitutive part of melanogenesis. Human and mouse agouti proteins, which inhibit basal and alpha MSH-induced melanogenesis in B16-F1 cells, both reduced alpha MSH-induced melanin production in G4F-15 cells but did not affect the constitutive melanogenesis. These results indicate that human MC1 receptor expressed in mouse B16-G4F cells induces constitutive activation of the signalling pathway controlling melanogenesis, most likely by tightly coupling to Gs alpha, in a similar manner to that reported for constitutively active receptor mutants in other systems.

Peptide Hormone Covalently Bound to Polyelectrolytes and Embedded into Multilayer Architectures Conserving Full Biological Activity

“…The melanin production on bare plastic and unmodified multilayers is due to a low constitutive activity of the MC1 receptor. 17 To verify that the decreasing response of the cells with CP1/2 (CP1 or CP2) position in the film, in the cAMP assay, was not due to degradation of the multilayers, we preincubated, for 12 h, different PLL-CP1 containing multilayers with cell supernatants. These supernatants were obtained from B16-F1 cells cultivated for 2 h in serum free MEM containing 0.2% BSA, the same culture conditions as those used for cAMP assay.…”

“…The experiments were done in the absence of phosphodiesterase inhibitors. 17 AFM Imaging. AFM height mode images of PEI(PSS/ PAH) 2 PGA/PLL-CP1(PGA/PLL) 2 multilayers on a silica surface were taken by using a Nanoscope III (Digital Instruments, Santa Barbara, CA) equipped with a silicon nitride cantilever (model MLCT-AUHW Park Scientific, Sunnyvale, CA) having a spring constant of 0.03 N/m.…”