Melanophore Based Receptor Functional Activity

Melanophore cells derived from the neural crest of Xenopus laevis offer a highly sensitive system for the screening of ligands for GPCRs. The amphibian mela-nophores contain pigmented organelles called melanosomes, which are filled with the dark pigment melanin. Many Gs- and Grcoupled receptors have been shown to influence pigment dispersion and aggregation after stimulation with the appropriate ligands.

The state of pigment distribution within a cell reflects activation of AC or phospholipase C.75 When melanophores are stimulated by agonists such as aMSH that elevate cAMP above basal levels, melanophore dispersion is induced in the cytoplasm and the cells will appear dark. Inhibition of cAMP production results in melanophore aggregation into cells and they appear light. Therefore, the melanophore system can be used as an endogenous signaling technique to mediate cell darkening or lightening due to pigment dispersion and aggregation, respectively. The pigment dispersion and aggregation can be determined in a microtiter plate by measuring absorbance or by imaging the cell response. The main advantage of this format is its application to Gai-coupled receptors. DeCamp et al. reported the expression of more than 100 GPCRs in melanophores.76 However, melanophores themselves express a range of endogenous GPCRs, and the specificity of compounds hitting the receptor of interest should be elucidated by other methods. This technology may have application for the study of orphan GPCRs for which functional expression in mammalian cells is difficult to achieve. The assay platform is simple, automatable, and miniaturizable to a 1536-well format, and one can efficiently screen for inverse agonists.77

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