Evidence for the interaction of protein kinase C and melanocortin 3-receptor signaling pathways

Abstract

The melanocortin-3 receptor, MC3-R, is abundant in the brain and is activated by γ-2-melanocyte stimulating hormone (γ-2-MSH). We have previously reported the translocation of protein kinase C (PKC) in spontaneous hypertensive rat (SHR) brain synaptosomes treated with γ-2-MSH. In this study, the expression of PKA and the related PKB in SHR brain synaptosomes was analyzed. PKA was detected in total synaptosomal fractions but not in particulate fractions, whereas PKB was not detected in either fraction. We next tested the hypothesis that the PKC pathway is involved in MC3-R signaling in a neuronal, CAD, cell line. Mobilization of intracellular Ca 2+ was analyzed by dual fluorescence imaging of Fura-2AM loaded MC3-R transfected cells. An increase in intracellular Ca 2+ was observed upon treatment with γ-2-MSH. A MC3-R-green fluorescent protein (GFP) fusion protein was expressed and shown to localize mainly to the plasma membrane in the soma and to neurites in differentiated CAD cells. Treatment with γ-2-MSH led to a punctate appearance and co-immunoprecipitation of the receptor fusion protein with protein kinase C-γ (PKC-γ). Differentiation of some neuronal cells has been shown to be associated with changes in the expression levels of protein kinase C isoenzymes. Induction of CAD cell differentiation was associated with down-regulation of the atypical PKC-ζ and protein kinase B (PKB/Akt1), that was less pronounced in MC3-R transfected cells. However, the levels of classical PKC isozymes, PKC-α, PKC-γ, and PKC-β were unchanged. These studies therefore indicate a role for PKC isozymes in γ-2-MSH/MC3-R receptor signaling and in neuronal cell differentiation.