RESUMO
The melanocortin 3-receptor is involved in regulating energy metabolism, body fluid composition and inflammatory responses. Melanocortin receptors function by activating membrane bound adenylate cyclase. However, the literature reports indicate that some G protein coupled receptors (GPCRs) can also activate mitogen activated protein kinase (MAPK) or phosphoinositide 3 kinase (PI3K) signaling pathways consequent to their endocytosis. These studies were undertaken to evaluate the role of these pathways in MC3R signaling in brain-stem neuronal cells. Recruitment of arrestins is implicated in the activation of secondary pathways by GPCRs and our data shows the colocalization of either arrestin B1 or B2 with MC3R in endosomes. An alteration in PKB phosphorylation pattern was observed in MC3R expressing cells independent of agonist stimulation. MC3R transfectants exhibited increased proliferation rates and inhibition of PKB pathway with triciribine abrogated cell proliferation in both vector control and MC3R transfectants. PKB is constitutively active in proliferating CAD cells but could be further activated by culturing the cells in differentiation medium. These studies suggest that the AKT/PKB pathway plays an important role in the proliferation of CAD cells and suggest a link between MC3R and cell growth pathways that may involve the alteration of AKT/PKB signaling pathway.
Assuntos
Arrestinas/biossíntese , Tronco Encefálico/metabolismo , Endossomos/metabolismo , Proteína Oncogênica v-akt/genética , Receptor Tipo 3 de Melanocortina/biossíntese , Androstadienos/farmacologia , Antineoplásicos/farmacologia , Arrestinas/genética , Western Blotting , Tronco Encefálico/citologia , Tronco Encefálico/ultraestrutura , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Endossomos/ultraestrutura , Humanos , Microscopia de Fluorescência , Proteína Oncogênica v-akt/fisiologia , Plasmídeos/genética , Receptor Tipo 3 de Melanocortina/genética , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Ribonucleosídeos/farmacologia , Sais de Tetrazólio , Tiazóis , Transfecção , Ubiquitina/farmacologia , Wortmanina , beta-ArrestinasRESUMO
Melanocortins play a central role in autonomic modulation of metabolism by acting through a family of highly homologous G protein-coupled receptors. Studies with gene knockout mice have implicated neural melanocortin receptors, MC3R and MC4R, in the etiology of obesity, insulin resistance, and salt-sensitive hypertension. In an attempt to better understand the mechanisms of function of these receptors, we expressed MC3R and MC4R in neuronal cells and demonstrated their co-localization to several membrane regions. We now show that in cultured neuronal cells, MC3R localizes to lipid rafts and undergoes endocytic internalization upon activation by gamma-MSH through a protein kinase-sensitive pathway. The appearance of the internalized receptor in lysosomes suggests that it is subsequently degraded. The expression of protein kinase A regulatory subunits and of c-Jun and c-Fos was analyzed by either immunoblotting or real-time PCR. No discernable changes were observed in the expression levels of these protein kinase A and protein kinase C responsive genes. Immunohistochemical studies showed a robust expression of MC3R protein in brain nuclei with relevance to cardiovascular function and fluid homeostasis further supporting the notion that the physiological effects of melanocortins on the cardiovascular system arise from effects on the central nervous system.
