MT1 melatonin receptor internalization underlies melatonin‐induced morphologic changes in Chinese hamster ovary cells and these processes are dependent on Gi proteins, MEK 1/2 and microtubule modulation

Abstract

Melatonin induces cellular differentiation in numerous cell types. Data show that multiple mechanisms are involved in these processes that are cell-type specific and may be receptor dependent or independent. The focus of this study was to specifically assess the role of human MT1 melatonin receptors in cellular differentiation using an MT1-Chinese hamster ovary (CHO) model; one that reproducibly produces measurable morphologic changes in response to melatonin. Using multiple approaches, we show that melatonin induces MT1-CHO cells to hyperelongate through a MEK 1/2, and ERK 1/2-dependent mechanism that is dependent upon MT1 receptor internalization, Gi protein activation, and clathrin-mediated endocytosis. Using immunoprecipitation analysis, we show that MT1 receptors form complexes with Gi(alpha) 2,3, Gq(alpha), beta-arrestin-2, MEK 1/2, and ERK 1/2 in the presence of melatonin. We also show that MEK and ERK activity that is induced by melatonin is dependent on Gi protein activation, clathrin-mediated endocytosis and is modulated by microtubules. We conclude from these studies that melatonin-induced internalization of human MT1 melatonin receptors in CHO cells is responsible for activating both MEK 1/2 and ERK 1/2 to drive these morphologic changes. These events, as mediated by melatonin, require Gi protein activation and endocytosis mediated through clathrin, to form MT1 receptor complexes with beta-arrestin-2/MEK 1/2 and ERK 1/2. The MT1-CHO model is invaluable to mapping out signaling cascades as mediated through MT1 receptors especially because it separates out MEK/ERK 1/2 activation by MT1 receptors from that of receptor tyrosine kinases.

Melatonin and Pancreatic Islets: Interrelationships between Melatonin, Insulin and Glucagon

“…The same authors suggest that hMT1 and Gi-, but not hMT1 and Gq-coupling, is affected by this mechanism. Another paper [91] also supports the idea that desensitization is linked to modulation of microtubuli aggregation and MT1 receptor internalization, as well as a change in cell morphology. Some data indicate that downregulation of the hMT1 is dependent on receptor density and/or constitutive activity [92].…”

Melatonin treatment induces apoptosis through regulating the nuclear factor-κB and mitogen-activated protein kinase signaling pathways in human gastric cancer SGC7901 cells

“…Although the mechanism of melatonin with respect to the downregulation of cell proliferation in cancer is not fully understood, it has been demonstrated that in ovarian CHO cells the ERK1/2 pathway can be activated by melatonin treatment through the MT1 receptor (44). Melatonin may affect the phosphorylation of JNK and p38 in hepatocellular carcinoma cells (45).…”

Melatonin and breast cancer: cellular mechanisms, clinical studies and future perspectives

“…Recently, microtubules have been implicated in melatonin receptor signalling through a direct effect on Gi activation and also through receptor trafficking and sequestration of MAPK signalling components (Refs 41, 42). …”