Receptor Trafficking via the Perinuclear Recycling Compartment Accompanied by Cell Division Is Necessary for Permanent Neurotensin Cell Sensitization and Leads to Chronic Mitogen-activated Protein Kinase Activation

Abstract

Most G protein-coupled receptors are internalized after interaction with their respective ligand, a process that subsequently contributes to cell desensitization, receptor endocytosis, trafficking, and finally cell resensitization. Although cellular mechanisms leading to cell desensitization have been widely studied, those responsible for cell resensitization are still poorly understood. We examined here the traffic of the high affinity neurotensin receptor (NT1 receptor) following prolonged exposure to high agonist concentration. Fluorescence and confocal microscopy of Chinese hamster ovary, human neuroblastoma (CHP 212), and murine neuroblastoma (N1E-115) cells expressing green fluorescent proteintagged NT1 receptor revealed that under prolonged treatment with saturating concentrations of neurotensin (NT) agonist, NT1 receptor and NT transiently accumulated in the perinuclear recycling compartment (PNRC). During this cellular event, cell surface receptors remained markedly depleted as detected by both confocal microscopy and 125 I-NT binding assays. In dividing cells, we observed that following prolonged NT agonist stimulation, NT1 receptors were removed from the PNRC, accumulated in dispersed vesicles inside the cytoplasm, and subsequently reappeared at the cell surface. This NT binding recovery allowed for constant cell sensitization and led to a chronic activation of mitogenactivated protein kinases p42 and p44. Under these conditions, the constant activation of NT1 receptor generates an oncogenic regulation. These observations support the potent role for neuropeptides, such as NT, in cancer progression. G protein-coupled receptors (GPCRs)1 and their agonists participate in numerous aspects of cellular and tissue regulation (1). Agonist interaction instantly leads to GPCR activation, phosphorylation, sequestration, and cell desensitization. Upon receptor endocytosis, the GPCRs may return to the cell surface directly from sorting endosomes (short cycle) (2). However, a long cycle involving the passage through the perinuclear recycling compartment (PNRC), before reaching the plasma membrane, has been suggested recently (2-4). In the present study, by examining the effect of prolonged agonist exposure on neurotensin receptor (NT1 receptor) intracellular trafficking, we determined the conditions leading to cell agonist re-sensitization, along with the physiological consequences of this recovery on intracellular signalization.Neurotensin (NT) is a brain and gastrointestinal peptide that fulfils many central and peripheral functions through its interaction with specific receptors (5, 6). Three subtypes of NT receptors have been cloned: NT1, NT2, and NT3 (7-9). NT1 and NT2 exhibit high (sub-nanomolar) and low (nanomolar) affinity for NT, respectively, and belong to the family of G proteincoupled receptors. NT3 is a single transmembrane domain receptor with 100% homology to gp95/sortilin (9). All three receptors are internalized after interaction with NT (10 -12). In the periphery, the bulk of NT is released into t...

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