Abstract

The role and signaling properties of the low-affinity neurotensin receptor (NTS2) are still controversial. In particular, it is unclear whether neurotensin acts as an agonist, inverse agonist, or antagonist at this site. In view of the growing evidence for a role of NTS2 in antinociception, the elucidation of the pharmacological and coupling properties of this receptor is particularly critical. In the present study, we demonstrate that in Chinese hamster ovary (CHO) cells expressing the rat NTS2 receptor, neurotensin (NT), levocabastine, neuromedin N, and the high-affinity NT receptor antagonist SR48692 [2-{[1-(-7-chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carbonyl]amino}adamantane-2-carboxylic acid] all bind to and activate the NTS2 receptor. This activation is followed by ligand-induced internalization of receptor-ligand complexes, as evidenced by confocal microscopy using a fluorescent NT analog. All compounds tested produced a rapid and sustained activation of extracellular signal-regulated kinases 1/2 (ERK1/2) but were without specific effect on Ca²⁺ mobilization. The agonist-induced activation of ERK1/2 was completely abolished by preincubation of the cells with the endocytosis inhibitors phenylarsine oxide and monodansylcadaverine as well as overexpression of a dominant-negative mutant of dynamin 1 (DynK44A), indicating that receptor internalization was required for ERK1/2 activation. NTS2-induced activation of ERK1/2 was not species-specific, because the same agonistic effects of NT and analogs were observed in CHO cells transfected with the human NTS2 receptor. In conclusion, this study demonstrates that NTS2 is a bona fide NT receptor and that activation of this receptor by NT or NT analogs results in an internalization-dependent activation of the ERK1/2 signaling cascade.