Differential Binding Profile and Internalization Process of Neurotensin via Neuronal and Glial Receptors

Abstract

Two G-protein-coupled receptors for the tridecapeptide neurotensin (NT) have been identified and cloned in mammalian brain: a high-affinity (K d ϭ 0.3 nM) receptor, sensitive to the antagonist SR 48692 but insensitive to levocabastine, and a lower-affinity (K d ϭ 2-4 nM) receptor, sensitive to levocabastine but with poor affinity for SR 48692. Although there is good evidence that the high-affinity site is predominantly expressed in neurons, little is known of the cellular localization of the low-affinity receptor. In the present study, we identify by confocal microscopy selective levocabastine-sensitive, SR 48692-resistant binding of a fluorescent derivative of NT (fluo-NT) to a subpopulation of glial fibrillary acidic protein-immunoreactive glial cells grown in culture from the midbrain and cerebral cortex of embryonic and neonatal rats, respectively. We also demonstrate, by combining fluo-NT detection with tyrosine hydroxylase immunofluorescence, that these glial binding sites are differentially regulated from the SR 48692-sensitive NT receptor expressed in the same cultures by mesencephalic dopamine neurons. Whereas the latter undergoes rapid ligandinduced internalization followed by centripetal mobilization of ligand-receptor complexes from processes to perikarya and from perikaryal periphery to cell center, the former induces the formation of cell-surface clusters that fail to internalize. It is concluded that NT may exert its effects on both neurons and astrocytes in the CNS. Whereas NT neural signaling is exerted through high-affinity receptors and may be partly effected through internalization of receptor-ligand complexes, glial signaling is exerted through low-affinity NT receptors and appears to be transduced exclusively at the level of the plasma membrane.

Presynaptic action of neurotensin on cultured ventral tegmental area dopaminergic neurones

“…However, the increase in spontaneous EPSC frequency caused by NT(8^13) is blocked by SR142948A, a compound with high a⁄nity for both type 1 (NTR1) and type 2 NT (NTR2) receptors Trudeau, L.-E., unpublished results). It has been proposed that dopaminergic neurones preferentially express NTR1 and not NTR2, which may be preferentially expressed in astrocytes (Nouel et al, 1997). It is thus likely that most e¡ects observed here were mediated by NTR1.…”

Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

“…Binding of fluo-NT was also assessed in cells naturally expressing the high-affinity neurotensin receptor (NT-1; SN17 neuron-neuroblastoma hybrid cells (26); primary neurons in culture (27)) or the low-affinity NT receptor (NT-2; astrocytes in culture (27)). Binding was carried out at 4 o C or at 37 o C in the presence of the endocytosis inhibitor, phenylarsine oxide (PAO), to prevent ligand sequestration and/or internalization.…”

Chronic, but not acute, dosing of antipsychotic drugs alters neurotensin binding in rat brain regions

“…Neurotensin has two receptors in the adult brain (Kitabgi et al, 1987), one which has a low anity for NT, termed neurotensin receptor-2 NTR-2 (Chalon et al, 1996), which has been shown to be involved in the formation of ligand-induced glial cell-surface receptor clusters that do not undergo subsequent internalization, the function of which has yet to be determined (Nouel et al, 1997). The second receptor has a high anity for NT and is called neurotensin receptor-1 (NTR-1; Tanaka et al, 1990) and is located presynaptically in the striatum and postsynaptically in the nucleus accumbens (Schotte et al, 1988).…”