General

The tridecapeptide neurotensin (NT) mediates its central and peripheral effects through interaction with three identified receptor subtypes, referred to as NTS₁, NTS₂ and NTS₃ (Sortilin 1). NTS₁ and NTS₂ belong to the 7 transmembrane domain/GPCR family, whereas Sortilin 1 is a single transmembrane domain receptor. Therefore, the following introduction summarises only the properties of the two GPCRs NTS₁ and NTS₂.

Neurotensin

Neurotensin, first isolated from bovine hypothalami by Carraway and Leeman in 1973 [9], was also found in the intestine. Classically, NT was synthesised from a precursor that contains another peptide, Neuromedin N, closely related to NT in terms of sequence and activity [11]. Cerebral administration of NT modulates dopaminergic transmission and leads to hypothermic and naloxone-independent analgesic responses. In the periphery, NT induces hypotension, decreases gastric acid secretion, activates lipid digestion and protects pancreatic beta cells from cytotoxic agent-induced apoptosis [8].

Receptor types

The existence of two distinct receptor sites for NT has been evidenced by ligand binding studies on rat brain synaptic membranes [19]: a high affinity NT binding site, later cloned as the NTS₁ receptor, and a lower affinity NT binding site (sensitive to the H₁ anti-histamine levocabastine), the NTS₂ receptor.

NTS₁ was first cloned from rat brain by Tanaka et al. [26]. Human NTS₁ was cloned from the colonic adenocarcinoma cell line HT29 and shares 84% homology with the rat NTS₁ receptor [27]. NT binds to these NTS₁ receptors with an affinity of 0.1-0.4 nM, in comparison with 2-5 nM for NTS₂. With regard to G protein-coupling, NTS₁ is a G_q-preferring receptor since in almost all systems examined the binding of NT to NTS₁ activated phospholipase C [1-2,13,16,21,25,29]. However, coupling of NTS₁ to G_i/o and to G_s has been observed in some expression systems [3,7,13,15,21,30].

Activation of NTS₁ is probably responsible for the observed effects of NT on cancer cell proliferation and food intake. However, the most convincing implication of NTS₁ is related to the NT-dopamine interactions in the brain. Indeed, NT modulates dopamine transmisssion in the nigro-striatal and mesocorticolimbic pathways through NTS₁, indicating that NT analogues specifically targeting this receptor might represent a new class of antipsychotic drugs.

NTS₂ was cloned from mouse and rat brain [10,18] with the use of a NTS₁ homology screening protocol. Human NTS₂ was further cloned [28] and is 82% identical to the rat and mouse counterparts. Pharmacologically, mouse and rat NTS₂ can be easily distinguished from other NT receptors by levocabastine, a histamine H₁ antagonist, which totally and selectively inhibits the binding of NT to NTS₂. Signal transduction of NTS₂ is unclear because it depends on the cell system of expression used and on the receptor species. For example, NT activates mouse NTS₂ when expressed in oocytes [5,18] but is unable to activate the same receptor expressed in HEK cells [4]. Rat NTS₂ has been shown to be involved in NT-induced Erk1/2 phosphorylation by a mechanism that is dependent on receptor internalisation [14,24]. In contrast, human NTS₂ can increase cytosolic calcium only when activated by the selective NTS₁ antagonist SR48692, and NT has been shown to inhibit this effect as an antagonist [5,28]. The difficulty in studying the signal transduction mechanism of NTS₂ certainly comes from its ability to be constitutively active [23].

NTS₂ contributes in the protective effect of NT on pancreatic beta cells [8]. NTS₂ has been described to be responsible for the analgesic response of centrally administered NT [12] . This observation is enhanced by the cerebral localisation of both its messenger RNA and its protein in structures implicated in the descending control of nociceptive imputs, especially in the periaqueductal gray and dorsal raphe. However, the expression of NTS₂ immunoreactivity in areas devoid of neurotensinergic inputs indicates that NT might not be the exclusive endogenous ligand for NTS₂.

Receptor structure

Although NTS₂ belongs to the rhodopsin-like GPCR family, it has an unusual feature in its structure compared with those of NTS₁ and other GPCRs. The Asp residue at position 2.50 in transmembrane helix 2 is a common characteristic of most rhodopsin-like GPCRs, including NTS₁. For NTS₂, the Asp residue is replaced by Ala⁷⁹ in rat and mouse and by Gly⁷⁹ in the human receptor. The absence of the Asp residue in helix 2 leads to a receptor that is much less sensitive to sodium ions [17]. Another special characteristic of the NTS₂ is the absence of N-glycosylation sites in extracellular sequences. A splice variant corresponding to a deletion of 181 base pairs has been identified in the mouse and the rat brain [6,22]. The expressed protein is truncated to 281 amino acids and bears only five transmembrane domains. The rat splice variant binds NT with very poor affinity (10 μM) [22].

Receptor regulation and expression

The differential regulation of the mRNA for NTS₁ by NT receptor agonists has been observed in several cell systems. The expression of NTS₁ was found to be differentially regulated at the transcriptional and post-transcriptional levels, depending on the duration of exposure and the concentration of the agonist. Levels of mRNA encoding NTS₂ are increased in astrocytes in response to a brain stab wound [20]. Agonist stimulation of NTS₂ results in desensitization in the oocyte expression system [18].

NTS₁ is expressed mainly in the brain and intestine. In the rat brain, high levels of NTS₁ are found in neurons of the diagonal band of Broca, medial septal nucleus, magnocellular preoptic area, suprachiasmatic nucleus, substantia nigra and ventral tegmental area. NTS₁ is selectively expressed by central dopamine neurons and is clearly associated with mesolimbic and mesocortical systems. NTS₂ is essentially expressed in the brain. High densities of NTS₂ are detected in the olfactory bulb, bed nucleus of the stria terminalis, amygdaloid complex, substantia nigra, ventral tegmental, and several brainstem nuclei. High levels of expression are also found in regions involved in descending control of nociception.

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