Neurotensin Agonist Induces Differential Regulation of Neurotensin Receptor mRNA

Abstract

The binding of neurotensin (NT) to specific receptors triggers the multiple functions that NT exerts in both periphery and brain. By studying the effect of the concentration and time of NT agonist exposure, two separate regulatory mechanisms were detected for the neurotensin receptor (NTR) gene in human colonic adenocarcinoma cells (HT-29).The incubation of cells for 6 h with the NT agonist, JMV 449, resulted in an increase of 270% in NTR mRNA levels. These changes were the direct result of new NTR gene transcription, as indicated by run-on and half-life experiments. In addition, the transcriptional activation of the NTR gene was dependent on NT-receptor complex internalization and de novo protein synthesis.A second response was detected with prolonged exposure to JMV 449. In this case, a decrease of 70% was detected in NTR mRNA levels. Unlike the initial phase, this change was mediated by a post-transcriptional event as the half-life of NTR mRNA from treated cells decreased by 50% as compared with control cells.NT agonist appears to regulate the synthesis of NTR mRNA. In HT-29 cells, this feedback is exerted by a biphasic response. These phases are apparently independent and mediated by two separate mechanisms. Neurotensin (NT)1 is a tridecapeptide, widely distributed in the central nervous system and peripheral tissues, exerting multiple functions (1). In the central nervous system, NT is a neurotransmitter as well as a neuromodulator of other neurotransmitters such as dopamine, acetylcholine, serotonin, and noradrenaline (2, 3). NT also possesses neuroendocrine actions inducing the release of several pituitary hormones (4). In the periphery, NT is secreted from mucosal endocrine cells of the small intestine into the circulation (5). In the gastrointestinal tract, NT causes many physiological effects including the stimulation of pancreatic secretion, the facilitation of colonic motility and fatty acid translocation, and tissue growth (6).In rat, NT actions are mediated by the stimulation of several specific receptors exhibiting high or low affinity for NT (7,8).The high affinity neurotensin receptor (NTR) is composed of 424 amino acids and belongs to the seven-transmembrane domain receptor family coupled to the G-proteins (7). The human NTR counterpart has also been cloned from human colonic adenocarcinoma cells (HT-29) (9). When HT-29 cells are challenged with a NT agonist, phosphatidylinositols are hydrolyzed leading to Ca 2ϩ mobilization (10). In contrast to N1E-115 cells, stimulation by NT in HT-29 cells is not associated with protein kinase C activation (10, 11).In addition to triggering cellular responses by specific ligands, receptors are often themselves regulated by their own agonists. In the case of NT, several studies have shown that variations in NTR expression were caused by changes in NT levels. For example, acute agonist stimulation of NTR induces desensitization and down-regulation of receptor in primary cultures of rat forebrain and HT-29 cells (12,13). Prolonged exposure of N1E-115 cells to N...

Activation of Receptor Gene Transcription Is Required to Maintain Cell Sensitization after Agonist Exposure

“…The ability to separate transcriptional up-regulation from post-transcriptional destabilization in HT-29 cells peaked our interest previously, because these experiments suggested that these mechanisms were entirely independent (20). In this paper, we confirm this interpretation because only one of the two mechanisms is present in the two cell lines.…”

“…The initial mechanism detectable after 6 h of agonist exposure, resulted in the activation of the NTR gene transcription. The second mechanism could only be detected after prolonged agonist exposure (72 h), and was post-transcriptional, resulting in the destabilization of NTR mRNA (20). Similar results are also found for the ␤2-adrenergic receptor where a short term agonist exposure stimulated the rate of the receptor gene transcription and long term agonist exposure induced the destabilization of the (38,39).…”

“…Both Modes of NTR mRNA Regulation Are Present in Vivo-We have previously shown that both types of NTR mRNA regulation, activation of NTR gene transcription and destabilization of NTR mRNA, could be present in the same cell line (20). It could be surmised that in vivo the cells possess one or both NTR mRNA regulatory mechanisms.…”

“…We have previously demonstrated that agonist-induced NTR expression is regulated at the transcriptional and post-transcriptional level (20) and that these mechanisms act independently of each other. In this study, our objective was to examine the effect of NTR expression variation due to NT agonist on cell sensitivity and on target gene stimulation (e.g.…”

Ligand-gated Ion Channels

“…A nonpeptide antagonist, SR142948A, shows high affinity (pKi~9) at both NTS1 and NTS2 receptors (Gully et al, 1997). Rank order of potency Neurotensin > neuromedin N (Hermans et al, 1997) Neurotensin = neuromedin N (Mazella et al, 1996) Selective agonists JMV449 (Souaze et al, 1997) Levocobastine (Mazella et al, 1996) Selective antagonists SR48692 (7.5-8.2; Gully et al, 1997) - Labbe-Jullie et al, 1995) -Neurotensin appears to be a low-efficacy agonist at the NTS2 receptor (Vita et al, 1998), while the NTS1 receptor antagonist SR48692 is an agonist at NTS2 receptors (Vita et al, 1998). An additional protein, provisionally termed NTS3 (also known as NTR3, gp95 and sortilin; ENSG00000134243), has been suggested to bind lipoprotein lipase and mediate its degradation (Nielsen et al, 1999).…”