Neurotensin Receptor Type Research Articles

The effect of neurotensin on insulin-induced proliferation of human fibroblasts

Neurotensin has been shown to influence growth in a number of cancerous and non-cancerous cells and to enhance the proliferative effects of growth factors without itself inducing proliferation. Here we show that neurotensin potentiates the proliferative effects of insulin on IMR90 human fibroblasts in a concentration and neurotensin receptor type 1-dependent manner. This potentiating effect of neurotensin was blocked by inhibitors of phospholipase C and protein kinase C, was accompanied by an increase in the level of soluble inositol phosphates and did not involve an autocrine factor. These results show that neurotensin can enhance insulin-dependent proliferation of human fibroblasts and suggest a possible role for neurotensin in tissue growth and repair.

Neurotensin depolarizes globus pallidus neurons in rats via neurotensin type-1 receptor

The globus pallidus is a major component in the indirect pathway of the basal ganglia. There is evidence that neurotensin receptors exist in this nucleus. To determine the electrophysiological effects of neurotensin on pallidal neurons, whole-cell patch-clamp recordings were performed in the acutely prepared brain slices. Under current-clamp recordings, neurotensin at 1 μM depolarized pallidal neurons. Voltage-clamp recordings also showed an inward current induced by neurotensin. The depolarizing effect of neurotensin could be mimicked by the C-terminal fragment, neurotensin (8–13), but not by the N-terminal fragment, neurotensin (1–8). Both SR 142948A, a non-selective neurotensin receptor type-1 and type-2 antagonist, and SR 48692, a selective type-1 receptor antagonist, blocked the depolarizing effect of neurotensin, and which themselves had no effect on membrane potential. Thus, neurotensin type-1 receptors appear to mediate the effect of neurotensin. The depolarization evoked by neurotensin persisted in the presence of tetrodotoxin, ionotropic and metabotropic glutamate and GABA receptor antagonists, indicating that neurotensin excited the pallidal neurons by activating the receptor expressed on the neurons recorded. Current-voltage relationship revealed that both the suppression of a potassium conductance and the activation of a cationic conductance are involved in the neurotensin-induced depolarization. Based on the action of neurotensin in the globus pallidus we hypothesize that alterations of the striatopallidal neurotensin system contribute to symptoms of basal ganglia motor disorders.

Neurotensin regulates DARPP-32 thr34 phosphorylation in neostriatal neurons by activation of dopamine D1-type receptors.

Neurotensin modulates dopaminergic transmission in the nigrostriatal system. DARPP-32, a dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa, is phosphorylated on Thr34 by cAMP-dependent protein kinase, resulting in its conversion into a potent inhibitor of protein phosphatase-1 (PP 1). Here, we examined the effect of neurotensin on DARPP-32 Thr34 phosphorylation using mouse neostriatal slices. Neurotensin stimulated DARPP-32 Thr34 phosphorylation by 4-7-fold with a K(0.5) of approximately 50 nM. The effect of neurotensin was antagonized by a combined neurotensin receptor type-1 (NTR1)/type-2 (NTR2) antagonist, SR142948. It was not antagonized by a NTR1 antagonist, SR48692 or by a NTR2 antagonist, levocabastine; neither was it antagonized by the two combined. Pretreatment with TTX or cobalt abolished the effect of neurotensin. The effect of neurotensin was antagonized by a dopamine D1 antagonist, SCH23390, and by ionotropic glutamate receptor antagonists, MK801 and CNQX. These results indicate that neurotensin stimulates the release of dopamine from nigrostriatal presynaptic terminals in an NMDA receptor- and AMPA receptor-dependent manner, leading to the increase in DARPP-32 Thr34 phosphorylation. Neurotensin stimulated the phosphorylation of Ser845 of the AMPA receptor GluR1 subunit in wild-type mice but not in DARPP-32 knockout mice. Thus, neurotensin, by stimulating the release of dopamine, activates the dopamine D1-receptor/cAMP/PKA/DARPP-32/PP 1 cascade.

Neurotensin induces mating in Saccharomyces cerevisiae cells that express human neurotensin receptor type 1 in place of the endogenous pheromone receptor.

Heterologous expression of the human neurotensin receptor type I (hNT1-R) has been achieved in the yeast Saccharomyces cerevisiae. Immunoanalysis of membranes prepared from cells expressing a c-myc-tagged version of hNT1-R revealed multiple c-myc cross-reacting polypeptides of high molecular mass, suggesting that hNT1-R was glycosylated in yeast. High-affinity binding sites for 125I-labeled-[monoiodo-Tyr3]neurotensin ([125I-Tyr3]NT) were detected on hNT1-R-expressing cells with Kd and Bmax values of 3.2 nM and of 500 receptors per cell, respectively. Competition binding studies of neurotensin with SR142948 and SR48692, two nonpeptidic antagonists of hNT1-R, indicated that the yeast-produced recombinant receptor displayed the same pharmacological properties as hNT1-R expressed in mammalian cells. Interestingly, neurotensin activated the pheromone pathway in hNT1-R-expressing cells in a dose-dependent fashion, as revealed by a beta-galactosidase activity assay with a pheromone-responsive Fus1:lacZ construct. Mutational inactivation of the SST2 and STE2 genes increased the level of beta-galactosidase activity in response to neurotensin by twofold. Recombinant hNT1-R-producing cells, which lacked the endogenous G-protein-coupled receptor for the alpha pheromone, mated with wild-type MATalpha haploid cells in response to neurotensin, leading to bona fide diploid zygote formation. This is the first report of a mammalian receptor that can replace the endogenous pheromone receptor when produced in yeast, by signaling a fully effective, agonist-induced, mating process.

Contulakin-G, an O-Glycosylated Invertebrate Neurotensin

We have purified contulakin-G, a 16-amino acid O-linked glycopeptide (pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu-OH, pGlu is pyroglutamate) from Conus geographus venom. The major glycosylated form of contulakin-G was found to incorporate the disaccharide beta-D-Galp-(1-->3)-alpha-D-GalpNAc-(1-->) attached to Thr10. The C-terminal sequence of contulakin-G shows a high degree of similarity to the neurotensin family of peptides. Synthetic peptide replicates of Gal(beta-->3) GalNAc(alpha-->)Thr10 contulakin-G and its nonglycosylated analog were prepared using an Fmoc (9-fluorenylmethoxycarbonyl) protected solid phase synthesis strategy. The synthetic glycosylated con- tulakin-G, when administered intracerebroventricular into mice, was found to result in motor control-associated dysfunction observed for the native peptide. Contulakín-G was found to be active at 10-fold lower doses than the nonglycosylated Thr10 contulakin-G analog. The binding affinities of contulakin-G and the nonglycosylated Thr10 contulakin-G for a number of neurotensin receptor types including the human neurotensin type 1 receptor (hNTR1), the rat neurotensin type 1 and type 2 receptors, and the mouse neurotensin type 3 receptor were determined. The binding affinity of the nonglycosylated Thr10 contulakin-G was approximately an order of magnitude lower than that of neurotensin1-13 for all the receptor types tested. In contrast, the glycosylated form of contulakin-G exhibited significantly weaker binding affinity for all of the receptors tested. However, both contulakin-G and nonglycosylated Thr10 contulakin-G were found to be potent agonists of rat neurotensin receptor type 1. Based on these results, we conclude that O-linked glycosylation appears to be a highly unusual strategy for increasing the efficacy of toxins directed against neurotransmitter receptors.