Agonism, Inverse Agonism, and Neutral Antagonism at the Constitutively Active Human Neurotensin Receptor 2

Abstract

Two G protein-coupled neurotensin (NT) receptors, termed NTR1 and NTR2, have been identified so far. In contrast to the NTR1, which has been extensively studied, little is known about the pharmacological and biological properties of the NTR2. In the course of characterizing NT analogs that exhibited binding selectivity for the NTR2, we discovered that this receptor constitutively activated inositol phosphate (IP) production. Here, we report on the constitutive activity of the human NTR2 (hNTR2) transfected in COS cells and on compounds that exhibit agonism, inverse agonism, and neutral antagonism at this receptor. IP levels increased linearly with time, whereas they remained constant in mock-transfected cells. Furthermore, IP production was proportional to the amount of hNTR2 present at the cell membrane. SR 48692, a nonpeptide antagonist of the NTR1, stimulated IP production, whereas levocabastine, a nonpeptide histamine H1 antagonist that binds the NTR2 but not the NTR1, behaved as a weak partial inverse agonist. NT analogs modified at position 11 of the NT molecule, in particular by the introduction of bulky aromatic D amino acids, exhibited binding selectivity at the hNTR2 and also behaved as partial inverse agonists, reversing constitutive IP production up to 50%. Finally, NT barely affected constitutive IP production but antagonized the effects of both agonist and inverse agonist compounds, thus behaving as a neutral antagonist. The unique pharmacological profile of the hNTR2 is discussed in the light of its sequence similarity with the NTR1 and the known binding site topology of NT and SR 48692 in the NTR1.

Interactions between neurotensin receptors and G proteins

“…However, NT was also found to induce inhibition of adenylyl cyclase and stimulation of arachidonic acid production through interaction with G i/o -type G proteins in selected systems, such as CHO cells transfected with rat or human NTS1 [20,53,56] and rat neuroblastoma N1E115 cell line [11]. Furthermore, this peptide stimulated adenylyl cyclase through interaction with G s in cells transfected with rat or human NTS1 [62,69,79] and in human pancreatic cancer cells endogenously expressing the receptor [31], but not in human colonic adenocarcinoma HT29 cells [1].…”

“…Another step was achieved through the demonstration that human NTS2 expressed in COS cells was constitutively active on InsP production [62]. SR 48692 retained agonist activity towards this transduction pathway, NT behaved as a neutral antagonist, and levocabastine as a weak partial inverse agonist [62].…”

“…SR 48692 retained agonist activity towards this transduction pathway, NT behaved as a neutral antagonist, and levocabastine as a weak partial inverse agonist [62]. When expressed in HEK-293 cells [30], human NTS2 constitutively activated serum response element (SRE) signaling through both pertussis-sensitive G proteins and Rho-GTPases, suggesting coupling to G i/o and G 12/13 -type G proteins.…”

“…It can be suggested that human NTS2 may interact with G q/11 [30,62,76], G i/o [30] and G 12/13 [30]. Whether interaction with G i/o might account for arachidonic acid release [76], as observed with NTS1 [53], remains to be established.…”

“…The constitutive activity of human NTS2 towards InsP production [62] and SRE signaling [30] suggested a ligand-independent activation of G q/11 , G i/o and perhaps G 12/13 . It is not clear whether constitutive activity towards InsP production does not occur in CHO cells or was just missed in previous studies [76].…”