Intermolecular cross-talk between NTR1 and NTR2 neurotensin receptor promotes intracellular sequestration and functional inhibition of NTR1 receptors

https://doi.org/10.1016/j.bbrc.2009.12.007Get rights and content

Abstract

G-protein-coupled receptors (GPCR) are now regarded as being able to acquire heterodimer conformations affecting their pharmacology, signaling and trafficking. In co-immunoprecipitation studies using differentially epitope-tagged receptors, we herein provide direct evidence for heterodimerization of human neurotensin type 1 receptor (hNTR1) and type 2 receptor (hNTR2). Using chimeric constructs, we also identified the hNTR2 transmembrane 2 (TM2) to TM4 region as crucial for the formation of the dimerization interface. At the functional level, we demonstrated that the co-expression of hNTR2 suppressed hNTR1-mediated adenylate cyclase/cAMP and phospholipase C activation. Finally, confocal microscopy revealed that whereas tagged hNTR1 expressed alone were localized to the plasma membrane, co-expression of hNTR2 caused the retention of hNTR1 in sub-cellular compartments, indicating that heterodimerization with hNTR2 interferes with the proper recruitment of hNTR1 to the plasma membrane. Overall, this study proposes a novel function of NTR2 in the regulation of NTR1 activity.

Introduction

Neurotensin (NT), a brain-gut tridecapeptide, fulfils a dual function of neurotransmitter or neuromodulator in the central nervous system (CNS) and of local hormone at the periphery [1], [2]. NT actions and related signal transduction depend on recognition of the peptide at the plasma membrane of target cells by three specific receptors referred to as NTR1, NTR2 and NTR3/sortilin. NTR1 and NTR2 belong to the family of G-protein-coupled receptors (GPCR) with seven transmembrane domains, whereas NTR3 is part of the Vps10p family of sorting receptors characterized by a single transmembrane domain [3], [4], [5].

NTR1 is known to link through Gi/o, Gq, and Gs protein, to a variety of signaling cascades, including formation of cAMP and cGMP, production of inositol phosphates through phospholipase C activation, activation/inhibition of mitogen-activated protein kinases (ERK1/2 and JNK) and serine/threonine protein kinase Akt [2]. In contrast to NTR1, the signaling properties of NTR2 are still controversial, exhibiting cell type- and species-dependent pharmacological properties [2], [5]. For instance, NT analogs were reported to act as an agonist, inverse agonist, or competitive antagonist at NTR2 sites depending on the heterologous expression system (oocytes, CHO, COS-7, and HEK-293 cells) [6], [7], [8], [9], [10], [11].

GPCRs have classically been assumed to exist and act as monomeric entities in neuronal and non-neuronal cells. However, a large number of data obtained using biochemical and functional approaches argue for the existence of these receptors as homo- or hetero-dimeric or even higher-structure oligomers [12]. Such intermolecular interactions are important for receptor function, including agonist binding, potency, efficacy, G-protein selectivity, trafficking to the plasma membrane, and agonist-promoted internalization [13]. The process of intramolecular cross-talk by which individual NT receptors interact to form heterodimeric or heteromultimeric complexes could be of particular importance in the NT family, since it is quite frequent that two NT receptors subtypes are co-expressed in NT-related brain regions, such as the dorsal raphe, substantia nigra and the ventral tegmental area [14] or in a given cell type [15], [16], [17]. In this context, the two structurally different receptors NTR1 and NTR3, co-express in several human cancer cell lines and interact physically to modulate both NT-induced phosphorylation of ERK1/2 and the phosphoinositide (PI) turnover mediated by NTR1 [18]. Similarly, NTR2 was shown to exist as homodimers either in vitro or in vivo and to form heterodimeric complexes with the truncated splice variant vNTR2 isoform [15], [16], [19].

The aim of this study was therefore to investigate whether physical interactions occurring between human NTR1 and NTR2 influence NTR1 coupling to second messenger signaling cascades and to identify the NTR2 structural domains responsible for NTR1/NTR2 functional heterodimerization. Furthermore, alterations in trafficking of NTR1 by physical interaction with NTR2 were determined.

Section snippets

Materials and methods

Plasmids and construction of chimeras. Human NTR1 (hNTR1), rat NTR1 (rNTR1), and hNTR2 constructs are generous gifts from Dr. J. Mazella (CNRS, Valbonne, France). The cDNAs for bullfrog NTR1 (bfNTR1) and NTR4 (bfNTR4) were previously developed in our laboratory [20]. For domain swapping between hNTR2 and bfNTR4, individual cDNA fragments of interest were amplified using overlapping PCR, as previously described [21]. The plasmid encoding luciferase containing four copies of the cAMP-response

hNTR2 exerts an inhibitory effect on NTR1-mediated signaling

With regards to G-protein coupling, NTR1 is a Gq- and Gs-preferring receptor [2]. In this study, we first examined whether NTR1 receptor stimulation resulted in adenylate cyclase/PKA and phospholipase C/PKC activation, by using CRE- and c-fos-luciferase (luc) assays, respectively [20]. In HeLa cells transiently expressing either hNTR1 or rNTR1, stimulation with NT for up to 6 h induced a pronounced increase in CRE-luc activity (Fig. 1A). Activation of the recently cloned bfNTR1 and bfNTR4 by

Discussion

The present study demonstrates that co-expression of hNTR2 is able to impede NTR1-mediated adenylyl cyclase/cAMP and phospholipase C activation. The lack of the effects observed after co-transfection of NTR1 with closely related GPCRs, GnRHR-II and bfNTR4 reinforces the specificity of this functional interaction. This inhibitory effect of hNTR2 is likely due to heterodimerization of hNTR1 and hNTR2, which, in turn, retains a large portion of hNTR1 in the cytoplasm.

hNTR1 and hNTR2 form stable

Disclosure statement

The authors have nothing to disclose.

Acknowledgments

This work was supported by Grants from the Korea Research Foundation (KRF-2006-005-J03001), the Ministry of Sciences and Technology (R01-2004-000-10163-0), and the Canadian Institutes of Health Research (CIHR, MOP-74618) awarded, respectively, to J.Y.S., H.B.K. and P.S. P.S. is a CIHR new investigator and member of the FRSQ-funded Centre de Recherche Clinique Étienne Lebel.

Cited by (0)

View full text