Internalization and trafficking of neurotensin via NTS3 receptors in HT29 cells

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Abstract

The neurotensin receptor-3, originally identified as sortilin, is unique among neuropeptide receptors in that it is a single trans-membrane domain, type I receptor. To gain insight into the functionality of neurotensin receptor-3, we examined the neurotensin-induced intracellular trafficking of this receptor in the human carcinoma cell line HT29, which expresses both neurotensin receptor-1 and -3 sub-types. At steady state, neurotensin receptor-3 was found by sub-cellular fractionation and electron microscopic techniques to be predominantly associated with intracellular elements. A small proportion (∼10%) was associated with the plasma membrane, but a significant amount (∼25%) was observed inside the nucleus. Following stimulation with neurotensin (NT), neurotensin/neurotensin receptor-3 complexes were internalized via the endosomal pathway. This internalization entailed no detectable loss of cell surface receptors, suggesting compensation through either recycling or intracellular receptor recruitment mechanisms. Internalized ligand and receptors were both sorted to the pericentriolar recycling endosome/Trans-Golgi Network (TGN), indicating that internalized neurotensin is sorted to this compartment via neurotensin receptor-3. Furthermore, within the Trans-Golgi Network, neurotensin was bound to a lower molecular form of the receptor than at the cell surface or in early endosomes, suggesting that signaling and transport functions of neurotensin receptor-3 may be mediated through different molecular forms of the protein. In conclusion, the present work suggests that the neurotensin receptor-3 exists in two distinct forms in HT29 cells: a high molecular weight, membrane-associated form responsible for neurotensin endocytosis from the cell surface and a lower molecular weight, intracellular form responsible for the sorting of internalized neurotensin to the Trans-Golgi Network.

Introduction

The tridecapeptide neurotensin (NT) is widely distributed in brain and periphery and is involved in a variety of neural, endocrine, cardiovascular, and digestive functions (for review, see Rostène & Alexander, 1997). Three different NT receptors, referred to as NTS1, NTS2, and NTS3 have so far been cloned (for reviews, see Mazella, 2001; Vincent, Mazella, & Kitabgi, 1999). Whereas, NTS1 and NTS2 are both seven trans-membrane domain receptors, NTS3 is a single trans-membrane domain type I receptor, which shows 100% homology with the sorting protein, sortilin (Mazella et al., 1998, Petersen et al., 1997). This receptor belongs to a new receptor family related to the yeast sorting receptor Vps10p and characterized by similar N-terminal luminal domains (Marcusson, Horazdovsky, Cereghino, Gharakhanian, & Emr, 1994). This receptor family includes, in addition to NTS3/sortilin, SorLA and SorCS-1, -2, and -3 (for review, see Hampe, Rezgaoui, Hermans-Borgmeyer, & Schaller, 2001). These receptors are produced as inactive precursors that are converted to mature forms upon cleavage by furin (Hampe et al., 2001, Petersen et al., 1999). Accordingly, NTS3 binds NT with considerably lower affinity when transfected in epithelial cell lines alone (Kd = 17 nM for hNTS3 and 45 nM for mNTS3) (Navarro et al., 2001) than together with furin (Kd = 0.3 nM for hNTS3) (Mazella et al., 1998). In addition to NT, NTS3/sortilin binds a variety of other ligands, including the receptor-associated protein (RAP) (Petersen et al., 1997, Tauris et al., 1998), lipoprotein lipase (LpL) (Nielsen, Jacobsen, Olivecrona, Gliemann, & Petersen, 1999), and the 44-amino-acid propeptide cleaved during maturation of the receptor precursor (Petersen et al., 1999).

In addition to its N-terminal and single trans-membrane domains, NTS3 comprises a short C-terminal cytoplasmic segment identical to the corresponding domain in the mannose 6-phosphate/insulin growth factor-II receptor (CI-M6PR) (Petersen et al., 1997). This C-terminal tail contains several sorting motifs, suggesting that NTS3/sortilin can engage in various types of trafficking, including endocytosis and transport between the Golgi and late endosomes (Nielsen et al., 2001). Indeed, a major pool of NTS3/sortilin was found to be associated with Golgi vesicles in adipocytes (Morris et al., 1998). Furthermore, only a small proportion of NTS3 appears to be present at the cell surface of either adipocytes or transfected cells under steady-state conditions (Morris et al., 1998, Navarro et al., 2001). However, in adipocytes, NTS3 is up-regulated at the plasma membrane upon stimulation with insulin, together with the glucose transporter GLUT4 (Lin, Pilch, & Kandror, 1997; Morris et al., 1998). Similarly, in neuronal cell cultures, NTS3 (identified at the time as a 100 kDa NT binding protein) was shown to be recruited at the cell membrane following stimulation with NT (Chabry, Gaudriault, Vincent, & Mazella, 1993).

The role of NTS3/sortilin in mediating central and/or peripheral effects of NT remains elusive. When transfected in epithelial cell lines, this receptor binds and efficiently internalizes NT (Navarro et al., 2001). Moreover, NT stimulates the growth of CHO cells stably transfected with NTS3, suggesting that the latter may behave as a signaling receptor regulating cell proliferation (Dal Farra et al., 2001). Accordingly, NTS3 was found to be expressed in a variety of human cancer cell lines derived from colon, pancreas and prostate (Dal Farra et al., 2001) upon which NT had been documented to exert a proliferative action (Iwase et al., 1996; Maoret et al., 1994; Seethalakshmi, Mitra, Dobner, Menon, & Carraway, 1997). However, the strongest evidence to date that NTS3 can behave as a true NT receptor lies with the recent demonstration that this protein may be involved in the NT-induced migration of human microglial cells, via stimulation of both MAP and Pi3-kinase-dependent pathways (Martin, Vincent, & Mazella, 2003).

Nonetheless, little is known regarding the effects of NT on endogenous NTS3 receptor trafficking. In particular, it remains to be determined whether endogenously expressed NTS3 receptors internalize NT and, if this is the case, to which intracellular compartments ligand and receptors are targeted. We recently demonstrated that NTS3 receptors are abundantly expressed in the human adenocarcinoma cell line HT29 (Martin, Navarro, Vincent, & Mazella, 2002), from which the human NTS1 receptor was originally cloned (Vita et al., 1993). Stimulation of HT29 cells with NT results in a robust down-regulation of cell surface NT binding sites and concomitant desensitization of the calcium mobilizing effects of NT (Turner, James-Kracke, & Camden, 1990). However, it is unclear whether this down regulation reflects a loss of NTS3 and/or NTS1 receptors from the cell surface and whether NT is internalized through NTS3 and/or NTS1 binding sites. The aim of the present study was therefore to address some of these issues by tracking NTS3 and NT-labeled NTS3 receptors in HT29 cells by combined biochemical and immunocytochemical techniques.

Section snippets

Materials

Neurotensin (NT) was purchased from Bachem (Voisins-le-Bretonneux, France) and NT(2-13) was synthesized by Neosystem (Strasbourg, France). 125I-Tyr3-NT, α-azidobenzoyl-125I-Tyr3-NT(2-13), and Nα-Bodipy-NT(2-13) were prepared and purified as described previously (Faure et al., 1995; Mazella, Kitabgi, & Vincent, 1985; Sadoul, Mazella, Amar, Kitabgi, & Vincent, 1984). Dulbecco’s modified Eagle’s medium (DMEM) was from Invitrogen, Inc. (Burlington, Ont., Canada) and fetal bovine serum (FBS) from

Sub-cellular fractionation studies

Western blot analysis of whole cell lysates prepared from non-stimulated HT29 cells revealed the presence of an intense NTS3-immunoreactive band at an apparent molecular weight of 105 kDa (Fig. 1A). In addition, in some, but not all preparations, a weakly immunoreactive band was detected at an apparent molecular weight of 135 kDa (Fig. 1A). This second band, which was previously shown to correspond to a differentially glycosylated form of NTS3/sortilin (Martin et al., 2002), accounted for

Discussion

The present study demonstrates that the neuropeptide, NT is internalized after binding to NTS3/sortilin receptors on the surface of HT29 cells. It also shows that internalized NT molecules are trafficked via different molecular forms of NTS3 from the cell surface to early endosomes and from early endosomes to the TGN/pericentriolar recycling endosome.

Acknowledgements

This work was supported by the Centre National de la Recherche Scientifique (CNRS) Grant PICS No. 2051, by Grant MT-7366 from the Canadian Institutes for Health Research (CIHR) to A. Beaudet, and by a France-Québec exchange program from the Fonds de la Recherche en Santé du Québec to A. Beaudet and J. Mazella. A. Morinville and S. Martin are the recipients of fellowships from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Association Pour la Recherche sur le

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