NTS2 modulates the intracellular distribution and trafficking of NTS1 via heterodimerization

Neurotensin (NT) receptors NTS1 and NTS2 are known to display considerable distributional overlap in mammalian central nervous system (CNS). Using co-immunoprecipitation approaches, we demonstrated here that NTS1 forms constitutive heterodimers with NTS2 in transfected COS-7 cells. We also showed that co-expression of NTS2 with NTS1 markedly decreases the cell surface density of NTS1 without affecting ERK1/2 MAPK activity or NT-induced NTS1 internalization. However, radioligand-binding studies indicated that upon prolonged NT stimulation, cell surface NTS1 receptors are more resistant to down-regulation in cells co-expressing NTS1 and NTS2 than in cells expressing NTS1 alone. Taken together, these data suggest that NTS1/NTS2 heterodimerization affects the intracellular distribution and trafficking of NTS1 by making it more similar to that of NTS2 as witnessed in cells expressing NTS2 alone. NTS1/NTS2 heterodimerization might therefore represent an additional mechanism in the regulation of NT-triggered responses mediated by NTS1 and NTS2 receptors.     

Distinct regions of C-terminus of the high affinity neurotensin receptor mediate the functional coupling with pertussis toxin sensitive and insensitive G-proteins

The functional coupling of C-terminally truncated mutants of the high affinity rat neurotensin (NT) receptor (NTS1) was characterized in transfected Chinese hamster ovary cells. On cells expressing NTRDelta372 (truncated NTS1 lacking the entire 52 amino acid C-terminus), NT failed to promote [(35)S]guanosine 5'-[gamma-(35)S]triphosphate binding whereas a robust pertussis toxin (PTx) sensitive response was observed in cells expressing a partially truncated receptor (NTRDelta401 lacking the last 23 residues). Similar results were obtained when measuring the ability of NT to induce the production of arachidonic acid. Since neither deletions impaired the NT-induced phosphoinositide hydrolysis, these results indicate that the membrane proximal region of the C-terminus is specifically involved in the functional coupling of the receptor with PTx sensitive G-proteins. This region was also found to be involved in the control of receptor internalization. However, PTx failed to impair internalization, indicating that these two properties are not directly related.     

A Nonneuronal Isoform of Cell Adhesion Molecule L1: Tissue-Specific Expression and Functional Analysis

Abstract: The cell adhesion molecule L1 is a multifunctional protein in the nervous system characterizing cell adhesion, migration, and neurite outgrowth. In addition to full-length L1, we found an alternatively spliced variant lacking both the KGHHV sequence in the extracellular part and the RSLE sequence in the cytoplasmic part of L1. This L1 variant was expressed exclusively in nonneuronal cells such as Schwann cells, astrocytes, and oligodendrocytes, in contrast to the expression of the full-length L1 in neurons and cells of neuronal origin. To investigate the functions of the L1 variant, we established cell lines transfected with a cytoplasmic short L1 (L1cs) cDNA that lacks only the 12-bp segment encoding for the RSLE sequence. The promoting activities of homophilic cell adhesion, neurite outgrowth, and neuronal cell migration of L1cs-transfected cells (L4-2) were similar to those of full-length L1-transfected cells (L3-1), but the cell migratory activity of L4-2 itself was clearly lower than that of L3-1. In conclusion, the short form of L1 is a nonneuronal type, in contrast to the neuronal type of the full-length L1. Deletion of the four amino acids RSLE in the cytoplasmic region of L1 markedly reduced cell migratory activity, suggesting an importance of the RSLE sequence for the signaling events of neuronal migration mediated by L1.     

Identification of a Family of Sorting Nexin Molecules and Characterization of Their Association with Receptors

Sorting nexin 1 (SNX1) is a protein that binds to the epidermal growth factor (EGF) receptor and is proposed to play a role in directing EGF receptors to lysosomes for degradation (R. C. Kurten, D. L. Cadena, and G. N. Gill, Science 272:1008–1010, 1996). We have obtained full-length cDNAs and deduced the amino acid sequences of three novel homologous proteins, which were denoted human sorting nexins (SNX2, SNX3, and SNX4). In addition, we identified a presumed splice variant isoform of SNX1 (SNX1A). These molecules contain a conserved domain of ~100 amino acids, which was termed the phox homology (PX) domain. Human SNX1 (522 amino acids), SNX1A (457 amino acids), SNX2 (519 amino acids), SNX3 (162 amino acids), and SNX4 (450 amino acids) are part of a larger family of hydrophilic molecules including proteins identified in Caenorhabditis elegans and Saccharomyces cerevisiae. Despite their hydrophilic nature, the sorting nexins are found partially associated with cellular membranes. They are widely expressed, although the tissue distribution of each sorting nexin mRNA varies. When expressed in COS7 cells, epitope-tagged sorting nexins SNX1, SNX1A, SNX2, and SNX4 coimmunoprecipitated with receptor tyrosine kinases for EGF, platelet-derived growth factor, and insulin. These sorting nexins also associated with the long isoform of the leptin receptor but not with the short and medium isoforms. Interestingly, endogenous COS7 transferrin receptors associated exclusively with SNX1 and SNX1A, while SNX3 was not found to associate with any of the receptors studied. Our demonstration of a large conserved family of sorting nexins that interact with a variety of receptor types suggests that these proteins may be involved in several stages of intracellular trafficking in mammalian cells.     

The β2-microglobulin-free heterodimerization of rhesus monkey MHC class I A with its normally spliced variant reduces the ubiquitin-dependent degradation of MHC class I A

The MHC class I (MHC I) molecules play a pivotal role in the regulation of immune responses by presenting antigenic peptides to CTLs and by regulating cytolytic activities of NK cells. In this article, we show that MHC I A in rhesus macaques can be alternatively spliced, generating a novel MHC I A isoform (termed "MHC I A-sv1") devoid of α(3) domain. Despite the absence of β2-microglobulin (β2m), the MHC I A-sv1 proteins reached the cell surface of K562-transfected cells as endoglycosidase H-sensitive glycoproteins that could form disulfide-bonded homodimers. Cycloheximide-based protein chase experiments showed that the MHC I A-sv1 proteins were more stable than the full-length MHC I A in transiently or stably transfected cell lines. Of particular interest, our studies demonstrated that MHC I A-sv1 could form β2m-free heterodimers with its full-length protein in mammalian cells. The formation of heterodimers was accompanied by a reduction in full-length MHC I A ubiquitination and consequent stabilization of the protein. Taken together, these results demonstrated that MHC I A-sv1 and MHC I A can form a novel heterodimeric complex as a result of the displacement of β2m and illustrated the relevance of regulated MHC I A protein degradation in the β2m-free heterodimerization-dependent control, which may have some implications for the MHC I A splice variant in the fine tuning of classical MHC I A/TCR and MHC I A/killer cell Ig-like receptor interactions.     

Cell-type-specific pathways of neurotensin endocytosis

The neurotensin receptor subtype 1 (NTS1) is a G-protein-coupled receptor (GPCR) mediating a large number of central and peripheral effects of neurotensin. Upon stimulation, NTS1 is rapidly internalized and targeted to lysosomes. This process depends on the interaction of the phosphorylated receptor with β–arrestin. Little is known about other accessory endocytic proteins potentially involved. Here, we investigated the involvement of dynamin, amphiphysin, and intersectin in the internalization of NTS1 receptor-ligand complexes in transfected COS-7 and HEK 293 cells, by using the transferrin receptor as an internal control for the constitutive endocytic pathway. We found that NTS1 endocytosis was not only arrestin–dependent, but also dynamin–dependent in both COS-7 and HEK 293 cells, whereas internalization of the transferrin receptor was independent of arrestin but required dynamin. Overexpression of the SH3 domain of amphiphysin II had no effect on receptor internalization in either cell type. By contrast, overexpression of full-length intersectin or of its SH3 domain (but not of its EH domain) inhibited NTS1 internalization in COS-7 but not in HEK 293 cells. This difference between COS-7 and HEK 293 cells was not attributable to differences in endogenous intersectin levels between the two cell lines. Indeed, the same constructs inhibited transferrin endocytosis equally well in COS-7 and HEK 293 cells. However, immunogold electron microscopy revealed that internalized NTS1 receptors were associated with clathrin-coated pits in COS-7 cells but with smooth vesicles in HEK 293 cells, suggesting that NTS1 internalization proceeds via different endocytic pathways in these two cell types. This work was supported by grants to A.B. from CIHR and FRSQ.     

Thermostabilization of the Neurotensin Receptor NTS1

Structural studies on G-protein-coupled receptors have been hampered for many years by their instability in detergent solution and by the number of potential conformations that receptors can adopt. Recently, the structures of the β₁ and β₂ adrenergic receptors and the adenosine A_2a receptor were determined in the antagonist-bound state, a receptor conformation that is thought to be more stable than the agonist-bound state. In contrast to these receptors, the neurotensin (NT) receptor NTS1 is much less stable in detergent solution. We have therefore used a systematic mutational approach coupled with activity assays to identify receptor mutants suitable for crystallization, both alone and in complex with the peptide agonist NT. The best receptor mutant NTS1-7m contained four point mutations. It showed increased stability compared to the wild-type receptor, in the absence of ligand, after solubilization with a variety of detergents. In addition, NTS1-7m bound to NT was more stable than unliganded NTS1-7m. Of the four thermostabilizing mutations, only one residue (A86L) is predicted to be in the lipid environment. In contrast, I260A appears to be buried within the transmembrane helix bundle, F342A may form a distant part of the putative ligand-binding site, whereas F358A is likely to be in a region that is important for receptor activation. NTS1-7m binds NT with a similar affinity for the wild-type receptor. However, agonist dissociation was slower, and NTS1-7m activated G-proteins poorly. The affinity of NTS1-7m for the antagonist SR48692 was also lower than that of the wild-type receptor. Thus, we have successfully stabilized NTS1 in an agonist-binding conformation that does not efficiently couple to G-proteins.     

Identification and characterization of a novel splice variant of beta3 subunit of GABA(A) receptor in rat testis and spermatozoa

Gamma-aminobutyric acid type A (GABA(A)) receptors are the major sites of inhibitory action of fast synaptic neurotransmission in the brain. Their receptors are also widely distributed in peripheral and endocrine tissues. A full-length cDNA encoding a novel splice variant of beta3 subunit of GABA(A) receptor, designated as beta3t, was identified in rat testis. This isoform contains a segment, having identical amino acid sequence as the beta3 subunit of neuronal GABA(A) receptors except for a section composed of 25 different amino acid sequence in the N-terminus. Northern blot shows that this isoform is found in rat testis. The beta3t isoform mRNA was detected in germ cells in the late step of spermatogenesis by in situ hybridization assay. Results of immunohistochemical and immunocytochemical assays indicate that the beta3t isoform is expressed in rat testis and spermatozoa. To determine a possible function of the N-terminal 25 amino acid segment, a recombinant plasmid of beta3t-EGFPC was constructed by fusing green fluorescent protein to the C-terminus of the beta3t isoform. The chimera product failed to be translocated unto the cell surface when expressed in HEK 293 cells; whereas, the beta3 subunit of rat brain is incorporated into the plasma membrane. In conclusion, the present results show that one variant of beta3 subunit of GABA(A) receptor, designated as beta3t, is found in germ cells of rat testis and sperm. The inability of the beta3t variant to target into the plasma membrane maybe a consequence of the unique 25 amino acid segment in the N-terminus.     

Molecular and cellular regulation of neurotensin receptor under acute and chronic agonist stimulation

Neurotensin is a tridecapteptide acting mostly in the brain and gastrointestinal tract. NT binds two G protein coupled receptors (GPCR), NTS1 and NTS2, and a single transmembrane domain receptor, NTS3/gp95/sortilin receptor. NTS1 mediates the majority of NT action in neurons and the periphery. Like many other GPCRs, upon agonist stimulation, NTS1 is internalized, endocytosed, and the cells are desensitized. It is tacitly acknowledged that the intensity and the lasting of cellular responses to NT are dependent on free and functional NTS1 at the cell surface. Understanding how NTS1 expression is regulated at the membrane should provide a better comprehension towards its function. This review analyzes and discusses the current cellular and molecular mechanisms affecting the expression of NTS1 at the cellular membrane upon acute and chronic NT stimulation.     

Intermolecular interactions between the neurotensin and the third extracellular loop of human neurotensin 1 receptor

Neurotensin (NT) is a tridecapeptide hormone in the periphery and neurotransmitter in the brain that principally activates three receptor subtypes, named NTS1, NTS2, and NTS3. Since little is known about its structure in the presence of its principal receptor NTS1, we determined it using the key domain of the receptor, i.e. the third extracellular loop. We conclude the following: (i) for the receptor fragment, NT binding modifies its central part, underlying the great flexibility and adaptability of this region; (ii) for bound NT, the extended conformation of its C-terminus is confirmed for the first time in experimental conditions and in the presence of a part of the receptor; and (iii) despite some substitutions, the human receptor residues that are involved in the interaction with NT could be similar to those of the rat receptor which play an important role in NT binding.     

Identification of a splice variant of mouse caveolin-2 mRNA encoding an isoform lacking the C-terminal domain

We identified a splice variant of mouse caveolin-2 mRNA having an intronic sequence in place of the third exon (Deltaex3). The entire sequence of full-length (FL) and Deltaex3 caveolin-2 mRNA was determined; their sizes were 2490 and 973 bp, respectively. The Deltaex3 mRNA encoded a putative isoform lacking the C-terminal 49 amino acids of the authentic caveolin-2. The expression level of Deltaex3 was lower than that of FL mRNA, but considerable in some culture cells and tissues. The isoform lacking the C-terminus localized to the endoplasmic reticulum, while the authentic caveolin-2 was distributed to the Golgi and the plasma membrane along with caveolin-1. The result confirmed the necessity of the C-terminal domain of caveolin-2 for the caveolar localization, and showed the existence of a novel caveolin-2 isoform, which is not recruited to caveolae even in the presence of caveolin-1.