Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes

Mills, Anthony D.; Demoliou-Mason, Catherine; Barnard, Eric A.

doi:10.1111/j.1471-4159.1988.tb02998.x

Cited by 22 publications

(12 citation statements)

References 31 publications

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“…The apparent increase in molecular mass of TC II-R due to disulfide bond disruption noted in this study is not a unique situation. Other receptors such as the cation-independent mannose 6-phosphate receptor (27), neurotensin receptor (28), and ␤-adrenergic receptor (29) behave similarly on nonreducing SDS-PAGE following their reductive alkylation.…”

Section: Discussionmentioning

confidence: 99%

Effect of Disulfide Bonds of Transcobalamin II Receptor on Its Activity and Basolateral Targeting in Human Intestinal Epithelial Caco-2 Cells

Bose¹,

Seetharam²

1997

Journal of Biological Chemistry

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Transcobalamin II-receptor (TC II-R)1 is a single chain glycoprotein with a molecular mass of 62 kDa (1) containing about 27% carbohydrate. It promotes plasma transport of cobalamin (Cbl; vitamin B 12 ) bound to transcobalamin II (TC II) (2). TC II-R functions as a noncovalent homodimer with a molecular mass of 124 kDa in tissue membranes (1), and its dimerization occurs not in the ER, where it is a monomer, but rather in the plasma membrane (3). In addition, the dimerization of TC II-R is a membrane fluidity-driven event requiring a highly ordered rigid bilayer (4). In polarized epithelial Caco-2 cells, TC II-R is predominantly present in basolateral membranes (5), where it functions in the delivery of Cbl to be utilized as coenzymes (6). Treatment of the basolateral surface of Caco-2 cells with TC II-R antiserum in vitro or circulating TC II-R antibody in vivo results in a failure of TC II-Cbl uptake, causing intracellular Cbl deficiency (6). These studies have suggested that plasma membrane delivery of TC II-R is extremely important for cells to obtain their Cbl from the circulation to maintain their normal metabolic, differentiation, and proliferation status. Despite the importance of plasma membrane expression of TC II-R in the tissue transport of Cbl, no information is available regarding its intracellular trafficking and plasma membrane expression.Recent studies have shown that formation of intracellular inter-or intrachain disulfide bonds and their final rearrangement by thiol-disulfide exchange reaction is one of the co/posttranslational modifications of some proteins (7,8). The correct formation of intrachain and, in the case of oligomeric proteins, interchain disulfide bonds is crucial for the proper assembly of secretory and plasma membrane proteins, which in turn determines their stability, intracellular transport, maturation, and function (9 -11). Inhibition of disulfide bond formation by DTT treatment has been shown to affect secretion of albumin and H1 subunit of asialoglycoprotein receptor from fibroblasts (12) and of gp80 (clusterin, apolipoprotein J) in polarized MadinDarby canine kidney cells (13). However, the secretion of nondisulfide bond-containing proteins such as ␣ 1 -antitrypsin (14) in HepG2 cells or of a 20-kDa peptide derived from osteoponin in Madin-Darby canine kidney cells (13) is not affected. These studies have proposed that the retention of proteins in the ER following treatment with DTT is due to a failure of these proteins to form disulfide bonds, which then fold improperly. As a consequence, these misfolded proteins fail to achieve a conformation essential for their function as well as their exit from the ER for further processing and targeting (15).Human TC II-R contains 10 half-cysteines (16), and recent immunoblot studies of placental plasma membrane proteins under nonreducing and reducing conditions (1) have shown that TC II-R dimers contain intramolecular disulfide bonds that, upon reduction, increase the apparent molecular mass of the dimer by 20 kDa. Taken together, ...

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Section: Discussionmentioning

confidence: 99%

Effect of Disulfide Bonds of Transcobalamin II Receptor on Its Activity and Basolateral Targeting in Human Intestinal Epithelial Caco-2 Cells

Bose¹,

Seetharam²

1997

Journal of Biological Chemistry

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“…The biochemical and pharmacological properties of these binding sites have been extensively studied using mammalian brain homogenates as well as membrane preparations from neuronal and certain non-neuronal cell lines [5,6]. It has been shown that the interaction with these receptors modulates intracellular levels of cGMP, CAMP and inositol phosphates [7,8].…”

Section: Introductionmentioning

confidence: 99%

Cloning and expression of a complementary DNA encoding a high affinity human neurotensin receptor

et al. 1993

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A human neurotensin receptor (hNTR) cDNA was cloned from the colonic adenocarcinoma cell line HT29. The cloned cDNA encodes a putative peptide of 418 amino acids with 7 transmembrane domains. The amino acid sequence of the hNTR is 84% identical to the rat NTR meuron, 4 (1990) 847-8541. Transfection of this cDNA into COS cells results in the expression of receptors with pharmacological properties similar to those found with HT29 cells. Northern blot analysis using the hNTR cDNA probe indicated a single transcript of 4 kb in the brain, the small intestine and blood mononuclear cells.

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“…Our (Ahmad et al, 1987). In canine intestinal smooth muscle as well as rat (Mazella et al, 1983) and bovine brain (Mills et al, 1988 we observed in the circular muscle may suggest occuancy of a low-affinity receptor, but resolution of such row-affinity binding sites must await further studies using higher concentrations of [lZ5I]-NT. Nevertheless, the consistency of our estimates with those of others for a high-affinity binding site for NT is noteworthy, since our experiments were conducted in a physiological medium.…”

Section: Discussionmentioning

confidence: 59%

Neurotensin binding sites in porcine jejunum: Biochemical Characterization and intramural Localization

Seybold¹,

Treder²,

Aaonsen

et al. 1990

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Neurotensin is present in high concentrations in the mammalian gut, especially in enteroendocrine cells of the mucosa. Exogenous neurotensin has been shown to alter ion transport by the mucosa and contractile activity of intestinal smooth muscle. The purpose of this study was to determine the distribution of neurotensin binding sites within the intestinal wall. Initially, biochemical characteristics of [125I]neurotensin binding sites were determined within two preparations of the distal porcine jejunum: (1) the mucosa and submucosa, and (2) the circular and longitudinal muscle with their intramural plexuses. Ligand binding data for the preparation including the mucosa and submucosa indicated that [125I]neurotensin bound specifically to two sites having apparent equilibrium dissociation constants of approximately 0.046 and 0.37 nM. A binding site with a dissociation constant of approximately 0.38 nM was confirmed for the preparation of muscle and associated intramural plexuses. Xenopsin and neurotensin were equipotent to neurotensin in competing for these binding sites; neuromedin N was approximately 40 times less potent in the preparation of mucosa and submucosa. Receptor autoradiography was used to determine the distribution of [125I]neurotensin binding sites within the wall of the jejunum. Autoradiograms of [125I]neurotensin bound to cross sections of the proximal and distal jejunum showed that the highest densities of silver grains were associated with the internal submucosal ganglia, external submucosal plexus and myenteric ganglia. A moderate density of silver grains was associated with the circular muscle. The localization of neurotensin binding sites to submucosal ganglia is consistent with observations that neurotensin effects on active anion secretion by the mucosa are blocked by tetrodotoxin. Immunohistochemical localization of neurotensin in the porcine jejunum demonstrated a limited population of neurotensin immunoreactive cells within the mucosal epithelium. It is possible that neurotensin released from these cells in the mucosa as well as neurotensin-related peptides released from enteric neurons may be the endogenous ligands for the binding sites visualized in this study.

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Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes

Cited by 22 publications

References 31 publications

Effect of Disulfide Bonds of Transcobalamin II Receptor on Its Activity and Basolateral Targeting in Human Intestinal Epithelial Caco-2 Cells

Effect of Disulfide Bonds of Transcobalamin II Receptor on Its Activity and Basolateral Targeting in Human Intestinal Epithelial Caco-2 Cells

Cloning and expression of a complementary DNA encoding a high affinity human neurotensin receptor

Neurotensin binding sites in porcine jejunum: Biochemical Characterization and intramural Localization

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