Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P01189 (beta-endorphin)
 21,003 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Neutral endopeptidase-24.11 (NEP; EC 3.4.24.11) is an abundant metalloendopeptidase of the brush border membrane of kidney proximal tubules. We have recently shown that NEP is delivered directly to the apical domain of the plasma membrane when expressed in polarized Madin-Darby canine kidney (MDCK) cells in culture (Jalal, F., Lemay, G., Zollinger, M., Berteloot, A., Boileau, G., and Crine, P. (1991) J. Biol. Chem. 266, 19826-19832). Here, a soluble form of NEP consisting of the signal peptide of pro-opiomelanocortin fused in-frame with the ectodomain of NEP has been expressed in MDCK cells. Enzymatic assays performed on apical and basolateral culture media of MDCK cells grown on semi-permeable supports indicated that the recombinant enzyme was predominantly released at the apical surface. In contrast, when the chimeric protein was expressed in NIH 3T3 cells or when pro-opiomelanocortin was expressed in MDCK cells, non-polarized secretion was observed into both the apical and basolateral compartments of the culture chamber. Our results suggest that the ectodomain of NEP is sufficient for directing the targeting of this protein to the apical membrane of polarized MDCK epithelial cells.
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PMID:Expression and polarized apical secretion in Madin-Darby canine kidney cells of a recombinant soluble form of neutral endopeptidase lacking the cytosolic and transmembrane domains. 173 74

Neutral endopeptidase (EC 3.4.24.11) is an integral membrane protein found at the plasma membrane of many cell types and is especially abundant at the apical "brush border" membrane of the kidney proximal tubules. The enzyme consists of a short amino-terminal cytosolic domain of 27 amino acids, a single hydrophobic sequence which is believed to be responsible for anchoring the enzyme in the plasma membrane, and a large extracellular domain containing the active site. This model is consistent with the proposed function of neutral endopeptidase, which is believed to play an important role in the inactivation of small regulatory peptides at the cell surface. Site-directed mutagenesis has allowed the identification of 1 glutamic acid and 2 histidine residues essential for catalysis. All are located near the COOH terminus of the protein. We do not know, however, whether other segments of the protein are involved in the structure of the active site. The exact role of the cytosolic and transmembrane domains is also unknown. In this report, we have induced the secretion of a soluble form of recombinant neutral endopeptidase in COS-1 cells by fusing in-frame, the cDNA encoding the signal peptide of a secreted protein (pro-opiomelanocortin) to the cDNA sequences of the complete ectodomain of neutral endopeptidase. Characterization of the secreted recombinant protein indicated that it has the same catalytic properties as the membrane-bound recombinant enzyme or as the enzyme extracted from kidney brush border membranes. Thus the extracellular domain alone is sufficient for conferring full catalytic activity to neutral endopeptidase.
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PMID:Fusion of a cleavable signal peptide to the ectodomain of neutral endopeptidase (EC 3.4.24.11) results in the secretion of an active enzyme in COS-1 cells. 267 Sep 43

LLC-PK1 cells were transfected with a cDNA encoding rabbit neutral endopeptidase (NEP; EC 3.4.24.11), an abundant enzyme of the kidney proximal brush border. Clones of cells expressing high levels of the protein were isolated. Selective biotinylation and radioimmunolabelling were used to determine that 85-95% of NEP was localized in the apical domain of filter-grown LLC-PK1 cells. Pulse-chase and selective biotinylation studies revealed that the majority (85%) of newly made NEP was directly targeted to the apical membrane. However, a soluble form of NEP was found to be secreted in approximately equal amounts from both sides of the monolayer when expressed in LLC-PK1 cells. Transfected pro-opiomelanocortin, a pituitary hormone precursor, was secreted almost exclusively into the basolateral medium, suggesting that the bulk flow is to the basolateral membrane. This behaviour contrasts with that observed in MDCK cells, where both the transmembrane and secreted forms of NEP are directly targeted to the apical membrane and where the secretion of pro-opiomelanocortin is unpolarized.
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PMID:Direct targeting of neutral endopeptidase (EC 3.4.24.11) to the apical cell surface of transfected LLC-PK1 cells and unpolarized secretion of its soluble form. 782 24

The human placenta secretes large amounts of corticotropin-releasing hormone (CRH) which was thought to exert a paracrine action in the placenta. We have recently characterized high-affinity binding sites for CRH in the human placenta. However, our studies utilized whole placental membranes, which did not identify the site of binding of CRH in the plasma membrane. In this study we investigated the characteristics of CRH binding to purified mother-facing, brush border membranes (BBM) and fetus-facing, basal plasma membranes (BPM) of the syncytiotrophoblast. The two membranes were separated by a series of differential and density-gradient centrifugations. The purity of the membranes was determined by measuring alkaline phosphatase, as a marker of BBM and Na+/K+ATPase as a marker of BPM. Each membrane showed specific and high-affinity binding. Scatchard analysis revealed a high-affinity binding site for CRH with Kd of 1.0 +/- 0.15 and 1.3 +/- 0.176 for BBM and BPM, respectively. The maximal number of binding sites was significantly different (P < 0.01) in the two plasma membranes: Bmax of 79 +/- 6.4 fmol/mg protein for BBM and 23 +/- 3.9 fmol/mg protein for BPM. Both the mother-facing and fetus-facing membranes of the syncytiotrophoblast contain binding proteins for CRH, with significantly more binding sites on the mother-facing membranes. The functional consequences of CRH binding could be different for the two polar membranes due to differential localization of second messenger systems between the two membrane types. It is proposed that partial purification of BBM and BPM provides a better system to study CRH action in the placenta, than whole placental membrane preparations.
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PMID:Corticotropin-releasing hormone binding to the syncytiotrophoblast membranes. 1116 50