scholarly journals The C-terminal Domain of Aminopeptidase A Is an Intramolecular Chaperone Required for the Correct Folding, Cell Surface Expression, and Activity of This Monozinc Aminopeptidase

2004 ◽  
Vol 279 (41) ◽  
pp. 43285-43295 ◽  
Author(s):  
Raphaël Rozenfeld ◽  
Laurent Muller ◽  
Said El Messari ◽  
Catherine Llorens-Cortes
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Terminal Domain ◽  
Aminopeptidase A ◽  
Expression And Activity

scholarly journals Characterization, cell-surface expression and ligand-binding properties of different truncated N-terminal extracellular domains of the ionotropic glutamate receptor subunit GluR1

1996 ◽  
Vol 315 (1) ◽  
pp. 217-225 ◽  
Author(s):  
R. A. Jeffrey McILHINNEY ◽  
Elek MOLNÁR
Keyword(s):  
Cell Surface ◽  
Ligand Binding ◽  
Glutamate Receptor ◽  
Cell Membranes ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Receptor Subunit ◽  
Binding Studies ◽  
Terminal Domain ◽  
Glutamate Receptor Subunit

To identify the location of the first transmembrane segment of the GluR1 glutamate receptor subunit artificial stop codons have been introduced into the N-terminal domain at amino acid positions 442, 510 and 563, namely just before and spanning the proposed first two transmembrane regions. The resultant truncated N-terminal fragments of GluR1, termed NT1, NT2 and NT3 respectively were expressed in Cos-7 cells and their cellular distribution and cell-surface expression analysed using an N-terminal antibody to GluR1. All the fragments were fully glycosylated and were found to be associated with cell membranes but none was secreted. Differential extraction of the cell membranes indicated that both NT1 and NT2 behave as peripheral membrane proteins. In contrast NT3, like the full subunit, has integral membrane protein properties. Furthermore only NT3 is expressed at the cell surface as determined by immunofluorescence and cell-surface biotinylation. Protease protection assays indicated that only NT3 had a cytoplasmic tail. Binding studies using the selective ligand [3H]α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate ([3H]AMPA) demonstrated that NT3 does not bind ligand. Together these results indicate that the first transmembrane domain of the GluR1 subunit lies between residues 509 and 562, that the N-terminal domain alone cannot form a functional ligand-binding site and that this domain can be targeted to the cell surface provided that it has a transmembrane-spanning region.

scholarly journals Cyclic AMP Increases Cell Surface Expression of Functional Na,K-ATPase Units in Mammalian Cortical Collecting Duct Principal Cells

2001 ◽  
Vol 12 (2) ◽  
pp. 255-264 ◽  
Author(s):  
Sandrine Gonin ◽  
Georges Deschênes ◽  
Frank Roger ◽  
Marcelle Bens ◽  
Pierre-Yves Martin ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Cell Surface ◽  
Atpase Activity ◽  
Cultured Cells ◽  
Collecting Duct ◽  
Brefeldin A ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Cortical Collecting Duct ◽  
Expression And Activity

Cyclic AMP (cAMP) stimulates the transport of Na+ and Na,K-ATPase activity in the renal cortical collecting duct (CCD). The aim of this study was to investigate the mechanism whereby cAMP stimulates the Na,K-ATPase activity in microdissected rat CCDs and cultured mouse mpkCCDc14 collecting duct cells. db-cAMP (10−3 M) stimulated by 2-fold the activity of Na,K-ATPase from rat CCDs as well as the ouabain-sensitive component of 86Rb+ uptake by rat CCDs (1.7-fold) and cultured mouse CCD cells (1.5-fold). Pretreatment of rat CCDs with saponin increased the total Na,K-ATPase activity without further stimulation by db-cAMP. Western blotting performed after a biotinylation procedure revealed that db-cAMP increased the amount of Na,K-ATPase at the cell surface in both intact rat CCDs (1.7-fold) and cultured cells (1.3-fold), and that this increase was not related to changes in Na,K-ATPase internalization. Brefeldin A and low temperature (20°C) prevented both the db-cAMP-dependent increase in cell surface expression and activity of Na,K-ATPase in both intact rat CCDs and cultured cells. Pretreatment with the intracellular Ca2+chelator bis-(o-aminophenoxy)-N,N,N′,N′-tetraacetic acid also blunted the increment in cell surface expression and activity of Na,K-ATPase caused by db-cAMP. In conclusion, these results strongly suggest that the cAMP-dependent stimulation of Na,K-ATPase activity in CCD results from the translocation of active pump units from an intracellular compartment to the plasma membrane.

scholarly journals The Amino Terminal Lectin-Like Domain of Thrombomodulin Is Required for Constitutive Endocytosis

Blood ◽  
1997 ◽  
Vol 89 (2) ◽  
pp. 652-661 ◽  
Author(s):  
Edward M. Conway ◽  
Saskia Pollefeyt ◽  
Desiré Collen ◽  
Marta Steiner-Mosonyi
Keyword(s):  
Cell Surface ◽  
Protein C ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Immunogold Electron Microscopy ◽  
Wild Type ◽  
Hydrophobic Region ◽  
Cos Cells ◽  
Amino Terminal ◽  
Terminal Domain

Abstract Thrombomodulin (TM) is a multidomain protein that serves as a cofactor in a major natural anticoagulant system. To further characterize the structure-function of TM, we have transfected COS cells with different truncated forms of TM. In the first form, COS cells expressing TM that lacks the putative signal peptide (17 residues); the lectin-like, hydrophobic N-terminal domain (226 residues); and 12 residues of the first epidermal growth factor (EGF )-like repeat (COSdel.238 cells) were found to function normally with respect to TM transport to the cell surface and thrombin-dependent protein C activation. However, in contrast to wild-type TM, as visually studied by immunofluorescence and immunogold electron microscopy, the COSdel.238 cells did not constitutively internalize anti-TM–TM or thrombin-TM complexes. To identify the region responsible for mediating the endocytic process, deletant forms of TM lacking either the lectin-like region (residues 2-155) or the hydrophobic region of the N-terminal domain (residues 161-202) were expressed in COS cells (COSdel.2-155 and COSdel.161-202, respectively). Protein C cofactor activity was maintained in both cells. Although the COSdel.161-202 cells behaved similarly to wild-type TM-transfected cells, visual studies showed a lack of constitutive internalization of thrombin-TM or anti-TM–TM complexes in the COSdel.2-155 cells. We conclude that the lectin-like domain of human TM serves to regulate cell surface expression of TM via the endocytic route and therefore may also play a major physiologic role in controlling intracellular and extracellular accumulation of thrombin in a variety of biologic systems.

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