scholarly journals Structurally distinct membrane-associated and soluble forms of GH-binding protein in the mouse

2002 ◽  
Vol 172 (2) ◽  
pp. 321-331 ◽  
Author(s):  
RJ Cerio ◽  
F Xing ◽  
RJ Fatula ◽  
DE Keith ◽  
X Yang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mouse Liver ◽  
Noncovalent Interactions ◽  
Binding Capacity ◽  
Binding Protein ◽  
Liver Microsomes ◽  
Cell Surface Receptor ◽  
Soluble Form ◽  
Surface Receptor ◽  
Arginine Glycine Aspartic Acid

It has previously been shown that the large increase in GH-binding capacity of mouse liver microsomes during pregnancy is due largely to an increase in the amount of GH-binding protein (GHBP), with a more modest increase in GH receptor (GHR). Here we show that mouse liver GHBP is predominantly present as a membrane-associated protein structurally distinct from the soluble form of GHBP present in serum. Liver GHBP is associated with both intracellular membranes and the plasma membrane. Membrane-associated GHBP and soluble GHBP appear to be identical polypeptides distinguished by the addition of different N-glycans to asparagine residues. The pattern of release of GHBP from membranes by various treatments indicates that GHBP associates with membranes through noncovalent interactions with one or more membrane protein, but not with GHR. Covalent crosslinking provides evidence for several GHBP-associated membrane polypeptides, with molecular masses ranging from 58 kDa to over 200 kDa. These studies in the mouse and similar studies in the rat suggest that GHBP is an important cell-surface receptor for GH in the liver of these species. We postulate that an arginine-glycine-aspartic acid sequence found on rat and mouse GHBP but absent in other species is responsible for the association of GHBP with the plasma membrane by binding to one or more integrins on the surface of liver cells.

scholarly journals gp96 Is a Human Colonocyte Plasma Membrane Binding Protein for Clostridium difficile Toxin A

2008 ◽  
Vol 76 (7) ◽  
pp. 2862-2871 ◽  
Author(s):  
Xi Na ◽  
Ho Kim ◽  
Mary P. Moyer ◽  
Charalabos Pothoulakis ◽  
J. Thomas LaMont
Keyword(s):  
Plasma Membrane ◽  
Clostridium Difficile ◽  
Binding Protein ◽  
Membrane Binding ◽  
Cell Surface Receptor ◽  
Toxin A ◽  
Surface Receptor ◽  
A Cell ◽  
Plasma Membrane Binding

ABSTRACT Clostridium difficile toxin A (TxA), a key mediator of antibiotic-associated colitis, requires binding to a cell surface receptor prior to internalization. Our aim was to identify novel plasma membrane TxA binding proteins on human colonocytes. TxA was coupled with biotin and cross-linked to the surface of HT29 human colonic epithelial cells. The main colonocyte binding protein for TxA was identified as glycoprotein 96 (gp96) by coimmunoprecipitation and mass spectrum analysis. gp96 is a member of the heat shock protein family, which is expressed on human colonocyte apical membranes as well as in the cytoplasm. TxA binding to gp96 was confirmed by fluorescence immunostaining and in vitro coimmunoprecipitation. Following TxA binding, the TxA-gp96 complex was translocated from the cell membrane to the cytoplasm. Pretreatment with gp96 antibody decreased TxA binding to colonocytes and inhibited TxA-induced cell rounding. Small interfering RNA directed against gp96 reduced gp96 expression and cytotoxicity in colonocytes. TxA-induced inflammatory signaling via p38 and apoptosis as measured by activation of BAK (Bcl-2 homologous antagonist/killer) and DNA fragmentation were decreased in gp96-deficient B cells. We conclude that human colonocyte gp96 serves as a plasma membrane binding protein that enhances cellular entry of TxA, participates in cellular signaling events in the inflammatory cascade, and facilitates cytotoxicity.

The transfer of transthyretin and receptor-binding properties from the plasma retinol-binding protein to the epididymal retinoic acid-binding protein

2002 ◽  
Vol 362 (2) ◽  
pp. 265-271 ◽  
Author(s):  
Manickavasagam SUNDARAM ◽  
Daan M. F. van AALTEN ◽  
John B. C. FINDLAY ◽  
Asipu SIVAPRASADARAO
Keyword(s):  
Retinoic Acid ◽  
Binding Protein ◽  
Binding Pocket ◽  
Retinol Binding Protein ◽  
Cell Surface Receptor ◽  
Acid Binding Protein ◽  
The Family ◽  
Surface Receptor ◽  
A Cell ◽  
Plasma Retinol

Members of the lipocalin superfamily share a common structural fold, but differ from each other with respect to the molecules with which they interact. They all contain eight β-strands (A—H) that fold to form a well-defined β-barrel, which harbours a binding pocket for hydrophobic ligands. These strands are connected by loops that vary in size and structure and make up the closed and open ends of the pocket. In addition to binding ligands, some members of the family interact with other macromolecules, the specificity of which is thought to be associated with the variable loop regions. Here, we have investigated whether the macromolecular-recognition properties can be transferred from one member of the family to another. For this, we chose the prototypical lipocalin, the plasma retinol-binding protein (RBP) and its close structural homologue the epididymal retinoic acid-binding protein (ERABP). RBP exhibits three molecular-recognition properties: it binds to retinol, to transthyretin (TTR) and to a cell-surface receptor. ERABP binds retinoic acid, but whether it interacts with other macromolecules is not known. Here, we show that ERABP does not bind to TTR and the RBP receptor, but when the loops of RBP near the open end of the pocket (L-1, L-2 and L-3, connecting β-strands A—B, C—D and E—F, respectively) were substituted into the corresponding regions of ERABP, the resulting chimaera acquired the ability to bind TTR and the receptor. L-2 and L-3 were found to be the major determinants of the receptor- and TTR-binding specificities respectively. Thus we demonstrate that lipocalins serve as excellent scaffolds for engineering novel biological functions.

Neurotransmitter Control of Secretion

1987 ◽  
Vol 66 (1_suppl) ◽  
pp. 628-632 ◽  
Author(s):  
B. J. Baum
Keyword(s):  
Plasma Membrane ◽  
Salivary Gland ◽  
Salivary Secretion ◽  
Cell Surface Receptor ◽  
Specific Cell ◽  
Neural Signal ◽  
N Protein ◽  
Transduction Mechanisms ◽  
Surface Receptor ◽  
Specific Cell Surface

It is very well established that the principal control of salivary secretion is derived from autonomic innervation. Transmission of a neural signal to a salivary gland acinar cell occurs chemically via neurotransmitters, the first messengers of a secretory response. Neurotransmitters bind to specific cell surface receptor proteins, an event which activates precise transduction mechanisms which then transfer the neural signal to the inside of the cell. There are two major transduction mechanisms operative in salivary gland acinar cells. One involves the generation of cAMP, the other involves the breakdown of plasma membrane polyphosphoinositides. For both mechanisms, the appropriate stimulated receptor activates a second plasma membrane protein, termed an N (or G) protein. The N protein requires GTP to activate an enzyme (adenylate cyclase or phospholipase C), which then catalyzes the formation of a second messenger (cAMP and inositol trisphosphatel diacylglyeerol, respectively). This action provides the intracellular signal for secretory events (protein, fluid, electrolyte secretion) to begin.

scholarly journals Subcellular Redistribution of Pit-2 PiTransporter/Amphotropic Leukemia Virus (A-MuLV) Receptor in A-MuLV-Infected NIH 3T3 Fibroblasts: Involvement in Superinfection Interference

2000 ◽  
Vol 74 (6) ◽  
pp. 2847-2854 ◽  
Author(s):  
Zsolt Jobbagy ◽  
Susan Garfield ◽  
Lisa Baptiste ◽  
Maribeth V. Eiden ◽  
Wayne B. Anderson
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Leukemia Virus ◽  
Cell Surface Receptor ◽  
3T3 Cells ◽  
Sodium Dependent ◽  
Surface Receptor ◽  
A Cell ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT Amphotropic murine leukemia virus (A-MuLV) utilizes the Pit-2 sodium-dependent phosphate transporter as a cell surface receptor to infect mammalian cells. Previous studies established that infection of cells with A-MuLV resulted in the specific down-modulation of phosphate uptake mediated by Pit-2 and in resistance to superinfection with A-MuLV. To study the mechanisms underlying these phenomena, we constructed plasmids capable of efficiently expressing ɛ epitope- and green fluorescent protein (GFP)-tagged human Pit-2 proteins in mammalian cells. Overexpression of ɛ-epitope-tagged Pit-2 transporters in NIH 3T3 cells resulted in a marked increase in sodium-dependent Pi uptake. This increase in Piuptake was specifically blocked by A-MuLV infection but not by infection with ecotropic MuLV (E-MuLV) (which utilizes a cationic amino acid transporter, not Pit-2, as a cell surface receptor). These data, together with the finding that the tagged Pit-2 transporters retained their A-MuLV receptor function, indicate that the insertion of epitope tags does not affect either retrovirus receptor or Pitransporter function. The overexpressed epitope-tagged transporters were detected in cell lysates, by Western blot analysis using both ɛ-epitope- and GFP-specific antibodies as well as with Pit-2 antiserum. Both the epitope- and GFP-tagged transporters showed almost exclusive plasma membrane localization when expressed in NIH 3T3 cells, as determined by laser scanning confocal microscopy. Importantly, when NIH 3T3 cells expressing these proteins were productively infected with A-MuLV, the tagged transporters and receptors were no longer detected in the plasma membrane but rather were localized to a punctate structure within the cytosolic compartment distinct from Golgi, endoplasmic reticulum, endosomes, lysosomes, and mitochondria. The intracellular Pit-2 pool colocalized with the virus in A-MuLV-infected cells. A similar redistribution of the tagged Pit-2 proteins was not observed following infection with E-MuLV, indicating that the redistribution of Pit-2 is not directly attributable to general effects associated with retroviral infection but rather is a specific consequence of A-MuLV–Pit-2 interactions.

scholarly journals CYRI-A regulates macropinocytic cup maturation and mediates integrin uptake, limiting invasive migration

2020 ◽  
Author(s):  
Anh Hoang Le ◽  
Tamas Yelland ◽  
Nikki Paul ◽  
Loic Fort ◽  
Savvas Nikolaou ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Ewing Sarcoma ◽  
Negative Regulator ◽  
Cell Surface Receptor ◽  
Actin Dynamics ◽  
Surface Receptor ◽  
Sarcoma Cells ◽  
Wave Complex

The Scar/WAVE complex is the major driver of actin nucleation at the plasma membrane, resulting in lamellipodia and membrane ruffles. While lamellipodia aid migration, membrane ruffles can generate macropinosomes - cup-like structures - important for nutrient uptake and regulation of cell surface receptor levels. How macropinosomes are formed and the role of the actin machinery in their formation and resolution is still not well understood. Mammalian CYRI-B is a recently described negative regulator of the Scar/WAVE complex by RAC1 sequestration, but its other paralogue, CYRI-A has not been characterised. Here we implicate CYRI-A as a key regulator of macropinocytosis maturation and integrin internalisation from the cell surface. We find that CYRI-A is recruited to nascent macropinosomes in a transient but distinct burst, downstream of PIP3-mediated RAC1 activation to regulate actin polymerisation. CYRI-A precedes RAB5A recruitment to engulfed macropinocytic cups and departs as RAB5A is recruited, consistent with a role for CYRI-A as a local suppressor of actin dynamics, enabling the resolution of the macropinocytic cup. The suppression of integrin a5b1 uptake caused by the co-depletion of CYRI-A and B in Ewing sarcoma cells, leads to an enhancement of surface integrin levels and enhanced invasion and anchorage-independent growth in 3D. Thus CYRI-A is a dynamic regulator of integrin uptake via macropinocytosis, functioning together with CYRI-B to regulate integrin homeostasis on the cell surface.

scholarly journals Binding, internalization, and lysosomal association of 125I-human growth hormone in cultured human lymphocytes: a quantitative morphological and biochemical study.

1980 ◽  
Vol 87 (2) ◽  
pp. 360-369 ◽  
Author(s):  
P Barazzone ◽  
M A Lesniak ◽  
P Gorden ◽  
E Van Obberghen ◽  
J L Carpentier ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Plasma Membrane ◽  
Human Growth Hormone ◽  
Incubation Medium ◽  
Biochemical Study ◽  
Human Lymphocytes ◽  
Human Growth ◽  
Cell Surface Receptor ◽  
Intracellular Compartment ◽  
Surface Receptor

125I-human growth hormone (125I-hGH) binds specifically to receptors on cultures human lymphocytes (IM-9). When this process is studied by use of quantitative EM radioautography, under conditions of incubation at 15 degrees C for 5 min, the ligand is localized to the plasma membrane of the cell. At 30 degrees and 37 degrees C, however, 125I-hGH is progressively internalized by the cell as a function of time. The internalized ligand is found predominantly in the Golgi region of the cells, with a five-fold preferential localization to membrane-bounded structures with the morphological and cytochemical characteristics of lysosomes. Up to 59% of these lysosome-like structures are positive for the acid phosphatase reaction under the conditions of incubation at 37 degrees C for 120 min. When the cell associated radioactivity after 15-120 min of incubation at 37 degrees C is extracted in 1 M acetic acid and filtered on a Sephadex G-100 column, 58-73% of the material elutes as intact hGH. When cells are incubated with 125I-hGH at 37 degrees C for 15-120 min, separated from the incubation medium, and washed and diluted 100-fold, the percent 125I-hGH dissociable decreases as a function of increasing time of incubation. When cells are incubated with 125I-hGH for 15 min at 37 degrees C and the radioactivity that dissociates from the cells during 15-90 min is studied, the labeled material appearing in the incubation medium is progressively degraded as a function of time of incubation. When the dissociation process is studied radioautographically, grains are found both in plasma membrane and intracelluar compartments after 30 min of association, but after 30 and 120 min of dissociation a higher proportion of grains are in the intracellular compartment. After 120 min of association, there is less dissociation from either compartment and a preferential increase of grains in the intracellular compartment. These data suggest that receptor-linked internalization of a polypeptide hormone provides a mechanism that couples degradation of the ligand with loss of the cell surface receptor.

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