scholarly journals Polarization of endocytosis and receptor topography on cultured macrophages.

1980 ◽  
Vol 86 (1) ◽  
pp. 199-211 ◽  
Author(s):  
R J Walter ◽  
R D Berlin ◽  
J R Pfeiffer ◽  
J M Oliver
Keyword(s):  
Concanavalin A ◽  
Binding Sites ◽  
Membrane Receptors ◽  
Macrophage Cell ◽  
Oil Emulsion ◽  
Opsonized Zymosan ◽  
Membrane Topography ◽  
Polarized Epithelia ◽  
Endocytic Activity ◽  
Insight Into

In the 1774.2 macrophage cell line, microtubule disassembly by colchicine causes the polarization of membrane functions ane structure. Colchicine-treated cells develop a bulge or protuberance that is bordered by microvillous membrane. The protuberance is the site of concanavalin A cap formation. The fluid pinocytosis of horseradish peroxidase and of fluorescein- and rhodamine-conjugated high molecular-weight dextrans, the adsorptive pinocytosis of concanavalin A, and the concentration and phagocytosis at 37 degrees C of a range of phagocytic particles (IgG- and complement-opsonized erythrocytes, complement-opsonized zymosan, latex shpres, albumin-stabilized oil droplets) are all similarly restricted to the protuberance. A reduction in the rate of dextran pinocytosis, determined by fluorimetry, and reductions in phagocytic rates for oil emulsion and IgG-opsonized erythrocytes accompany the polarization of endocytic activity in colchicine-trated 1774.2 macrophages. Membrane receptors for phagocytic particles are not confined to the protuberance but rather may display their own unique topographical asymmetry. The inherent topography of receptors was inferred from particle distribution under conditions that limit particle-receptor redistribution (after labeling at 4 degrees C or a very brief incubation at 37 degrees C). Under these restrictive conditions, latex binding sites were detected over the whole membrane whereas receptors for IgG-opsonized erythrocytes, aggregated IgG, complement-opsonized erythrocytes, and complement-opsonized zymosan were excluded from the protuberance. Thus, functional (endocytosis) and structural (inherent receptor distribution) analyses of membrane topography define different patterns of asymmetry in protuberant cells. The asymmetry induced in 1774.2 macrophages by colchicine is highly analogous to the functional and structural polarity of epithelial cells. Exploration of this analogy may provide insight into the development of polarized epithelia and, more generally, into mechanisms by which specialized areas of membrane are established.

scholarly journals Quantifying the cooperative subunit action in a multimeric membrane receptor

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Nisa Wongsamitkul ◽  
Vasilica Nache ◽  
Thomas Eick ◽  
Sabine Hummert ◽  
Eckhard Schulz ◽  
...  
Keyword(s):  
Ion Channels ◽  
Ligand Binding ◽  
Cyclic Nucleotide ◽  
Binding Sites ◽  
Membrane Receptor ◽  
Membrane Receptors ◽  
Cooperative Action ◽  
The Individual ◽  
Insight Into

Abstract In multimeric membrane receptors the cooperative action of the subunits prevents exact knowledge about the operation and the interaction of the individual subunits. We propose a method that permits quantification of ligand binding to and activation effects of the individual binding sites in a multimeric membrane receptor. The power of this method is demonstrated by gaining detailed insight into the subunit action in olfactory cyclic nucleotide-gated CNGA2 ion channels.

scholarly journals Putative ligand binding sites of two functionally characterized bark beetle odorant receptors

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Jothi K. Yuvaraj ◽  
Rebecca E. Roberts ◽  
Yonathan Sonntag ◽  
Xiao-Qing Hou ◽  
Ewald Grosse-Wilde ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Bark Beetle ◽  
Pest Control ◽  
Binding Sites ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
Odorant Receptors ◽  
Functional Evolution ◽  
General Importance ◽  
Insight Into

Abstract Background Bark beetles are major pests of conifer forests, and their behavior is primarily mediated via olfaction. Targeting the odorant receptors (ORs) may thus provide avenues towards improved pest control. Such an approach requires information on the function of ORs and their interactions with ligands, which is also essential for understanding the functional evolution of these receptors. Hence, we aimed to identify a high-quality complement of ORs from the destructive spruce bark beetle Ips typographus (Coleoptera, Curculionidae, Scolytinae) and analyze their antennal expression and phylogenetic relationships with ORs from other beetles. Using 68 biologically relevant test compounds, we next aimed to functionally characterize ecologically important ORs, using two systems for heterologous expression. Our final aim was to gain insight into the ligand-OR interaction of the functionally characterized ORs, using a combination of computational and experimental methods. Results We annotated 73 ORs from an antennal transcriptome of I. typographus and report the functional characterization of two ORs (ItypOR46 and ItypOR49), which are responsive to single enantiomers of the common bark beetle pheromone compounds ipsenol and ipsdienol, respectively. Their responses and antennal expression correlate with the specificities, localizations, and/or abundances of olfactory sensory neurons detecting these enantiomers. We use homology modeling and molecular docking to predict their binding sites. Our models reveal a likely binding cleft lined with residues that previously have been shown to affect the responses of insect ORs. Within this cleft, the active ligands are predicted to specifically interact with residues Tyr84 and Thr205 in ItypOR46. The suggested importance of these residues in the activation by ipsenol is experimentally supported through site-directed mutagenesis and functional testing, and hydrogen bonding appears key in pheromone binding. Conclusions The emerging insight into ligand binding in the two characterized ItypORs has a general importance for our understanding of the molecular and functional evolution of the insect OR gene family. Due to the ecological importance of the characterized receptors and widespread use of ipsenol and ipsdienol in bark beetle chemical communication, these ORs should be evaluated for their potential use in pest control and biosensors to detect bark beetle infestations.

scholarly journals Structures of the somatotropin receptor and prolactin receptor on rat hepatocytes characterized by affinity labelling

1984 ◽  
Vol 220 (2) ◽  
pp. 361-369 ◽  
Author(s):  
K Yamada ◽  
D B Donner
Keyword(s):  
Binding Sites ◽  
Prolactin Receptor ◽  
Rat Hepatocytes ◽  
Membrane Receptors ◽  
Bovine Somatotropin ◽  
Male Rats ◽  
Disulphide Bonds ◽  
Low Concentrations ◽  
Affinity Labelling ◽  
Structural Descriptions

Human somatotropin competed for 125I-human somatotropin binding to hepatocytes from female or male rats. Bovine somatotropin and prolactin each inhibited part, but not all, of the uptake of 125I-human somatotropin. The binding of 125I-prolactin was inhibited by human somatotropin and prolactin, but not by bovine somatotropin. Bovine somatotropin and human somatotropin, but not prolactin, competed for 125I-bovine somatotropin binding sites. 125I-labelled hormones were covalently coupled to membrane receptors with higher efficiency on hepatocytes from female than from male rats, allowing structural descriptions of lactogenic and somatogenic binding sites that had not been possible previously. Disuccinimidyl suberate covalently coupled 125I-human somatotropin into saturable complexes of Mr 300 000, 220 000, 130 000, 65 000 and 50 000. Bovine somatotropin inhibited the incorporation of 125I-human somatotropin into complexes of Mr 300 000, 220 000 and 130 000, whereas low concentrations of prolactin competed for incorporation into the 65 000- and 50 000-Mr species. 125I-bovine somatotropin was incorporated into complexes of Mr 300 000, 220 000 and 130 000. Human somatotropin and bovine somatotropin, but not prolactin, inhibited the production of these complexes. 125I-prolactin binding produced complexes of Mr 65 000 and 50 000. Native prolactin and human somatotropin, but not bovine somatotropin, inhibited uptake of 125I-prolactin into these species. Thus direct affinity labelling, as well as competition for covalent coupling, suggests that the 300 000-, 220 000- and 130 000-Mr species are components of the somatotropin receptor and that the 65 000- and 50 000-Mr complexes result from hormone binding to the prolactin receptor. By subtracting the Mr of prolactin, it was calculated that the hormone was bound to species of Mr 43 000 and 28 000. These Mr values were not affected by reduction of solubilized membranes, suggesting that the structure of the prolactin receptor is not stabilized by interchain disulphide bonds between subunits. Subtracting the Mr of somatotropin from somatogenic complexes indicated that the hormone had bound to species of Mr 280 000, 200 000 and 100 000. The 300 000- and 220 000-Mr complexes were not isolated from reduced membranes, whereas the amount of the 130 000-Mr species was augmented. These observations could suggest that a major component of the somatotropin receptor is a trimeric aggregate in which some subunits are retained in a larger complex by interchain disulphide bonds.

scholarly journals Visualization of intracellular concanavalin A binding sites in retinal photoreceptors.

1978 ◽  
Vol 26 (10) ◽  
pp. 822-828 ◽  
Author(s):  
I Nir
Keyword(s):  
Concanavalin A ◽  
Binding Sites ◽  
Photoreceptor Cells ◽  
Con A ◽  
Thin Sections ◽  
Glycol Methacrylate ◽  
Outer Segments ◽  
Retinal Photoreceptors ◽  
Carbohydrate Components ◽  
The Relationship

Localization of carbohydrate components in retinal photoreceptor cells and membranes was studied. Frog and rat retinas were fixed with glutaraldehyde and embedded in glycol methacrylate or in a mixture of glycol methacrylate, glutaraldehyde and urea. Thin sections were incubated with ferritin-labeled concanavalin A (F-Con A) and stained with osmium vapors. Intensive binding was observed in both rod and cone outer segments. In the rod inner segment, differential binding of F-Con A was demonstrated. While numerous ferritin granules were observed in the myoid zone, only a few were seen in the ellipsoid zone, except for a local accumulation along the plasma membrane. In the rod outer segment, Con A binding sites were closely associated with the disk membranes. Ferritin granules were observed on both sides of the membranes. The relationship between the localization of Con A binding sites and the orientation of visual pigment molecules within the rod outer segments disk membranes was discussed.

Nuclear location of PLA2-I in proliferative cells

1998 ◽  
Vol 111 (7) ◽  
pp. 985-994 ◽  
Author(s):  
J.M. Fayard ◽  
C. Tessier ◽  
J.F. Pageaux ◽  
M. Lagarde ◽  
C. Laugier
Keyword(s):  
Binding Sites ◽  
Culture Medium ◽  
Aristolochic Acid ◽  
Direct Interaction ◽  
Nuclear Level ◽  
Proliferative Effect ◽  
Stromal Cell Line ◽  
Normal Rat ◽  
Nuclear Location ◽  
Insight Into

We have previously demonstrated that pancreatic PLA2 (PLA2-I) stimulates the proliferation of UIII cells, a stromal cell line derived from normal rat uterus. In order to gain further insight into the mechanism of action of PLA2-I, we have investigated the intracellular processing of PLA2-I. Either highly proliferative or growth arrested UIII cells were analyzed. Growth arrested cells were obtained from a contact inhibited monolayer or from aristolochic acid-treated cultures. Using cellular fractionation, western blotting, immunocytochemistry and confocal microscopy, we demonstrate that endogenous PLA2-I was mainly located in the nucleus in highly proliferative cells whereas its location was cytoplasmic in non proliferative cells. When non confluent UIII cells were incubated with nanomolar amounts of exogenous PLA2-I, the enzyme was internalized and, in the majority of cells, appeared within the nucleus. Both internalization and nuclear location of exogenous PLA2-I were suppressed by the addition of aristolochic acid to the culture medium. Binding experiments performed on purified nuclear preparations showed the presence of specific cooperative binding sites for PLA2-I. Collectively our data suggest that the proliferative effect exerted by pancreatic PLA2 in UIII cells is mediated by a direct interaction of the enzyme at the nuclear level. Putative mechanisms and targets are discussed.

Mapping of ligand binding sites provides insight into the function of factor H-related protein 5

2017 ◽  
Vol 89 ◽  
pp. 145
Author(s):  
Alexandra Papp ◽  
Marcell Cserhalmi ◽  
Ádám I. Csincsi ◽  
Barbara Uzonyi ◽  
David Ermert ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Factor H ◽  
Related Protein ◽  
Ligand Binding Sites ◽  
Insight Into

Participation of concanavalin A binding sites in the interaction between Trypanosoma cruzi and macrophages

1983 ◽  
Vol 62 (1) ◽  
pp. 287-299
Author(s):  
M.N. Meirelles ◽  
A. Martinez-Palomo ◽  
T. Souto-Padron ◽  
W. De Souza
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Trypanosoma Cruzi ◽  
Uniform Distribution ◽  
Concanavalin A ◽  
Binding Sites ◽  
Peritoneal Macrophages ◽  
Untreated Mouse ◽  
Mouse Peritoneal Macrophages

Untreated mouse peritoneal macrophages as well as macrophages treated with concanavalin A (ConA) were incubated in the presence of untreated or ConA-treated epimastigotes and trypomastigotes of Trypanosoma cruzi. Treatment of epimastigotes or trypomastigotes with ConA increased or decreased their uptake by macrophages, respectively. Treatment of their macrophages with ConA reduced by 70% and increased by five times the ingestion of epimastigotes and trypomastigotes, respectively. These results are discussed in relation to previous studies on the mobility of ConA receptors in the membrane of the parasite. Using fluorescein- or ferritin-labelled ConA we observed that ConA binding sites located on the plasma membrane of macrophages are internalized during endocytosis of T. cruzi, and observed in association with the membrane of the endocytic vacuole. Vacuoles without parasites showed a uniform distribution of ConA binding sites, while these sites were distributed in patches in vacuoles containing parasites. These results, in association with others previously reported, suggest the involvement of glycoproteins and/or glycolipids localized on the cell surface of T. cruzi and macrophages during the T. cruzi-macrophage interaction.

Differences in density of Concanavalin A-binding sites due to differences in surface morphology of suspended normal and transformed 3T3 fibroblasts

1975 ◽  
Vol 19 (1) ◽  
pp. 21-32
Author(s):  
J.G. Collard ◽  
J.H. Temmink
Keyword(s):  
Surface Morphology ◽  
Concanavalin A ◽  
Binding Sites ◽  
3T3 Cells ◽  
Con A ◽  
3T3 Fibroblasts ◽  
Transformed Fibroblasts ◽  
Transmission Electron ◽  
The Difference ◽  
Electron Microscopes

Calculations of the density of Concanavalin A (Con A)-binding sites on normal and transformed fibroblasts have, as yet, been based on the unproven assumption that suspended cells are smooth spheres. We studied the surface morphology of suspended normal and transformed fibroblasts with scanning and transmission electron microscopes, and found a large difference in surface morphology between suspended normal and transformed 3T3 cells. When this difference in surface morphology was taken into account, the estimated cell surface area of normal 3T3 cells was approximately seven times larger than that of transformed 3T3 cells. Since equal numbers of 3H-Con A molecules are bound on normal and transformed cells, the density of Con A-binding sites is approximately seven times greater on transformed than on normal 3T3 cells. The difference in density of Con A-binding sites between normal and transformed fibroblasts might be sufficient to explain the difference in agglutination response, as originally suggested by Burger, and may also be the cause of the different degrees of clustering of Con A-binding sites on the plasma membrane of these cells.

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