scholarly journals Ligand affinity of the 67-kD elastin/laminin binding protein is modulated by the protein's lectin domain: visualization of elastin/laminin-receptor complexes with gold-tagged ligands.

1991 ◽  
Vol 113 (1) ◽  
pp. 187-194 ◽  
Author(s):  
R P Mecham ◽  
L Whitehouse ◽  
M Hay ◽  
A Hinek ◽  
M P Sheetz
Keyword(s):  
Cell Surface ◽  
Laminin Receptor ◽  
Live Cells ◽  
Receptor Interaction ◽  
Ligand Complex ◽  
Lectin Domain ◽  
Receptor Complexes ◽  
Laminin Binding Protein

Video-enhanced microscopy was used to examine the interaction of elastin- or laminin-coated gold particles with elastin binding proteins on the surface of live cells. By visualizing the binding events in real time, it was possible to determine the specificity and avidity of ligand binding as well as to analyze the motion of the receptor-ligand complex in the plane of the plasma membrane. Although it was difficult to interpret the rates of binding and release rigorously because of the possibility for multiple interactions between particles and the cell surface, relative changes in binding have revealed important aspects of the regulation of affinity of ligand-receptor interaction in situ. Both elastin and laminin were found to compete for binding to the cell surface and lactose dramatically decreased the affinity of the receptor(s) for both elastin and laminin. These findings were supported by in vitro studies of the detergent-solubilized receptor. Further, immobilization of the ligand-receptor complexes through binding to the cytoskeleton dramatically decreased the ability of bound particles to leave the receptor. The changes in the kinetics of ligand-coated gold binding to living cells suggest that both laminin and elastin binding is inhibited by lactose and that attachment of receptor to the cytoskeleton increases its affinity for the ligand.

scholarly journals Trace Amine-Associated Receptor 1 Trafficking to Cilia of Thyroid Epithelial Cells

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1518
Author(s):  
Maria Qatato ◽  
Vaishnavi Venugopalan ◽  
Alaa Al-Hashimi ◽  
Maren Rehders ◽  
Aaron D. Valentine ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Human Thyroid ◽  
Apical Plasma Membrane ◽  
Thyroid Cell ◽  
Thyroid Cells ◽  
Trace Amine ◽  
Rat Thyroid

Trace amine-associated receptor 1 (rodent Taar1/human TAAR1) is a G protein-coupled receptor that is mainly recognized for its functions in neuromodulation. Previous in vitro studies suggested that Taar1 may signal from intracellular compartments. However, we have shown Taar1 to localize apically and on ciliary extensions in rodent thyrocytes, suggesting that at least in the thyroid, Taar1 may signal from the cilia at the apical plasma membrane domain of thyrocytes in situ, where it is exposed to the content of the follicle lumen containing putative Taar1 ligands. This study was designed to explore mouse Taar1 (mTaar1) trafficking, heterologously expressed in human and rat thyroid cell lines in order to establish an in vitro system in which Taar1 signaling from the cell surface can be studied in future. The results showed that chimeric mTaar1-EGFP traffics to the apical cell surface and localizes particularly to spherical structures of polarized thyroid cells, procilia, and primary cilia upon serum-starvation. Moreover, mTaar1-EGFP appears to form high molecular mass forms, possibly homodimers and tetramers, in stably expressing human thyroid cell lines. However, only monomeric mTaar1-EGFP was cell surface biotinylated in polarized human thyrocytes. In polarized rat thyrocytes, mTaar1-EGFP is retained in the endoplasmic reticulum, while cilia were reached by mTaar1-EGFP transiently co-expressed in combination with an HA-tagged construct of the related mTaar5. We conclude that Taar1 trafficking to cilia depends on their integrity. The results further suggest that an in vitro cell model was established that recapitulates Taar1 trafficking in thyrocytes in situ, in principle, and will enable studying Taar1 signaling in future, thus extending our general understanding of its potential significance for thyroid autoregulation.

scholarly journals Human macrophage maturation and heterogeneity: analysis with a newly generated set of monoclonal antibodies to differentiation antigens

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1257-1264 ◽  
Author(s):  
R Andreesen ◽  
KJ Bross ◽  
J Osterholz ◽  
F Emmrich
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Cell Lineage ◽  
Culture Conditions ◽  
Human Macrophage ◽  
Blood Monocytes ◽  
Differentiation Antigens

We have analyzed the expression of late differentiation antigens during terminal in vitro maturation of human macrophages (M phi) from blood monocytes (MO) in comparison to their distribution among mature M phi residing in various tissue sites. By immunizing mice with M phi derived from blood MO by culture on hydrophobic Teflon foils, monoclonal antibodies (mAbs) were developed (MAX.1, MAX.2, MAX.3, MAX.11) that reacted with lineage-restricted differentiation antigens. These antigens were expressed exclusively on M phi or were markedly increased after in vitro differentiation. The only overlap to another hemopoietic cell lineage was observed with MAX.3, which is shared by platelets and megakaryocytes. In the course of M phi maturation in vitro, the MAX.1 and MAX.3 antigens are detected within the cytoplasm two days before they appear on the cell surface. In contrast, the MAX.11 antigen is expressed simultaneously in the cytoplasm and at the cell surface, is found in varying degrees on a minor portion of blood MO and U937 cells, and is expressed rapidly at high density during early M phi differentiation in vitro. Among conventional mAbs that do not react with MO we found those against the transferrin (TF)-receptor, the BA-2, and the PCA1 antigen to label M phi. M phi matured in vivo and isolated from body fluids were positive with some but not all MAX mAbs. Distinctive patterns were observed with pulmonary M phi, exudate M phi from pleural and peritoneal effusions, synovial fluids, and early lactation milk. M phi from the alveolar space, for example, constantly expressed the MAX.2 antigen but not the MAX.3 antigen. Pleural effusion M phi, however, did not react with the MAX.1 mAb, but in most cases, it did react with the MAX.3 mAb. The detection of novel differentiation antigens, all expressed on monocyte-derived M phi but differently expressed on site-specific M phi in situ, underlines the remarkable heterogeneity among human M phi. The expression of these antigens is flexible because those MAX antigens that were not expressed in situ could be induced if cells from distinct tissue sites were cultured in vitro for several days. MAX mAbs may be of potential value to study both the sequential stages of maturation within the M phi lineage as well as differential developments induced by various culture conditions in parallel to environmental factors in vivo.

scholarly journals Stimulus-secretion coupling in the developing exocrine pancreas: secretory responsiveness to cholecystokinin.

1986 ◽  
Vol 103 (6) ◽  
pp. 2353-2365 ◽  
Author(s):  
A Chang ◽  
J D Jamieson
Keyword(s):  
Cell Surface ◽  
Exocrine Pancreas ◽  
Cell Surface Expression ◽  
Receptor Interaction ◽  
Amylase Secretion ◽  
Ionophore A23187 ◽  
Amylase Release ◽  
Fetal Pancreas ◽  
45Ca Efflux

We have studied the onset of secretory responsiveness to cholecystokinin (CCK) during development of the rat exocrine pancreas. Although acinar cells of the fetal pancreas (1 d before birth) are filled with zymogen granules containing the secretory protein, alpha-amylase, the rate of amylase secretion from pancreatic lobules incubated in vitro was not increased in response to CCK. In contrast, the rate of CCK-stimulated amylase discharge from the neonatal pancreas (1 d after birth) was increased four- to eightfold above that of the fetal gland. The postnatal amplification of secretory responsiveness was not associated with an increase in the number or cell surface expression of 125I-CCK binding sites. When 125I-CCK-33 binding proteins were analyzed by affinity crosslinking, two proteins of Mr 210,000 and 100,000-160,000 were labeled specifically in both fetal and neonatal pancreas. To determine if cell surface receptors for CCK in the fetal pancreas are functional and able to generate a rise in the cytosolic [Ca++], we measured 45Ca++ efflux from tracer-loaded lobules. 45Ca++ efflux from both fetal and neonatal pancreas was comparably increased by CCK, indicating CCK-induced Ca++ mobilization and elevated cytosolic [Ca++]. The Ca++ ionophore A23187 also stimulated the rate of 45Ca++ extrusion from pancreas of both ages. Increased amylase secretion occurred concurrently with A23187-stimulated 45Ca++ efflux in neonatal pancreas, but not in the fetal gland. A23187 in combination with dibutyryl cAMP potentiated amylase release from the neonatal gland, but not from fetal pancreas. Similarly, the protein kinase C activator, phorbol dibutyrate, did not increase the rate of secretion from the fetal gland when added alone or in combination with A23187 or CCK. We suggest that CCK-receptor interaction in the fetal pancreas triggers intracellular Ca++ mobilization. However, one or more signal transduction events distal to Ca++ mobilization have not yet matured. The onset of secretory response to CCK that occurs postnatally may depend on amplification of these transduction events.

scholarly journals α-Dystroglycan Is a Laminin Receptor Involved in Extracellular Matrix Assembly on Myotubes and Muscle Cell Viability

1999 ◽  
Vol 145 (6) ◽  
pp. 1325-1340 ◽  
Author(s):  
Federica Montanaro ◽  
Michael Lindenbaum ◽  
Salvatore Carbonetto
Keyword(s):  
Acetylcholine Receptors ◽  
Laminin Receptor ◽  
Matrix Assembly ◽  
Selection Procedures ◽  
Glycoprotein Complex ◽  
Gradual Loss ◽  
Expression Construct ◽  
Muscle Specific Kinase ◽  
Laminin Binding Protein

α-Dystroglycan (α-DG) is a laminin-binding protein and member of a glycoprotein complex associated with dystrophin that has been implicated in the etiology of several muscular dystrophies. To study the function of DG, C2 myoblasts were transfected stably with an antisense DG expression construct. Myotubes from two resulting clones (11F and 11E) had at least a 40–50% and 80–90% reduction, respectively, in α-DG but normal or near normal levels of α-sarcoglycan, integrin β1 subunit, acetylcholine receptors (AChRs), and muscle-specific kinase (MuSK) when compared with parental C2 cells or three clones (11A, 9B, and 10C) which went through the same transfection and selection procedures but expressed normal levels of α-DG. Antisense DG-expressing myoblasts proliferate at the same rate as parental C2 cells and differentiate into myotubes, however, a gradual loss of cells was observed in these cultures. This loss correlates with increased apoptosis as indicated by greater numbers of nuclei with condensed chromatin and more nuclei labeled by the TUNEL method. Moreover, there was no sign of increased membrane permeability to Trypan blue as would be expected with necrosis. Unlike parental C2 myotubes, 11F and 11E myotubes had very little laminin (LN) on their surfaces; LN instead tended to accumulate on the substratum between myotubes. Exogenous LN bound to C2 myotubes and was redistributed into plaques along with α-DG on their surfaces but far fewer LN/α-DG plaques were seen after LN addition to 11F or 11E myotubes. These results suggest that α-DG is a functional LN receptor in situ which is required for deposition of LN on the cell and, further, implicate α-DG in the maintenance of myotube viability.

scholarly journals Chemical profiling of DNA G-quadruplex-interacting proteins in live cells

2021 ◽  
Author(s):  
Xiaoyun Zhang ◽  
Jochen Spiegel ◽  
Sergio Martínez Cuesta ◽  
Santosh Adhikari ◽  
Shankar Balasubramanian
Keyword(s):  
Protein Interactions ◽  
Protein Profiling ◽  
Live Cells ◽  
Interacting Proteins ◽  
Chemical Profiling ◽  
G Quadruplex ◽  
Functional Classes ◽  
Chemical Strategy

AbstractDNA–protein interactions regulate critical biological processes. Identifying proteins that bind to specific, functional genomic loci is essential to understand the underlying regulatory mechanisms on a molecular level. Here we describe a co-binding-mediated protein profiling (CMPP) strategy to investigate the interactome of DNA G-quadruplexes (G4s) in native chromatin. CMPP involves cell-permeable, functionalized G4-ligand probes that bind endogenous G4s and subsequently crosslink to co-binding G4-interacting proteins in situ. We first showed the robustness of CMPP by proximity labelling of a G4 binding protein in vitro. Employing this approach in live cells, we then identified hundreds of putative G4-interacting proteins from various functional classes. Next, we confirmed a high G4-binding affinity and selectivity for several newly discovered G4 interactors in vitro, and we validated direct G4 interactions for a functionally important candidate in cellular chromatin using an independent approach. Our studies provide a chemical strategy to map protein interactions of specific nucleic acid features in living cells.

scholarly journals Cell surface galactosyltransferase mediates the initiation of neurite outgrowth from PC12 cells on laminin.

1990 ◽  
Vol 110 (2) ◽  
pp. 461-470 ◽  
Author(s):  
P C Begovac ◽  
B D Shur
Keyword(s):  
Cell Surface ◽  
Neurite Outgrowth ◽  
Enzymatic Reaction ◽  
Mesenchymal Cell ◽  
Laminin Receptor ◽  
Extracellular Matrix Molecule ◽  
Neurite Initiation ◽  
Neurite Formation ◽  
And Migration

Neurite outgrowth from PC12 pheochromocytoma cells, as well as from peripheral and central nervous system neurons in vitro, is mediated by the extracellular matrix molecule, laminin. We have recently shown that mesenchymal cell spreading and migration on laminin is mediated, in part, by the cell surface enzyme, beta 1,4 galactosyltransferase (GalTase). GalTase is localized on lamellipodia of migrating cells where it functions as a laminin receptor by binding to specific N-linked oligosaccharides in laminin (Runyan et al., 1988; Eckstein and Shur, 1989). In the present study, we examined whether GalTase functions similarly during neutrite outgrowth on laminin using biochemical and immunological analyses. PC12 neurite outgrowth was inhibited by reagents that perturb cell surface GalTase activity, including anti-GalTase IgG and Fab fragments, as well as the GalTase modifier protein alpha-lactalbumin. Control reagents had no effect on neurite outgrowth. Furthermore, blocking GalTase substrates on laminin matrices by earlier galactosyltion or enzymatic removal of GalTase substrates also inhibited neurite outgrowth. Conversely, neurite outgrowth was enhanced by the addition of UDP-galactose, which completes the GalTase enzymatic reaction, while inappropriate sugar nucleotides had no effect. The effects of all these treatments were dose and/or time dependent. Surface GalTase was shown to function during both neurite initiation and elongation, although the effects of GalTase perturbation were most striking during the initiation stages of neurite formation. Consistent with this, surface GalTase was localized by indirect immunofluorescence to the growth cone and developing neurite. Collectively, these results demonstrate that GalTase mediates the initiation of neurite outgrowth on laminin, and to a lesser extent, neurite elongation. Furthermore, this study demonstrates that process extension from both mesenchymal cells and neuronal cells is partly dependent upon specific oligosaccharide residues in laminin.

scholarly journals Immunocytochemical visualization of luteinizing hormone-human chorionic gonadotropin (LH-hCG) receptors in Leydig cells in primary culture.

1983 ◽  
Vol 31 (7) ◽  
pp. 898-904 ◽  
Author(s):  
M Begeot ◽  
C Mombrial ◽  
P M Dubois ◽  
M P Dubois ◽  
A Dazord ◽  
...  
Keyword(s):  
Luteinizing Hormone ◽  
Cell Surface ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Leydig Cells ◽  
Defined Medium ◽  
Short Exposure ◽  
Receptor Complexes ◽  
Immunocytochemical Reaction

Visualization of human chorionic gonadotropin (hCG) binding sites was obtained by immunocytochemical reaction on Leydig cells cultured in chemically defined medium. After a short in vitro incubation with hCG or luteinizing hormone (LH), the cells were fixed and the bound molecules were revealed using anti-hCG or anti-LH antisera. In both cases the immunocytochemical reaction appeared as granulations at the cell surface. After prolonged (48 hr) culture in the presence of 0.5, 5, or 50 ng/ml of hCG, the hormone receptor complex is still visible. Similarly, following short exposure to hCG (0.5 or 50 ng/ml) and one or two days of culture without hCG, the immunocytochemical reaction is still present. These observations suggest that the half-life of the bound hormone is very long. This in vitro system in which the amount and time of hormone exposure are precisely defined provides arguments in favor of a long-term maintenance of the receptor complexes at the cell surface.

Specific receptor for hydrazine: mapping the in situ release of hydrazine in live cells and in an in vitro enzymatic assay

2016 ◽  
Vol 52 (36) ◽  
pp. 6166-6169 ◽  
Author(s):  
Firoj Ali ◽  
Anila H. A. ◽  
Nandaraj Taye ◽  
Devraj G. Mogare ◽  
Samit Chattopadhyay ◽  
...  
Keyword(s):  
Hepg2 Cells ◽  
Enzymatic Assay ◽  
Live Cells ◽  
Specific Receptor ◽  
Specific Detection ◽  
Physiological Conditions

New chemodosimetric reagent for the specific detection of hydrazine in physiological conditions as well as for the mapping of its in situ generation in live Hct116 and HepG2 cells by enzymatic transformations.

scholarly journals Ligand-mediated autophosphorylation activity of the epidermal growth factor receptor during internalization.

1989 ◽  
Vol 109 (6) ◽  
pp. 2751-2760 ◽  
Author(s):  
W H Lai ◽  
P H Cameron ◽  
J J Doherty ◽  
B I Posner ◽  
J J Bergeron
Keyword(s):  
Electron Microscope ◽  
Cell Surface ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Receptor Complexes ◽  
Diminished Capacity ◽  
Epidermal Growth ◽  
Polyethylene Glycol Precipitation

The association of EGF with its receptor in endosomes isolated from rat liver homogenates was assessed biochemically by polyethylene glycol precipitation and morphologically by electron microscope radioautography. The proportion of receptor-bound ligand in endosomes at 15 min after the injection of doses of 0.1 and 1 microgram EGF/100 g body weight was 57%. This value increased to 77% for the dose of 10 micrograms EGF injected. Quantitative electron microscope radioautography carried out on endosomes isolated at 15 min after the injection of 10 micrograms 125I-EGF demonstrated that most radiolabel was over the endosomal periphery thereby indicating that ligand-receptor complexes were in the bounding membrane but not in intraluminal vesicles of the content. EGF receptor autophosphorylation activity during internalization was evaluated in plasmalemma and endosome fractions. This activity was markedly but transiently reduced on the cell surface shortly after the administration of saturating doses of EGF. The same activity, however, was augmented and prolonged in endosomes for up to 30 min after EGF injection. The transient desensitization of cell surface activity was not due to prior in vivo phosphorylation since receptor dephosphorylation in vitro failed to restore autophosphorylation activity. Transient desensitization of cell surface autophosphorylation activity coincided with a diminished capacity for endocytosis of 125I-EGF with endocytosis returning to normal after the restoration of cell surface autophosphorylation activity. The inhibition of cell surface autophosphorylation activity and the activation of endosomal autophosphorylation activity coincident with downregulation suggest that EGF receptor traffic is governed by ligand-regulated phosphorylation activity.

Sign in / Sign up

Export Citation Format

Share Document