Cloning of the J gene of bacteriophage lambda, expression and solubilization of the J protein: first in vitro studies on the interactions between J and LamB, its cell surface receptor

1998 ◽  
Vol 149 (9) ◽  
pp. 611-624 ◽  
Author(s):  
J. Wang ◽  
V. Michel ◽  
M. Hofnung ◽  
A. Charbit
Keyword(s):  
Cell Surface ◽  
Bacteriophage Lambda ◽  
In Vitro Studies ◽  
Cell Surface Receptor ◽  
Surface Receptor ◽  
J Protein ◽  
Lambda Expression

Interaction of Bacteriophage Lambda with Its Cell Surface Receptor:  An in Vitro Study of Binding of the Viral Tail Protein gpJ to LamB (Maltoporin)†

Biochemistry ◽  
2006 ◽  
Vol 45 (8) ◽  
pp. 2708-2720 ◽  
Author(s):  
Emir Berkane ◽  
Frank Orlik ◽  
Johannes F. Stegmeier ◽  
Alain Charbit ◽  
Mathias Winterhalter ◽  
...  
Keyword(s):  
Cell Surface ◽  
Bacteriophage Lambda ◽  
In Vitro Study ◽  
Vitro Study ◽  
Cell Surface Receptor ◽  
Surface Receptor ◽  
Tail Protein

scholarly journals ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis

2018 ◽  
Vol 19 (10) ◽  
pp. 3089 ◽  
Author(s):  
Marie Hlavničková ◽  
Milan Kuchař ◽  
Radim Osička ◽  
Lucie Vaňková ◽  
Hana Petroková ◽  
...  
Keyword(s):  
Cell Surface ◽  
Clinical Manifestations ◽  
Cell Surface Receptor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Interleukin 17 ◽  
Normal Human Skin ◽  
High Affinity ◽  
Th 17 ◽  
Surface Receptor

Interleukin 17 (IL-17) and its cognate receptor A (IL-17RA) play a crucial role in Th17 cells-mediated pro-inflammatory pathway and pathogenesis of several autoimmune disorders including psoriasis. IL-17 is mainly produced by activated Th-17 helper cells upon stimulation by IL-23 and, via binding to its receptors, mediates IL-17-driven cell signaling in keratinocytes. Hyper-proliferation of keratinocytes belongs to major clinical manifestations in psoriasis. To modulate IL-17-mediated inflammatory cascade, we generated a unique collection of IL-17RA-targeting protein binders that prevent from binding of human IL-17A cytokine to its cell-surface receptor. To this goal, we used a highly complex combinatorial library derived from scaffold of albumin-binding domain (ABD) of streptococcal protein G, and ribosome display selection, to yield a collection of ABD-derived high-affinity ligands of human IL-17RA, called ARS binders. From 67 analyzed ABD variants, 7 different sequence families were identified. Representatives of these groups competed with human IL-17A for binding to recombinant IL-17RA receptor as well as to IL-17RA-Immunoglobulin G chimera, as tested in enzyme-linked immunosorbent assay (ELISA). Five ARS variants bound to IL-17RA-expressing THP-1 cells and blocked binding of human IL-17 cytokine to the cell surface, as tested by flow cytometry. Three variants exhibited high-affinity binding with a nanomolar Kd value to human keratinocyte HaCaT cells, as measured using Ligand Tracer Green Line. Upon IL-17-stimulated activation, ARS variants inhibited secretion of Gro-α (CXCL1) by normal human skin fibroblasts in vitro. Thus, we identified a novel class of inhibitory ligands that might serve as immunosuppressive IL-17RA-targeted non-IgG protein antagonists.

Thranbospondin Promotes Cell-and Platelet-Substratum Adhesion

1987 ◽  
Author(s):  
George P Tuszynski ◽  
Vicki L Rothman ◽  
Andrew Murphy ◽  
Katherine Siegler ◽  
Linda Smith ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cell Types ◽  
Preliminary Evidence ◽  
Human Platelets ◽  
Cell Surface Receptor ◽  
Binding Assays ◽  
Surface Receptor

Thrombospondin (TSP), isolated from human platelets, promotes the in vitro, calcium-specific adhesion of a variety of cells, including platelets, melanoma cells, muscle cells, endothelial cells, fibroblasts, and epithelial cells. The cell adhesion-promoting activity of TSP is species independent since human, bovine, pig, rat and mouse cells all adhered to TSP. Furthermore, the cell adhesion-promoting activity of TSP is specific and not due to a nonspecific protein effect or to contamination by fibronectin, vitronectin, or laminin. That is, neither bovine serum albumin nor TSP preparations treated with a monospecific anti-TSP antibody support cell adhesion. As analyzed by polyacrylamide-gel electrophoresis and specific antibody binding assays, the TSP preparations used in these studies contained no detectable fibronectin or laminin and less than 0.04% vitronectin. The cell surface receptor for TSP appears distinct frcm that of fibronectin since an antiserum that blocks cell adhesion to fibronectin has no effect on adhesion to TSP. In addition, The platelet cell surface receptor for TSP appears distinct, frcm that of fibrinogen since thrcmbasthenic platelets adhere to TSP as well as control platelets. Antibodies to the GPIIb-GPIIIa complex block platelet adhesion to fibrinogen but have no effect on adhesion to TSP. Initial characterization of the cell surface receptor for TSP shows it to be protein in nature since cells treated with trypsin fail to adhere to TSP. In summary, our results provide the first clear evidence that TSP specifically promotes cell-substratum adhesion of a variety of cell types independent of the animal species. Our preliminary evidence suggests that the cell-surface receptor(s) for TSP is protein and that it is distinct for the receptor for fibronectin and fibrinogen. Our data suggest that TSP may play a central role in normal adhesive events mediated by platelets and other cells, such as those involved in hemostasis and wound healing. In addition, TSP may be involved in pathological adhesive events mediated by platelets and tumor cells, such as those involved in cardiovascular disease and tumor cell metastasis.

In vitro biosynthesis of the human cell surface receptor for transferrin

FEBS Letters ◽  
1983 ◽  
Vol 158 (2) ◽  
pp. 259-264 ◽  
Author(s):  
C. Schneider ◽  
U. Asser ◽  
D.R. Sutherland ◽  
M.F. Greaves
Keyword(s):  
Cell Surface ◽  
Human Cell ◽  
Cell Surface Receptor ◽  
Surface Receptor ◽  
In Vitro Biosynthesis

Inhibition of bovine retinal microvascular pericyte proliferation in vitro by adenosine

1992 ◽  
Vol 263 (2) ◽  
pp. H634-H640 ◽  
Author(s):  
J. A. Jackson ◽  
E. C. Carlson
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Adenosine Receptor ◽  
Inhibitory Effect ◽  
Cell Surface Receptor ◽  
Dual Function ◽  
Receptor Subtypes ◽  
Retinal Pericytes ◽  
Surface Receptor

Adenosine acts on bovine retinal microvascular pericytes through one or more adenosine receptor subtypes present on the cell surface. Retinal pericytes cultured in medium containing adenosine at concentrations from 10(-6) to 10(-4) M showed significant reduction in proliferation following several days in vitro compared with control cultures. The effects of adenosine were mimicked by polyadenylic acid and inhibited by 8-phenyltheophylline, indicating involvement of a cell surface receptor. Metabolites of adenosine had no effect on pericyte proliferation. An A2 adenosine receptor-specific analogue also inhibited pericyte growth, suggesting that inhibition by adenosine is mediated by A2-receptors and might involve a transient increase in adenosine 3',5'-cyclic monophosphate levels. The results of the present study demonstrate that in addition to demonstrated stimulatory effects on capillary endothelial cells, adenosine also has a direct inhibitory effect on retinal pericytes. We hypothesize a dual function of adenosine within the capillary wall resulting in loss of inhibition of endothelial cells and suggest a role for this nucleoside in pathological neovascularization processes such as proliferative diabetic retinopathy.

scholarly journals Expression levels of functional folate receptors α and β are related to the number of N-glycosylated sites

1997 ◽  
Vol 327 (3) ◽  
pp. 759-764 ◽  
Author(s):  
Feng SHEN ◽  
Huiquan WANG ◽  
Xuan ZHENG ◽  
Manohar RATNAM
Keyword(s):  
Folic Acid ◽  
Cell Surface ◽  
Folate Receptor ◽  
Glycosylation Site ◽  
Cell Surface Receptor ◽  
Wild Type ◽  
Mutant Proteins ◽  
Surface Receptor ◽  
And Function

In a previous study with inhibitors of N-glycosylation, it was proposed that core glycosylation of the folate receptor (FR) is required for the proper folding of the protein [Luhrs (1991) Blood 77, 1171-1180]. The human FR isoforms type α and type β have three and two candidate sites for N-glycosylation respectively, only one of which is conserved. The significance of N-glycosylation at each of these loci in the expression and function of FR was examined by eliminating the sites both individually and in combination by introducing Asn → Gln substitutions. Translation experiments in vitro showed that the mutations did not alter the synthetic rates of the polypeptides. The recombinant proteins were expressed in human 293 fibroblasts. Treatment with N-glycanase and analysis by Western blotting of the wild-type and mutant proteins revealed that all of the candidate sites in both FR-α and FR-β are glycosylated. When all of the N-glycosylation sites were abolished, 2% and 8% of FR-α and FR-β respectively were expressed on the cell surface compared with the corresponding wild-type proteins; the residual FR polypeptides in the cell lysates were unable to bind [3H]folic acid. In both the proteins, the inclusion of each additional N-glycosylation site partly contributed to restoration of cell surface [3H]folic acid binding and receptor-mediated folate transport. Further, in FR-β the introduction of an additional unnatural site of N-glycosylation resulted in the enhancement of the expression of the cell surface receptor compared with the wild-type protein. The results indicate that the total mass of N-glycosylation, not a specific locus of the modification, is critical for the efficient folding and optimal expression of functional FR-α and FR-β.

scholarly journals The Cell Surface Receptor Tartan Is a Potential In Vivo Substrate for the Receptor Tyrosine Phosphatase Ptp52F

2009 ◽  
Vol 29 (12) ◽  
pp. 3390-3400 ◽  
Author(s):  
Lakshmi Bugga ◽  
Anuradha Ratnaparkhi ◽  
Kai Zinn
Keyword(s):  
Cell Surface ◽  
Axon Guidance ◽  
Motor Nerve ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Cell Surface Receptor ◽  
Wild Type ◽  
Surface Receptor

ABSTRACT Receptor-linked protein-tyrosine phosphatases (RPTPs) are essential regulators of axon guidance and synaptogenesis in Drosophila, but the signaling pathways in which they function are poorly defined. We identified the cell surface receptor Tartan (Trn) as a candidate substrate for the neuronal RPTP Ptp52F by using a modified two-hybrid screen with a substrate-trapping mutant of Ptp52F as “bait.” Trn can bind to the Ptp52F substrate-trapping mutant in transfected Drosophila S2 cells if v-Src kinase, which phosphorylates Trn, is also expressed. Coexpression of wild-type Ptp52F causes dephosphorylation of v-Src-phosphorylated Trn. To examine the specificity of the interaction in vitro, we incubated Ptp52F-glutathione S-transferase (GST) fusion proteins with pervanadate-treated S2 cell lysates. Wild-type Ptp52F dephosphorylated Trn, as well as most other bands in the lysate. GST “pulldown” experiments demonstrated that the Ptp52F substrate-trapping mutant binds exclusively to phospho-Trn. Wild-type Ptp52F pulled down dephosphorylated Trn, suggesting that it forms a stable Ptp52F-Trn complex that persists after substrate dephosphorylation. To evaluate whether Trn and Ptp52F are part of the same pathway in vivo, we examined motor axon guidance in mutant embryos. trn and Ptp52F mutations produce identical phenotypes affecting the SNa motor nerve. The genes also display dosage-dependent interactions, suggesting that Ptp52F regulates Trn signaling in SNa motor neurons.

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