scholarly journals The N-terminal thrombin receptor fragment SFLLRN, but not catalytically inactive thrombin-derived agonists, activate U937 human monocytic cells: evidence for receptor hydrolysis in thrombin-dependent signalling

1993 ◽  
Vol 290 (2) ◽  
pp. 571-577 ◽  
Author(s):  
S Joseph ◽  
J MacDermot
Keyword(s):  
Amino Acid ◽  
Receptor Activation ◽  
Cell Surface Receptor ◽  
Single Amino Acid ◽  
Thrombin Receptor ◽  
Similar Response ◽  
U937 Cells ◽  
Monocytic Cells ◽  
Murine Macrophages ◽  
Tethered Ligand

It has previously been reported that murine macrophages can respond chemotactically and mitogenically to the serine proteinase thrombin. There is a similar response in these macrophages to catalytically inactivated thrombin or to peptide fragments of the thrombin B-chain [Bar-Shavit, Kahn, Mann and Wilner (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 976-980]. However, the existence of a non-proteolytic mechanism of thrombin receptor activation in mononuclear cells was not evident in the present study using U937 human monocytic cells. The ability of thrombin to stimulate intracellular Ca2+ mobilization, actin polymerization or cell proliferation was not mimicked by N alpha-tosyl-L-lysine chloromethyl ketone (TLCK)-treated thrombin or by a synthetic 14-amino-acid peptide (single amino acid letter code YPPWNKNFTENDLL) corresponding to a part of the B-chain of thrombin which was reported to be mitogenic in murine macrophages. Evidence was obtained, however, in U937 cells for the presence of proteolytic-dependent thrombin receptor similar to the thrombin receptor expressed in platelets, which following thrombin cleavage exposes a new N-terminal tethered ligand. In support of this, a thrombin-receptor-derived hexapeptide (TRP; sequence SFLLRN), corresponding to a part of the thrombin receptor tethered ligand, mimicked all the actions of thrombin in U937 cells. Further, TRP and thrombin cross-desensitized U937 cells to subsequent stimulation with either TRP or thrombin, suggesting that TRP acted through the same U937 cell surface receptor as did thrombin. Thrombin activation of U937 monocytic cells can therefore be accounted for entirely by a proteolytic mechanism of thrombin receptor activation.

scholarly journals Immunologic analysis of the cloned platelet thrombin receptor activation mechanism: evidence supporting receptor cleavage, release of the N-terminal peptide, and insertion of the tethered ligand into a protected environment

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2125-2136 ◽  
Author(s):  
KJ Norton ◽  
RM Scarborough ◽  
JL Kutok ◽  
MA Escobedo ◽  
L Nannizzi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Cleavage Site ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Thrombin Receptor ◽  
Thrombin Cleavage ◽  
Thrombin Activation ◽  
Tethered Ligand

The recently cloned functional thrombin receptor is thought to be activated by thrombin cleavage of the bond between R41 and S42, followed by the insertion of the new N-terminal region (“tethered ligand”) into an unknown site in the receptor. Antibodies to peptides at or near the cleavage site have been reported to inhibit thrombin- induced platelet activation to varying extents, but the precise mechanism(s) of their inhibition is unknown. We have produced: (1) a polyclonal antibody in rabbits to a peptide containing amino acids 34 to 52 (anti-TR34–52); enzyme-linked immunosorbent assays (ELISA) indicate that anti-TR34–52 contains antibodies to regions on both sides of the thrombin cleavage site; (2) two murine monoclonal antibodies (MoAbs) to a peptide containing amino acids 29 to 68; one antibody reacts primarily with residues N-terminal to the thrombin cleavage site, and the other reacts primarily with residues C-terminal to the cleavage site; and (3) a polyclonal rabbit antibody to a peptide containing amino acids 83 to 94 (anti-TR83–94). Anti-TR34–52 binds to platelets as judged by flow cytometry, and pretreating platelets with a thrombin receptor peptide ligand does not lead to loss of antibody reactivity, suggesting that platelet activation does not initiate redistribution or internalization of surface thrombin receptors. In contrast, pretreating platelets with thrombin leads to complete loss of anti-TR34–52 binding. Similarly, the binding of both MoAbs to platelets is dramatically reduced by pretreatment with thrombin. However, the binding of anti-TR83–94 is not decreased by thrombin activation, confirming that the receptor is not internalized. Anti-TR34–52 profoundly inhibits low dose thrombin-induced platelet shape change and aggregation, but the inhibition can be overcome with higher thrombin doses. However, anti-TR34–52 does not inhibit platelet aggregation induced by tethered ligand peptides. The TR34–52 peptide is a thrombin substrate, with cleavage occurring at the R41-S42 bond as judged by high performance liquid chromatography (HPLC) and platelet aggregation analysis. Anti-TR34–52 prevented cleavage of the TR34–52 peptide, suggesting that the antibody prevents platelet activation, at least in part, by preventing cleavage of the thrombin receptor. These data, although indirect, provide additional support for a thrombin activation mechanism involving thrombin cleavage of the receptor; in addition, they provide new evidence indicating that receptor cleavage is followed by loss of the N-terminal peptide, and insertion of the tethered ligand into a protected domain.

Different Roles of Two Consecutive Leucine Residues in a Receptor-Tethered Ligand Peptide (SFLLRNP) in Thrombin Receptor Activation

1995 ◽  
Vol 68 (9) ◽  
pp. 2695-2698 ◽  
Author(s):  
Takeru Nose ◽  
Yasuyuki Shimohigashi ◽  
Mika Okazaki ◽  
Yusuke Satoh ◽  
Tommaso Costa ◽  
...  
Keyword(s):  
Receptor Activation ◽  
Thrombin Receptor ◽  
Tethered Ligand

scholarly journals Generation of Novel Syncytium-Inducing and Host Range Variants of Ecotropic Moloney Murine Leukemia Virus in Mus spicilegus

2003 ◽  
Vol 77 (9) ◽  
pp. 5065-5072 ◽  
Author(s):  
Yong Tae Jung ◽  
Christine A. Kozak
Keyword(s):  
Amino Acid ◽  
Host Range ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Wild Mouse ◽  
Cell Surface Receptor ◽  
Single Amino Acid ◽  
Mus Spicilegus ◽  
Ecotropic Virus ◽  
Murine Leukemia

ABSTRACT Mus spicilegus is an Eastern European wild mouse species that has previously been reported to harbor an unusual infectious ecotropic murine leukemia virus (MLV) and proviral envelope genes of a novel MLV subgroup. In the present study, M. spicilegus neonates were inoculated with Moloney ecotropic MLV (MoMLV). All 17 inoculated mice produced infectious ecotropic virus after 8 to 14 weeks, and two unusual phenotypes distinguished the isolates from MoMLV. First, most of the M. spicilegus isolates grew to equal titers on M. dunni and SC-1 cells, although MoMLV does not efficiently infect M. dunni cells. The deduced amino acid sequence of a representative clone differed from MoMLV by insertion of two serine residues within the VRA of SUenv. Modification of a molecular clone of MoMLV by the addition of these serines produced a virus that grows to high titer in M. dunni cells, establishing a role for these two serine residues in host range. A second unusual phenotype was found in only one of the M. spicilegus isolates, Spl574. Spl574 produces large syncytia of multinucleated giant cells in M. dunni cells, but its replication is restricted in other mouse cell lines. Sequencing and mutagenesis demonstrated that syncytium formation could be attributed to a single amino acid substitution within VRA, S82F. Thus, viruses with altered growth properties are selected during growth in M. spicilegus. The mutations associated with the host range and syncytium-inducing variants map to a key region of VRA known to govern interactions with the cell surface receptor, suggesting that the associated phenotypes may result from altered interactions with the unusual ecotropic virus mCAT1 receptor carried by M. dunni.

scholarly journals Self-Compatible B Mutants in Coprinus With Altered Pheromone-Receptor Specificities

Genetics ◽  
2000 ◽  
Vol 156 (3) ◽  
pp. 1025-1033
Author(s):  
Natalie S Olesnicky ◽  
Andrew J Brown ◽  
Yoichi Honda ◽  
Susan L Dyos ◽  
Simon J Dowell ◽  
...  
Keyword(s):  
Amino Acid ◽  
Transmembrane Domain ◽  
Gene Mutations ◽  
Receptor Activation ◽  
Amino Acid Replacement ◽  
Single Amino Acid ◽  
Type Locus ◽  
Yeast Assay ◽  
Domain Iii ◽  
G Protein Coupled

Abstract A successful mating in the mushroom Coprinus cinereus brings together a compatible complement of pheromones and G-protein-coupled receptors encoded by multiallelic genes at the B mating-type locus. Rare B gene mutations lead to constitutive activation of B-regulated development without the need for mating. Here we characterize a mutation that arose in the B6 locus and show that it generates a mutant receptor with a single amino acid substitution (R96H) at the intracellular end of transmembrane domain III. Using a heterologous yeast assay and synthetic pheromones we show that the mutation does not make the receptor constitutively active but permits it to respond inappropriately to a normally incompatible pheromone encoded within the same B6 locus. Parallel experiments carried out in Coprinus showed that a F67W substitution in this same pheromone enabled it to activate the normally incompatible wild-type receptor. Together, our experiments show that a single amino acid replacement in either pheromone or receptor can deregulate the specificity of ligand-receptor recognition and confer a self-compatible B phenotype. In addition, we use the yeast assay to demonstrate that different receptors and pheromones found at a single B locus belong to discrete subfamilies within which receptor activation cannot normally occur.

Activation of vascular thrombin receptors mediates cardiac response to alpha-thrombin in isolated, perfused guinea pig heart

1996 ◽  
Vol 270 (5) ◽  
pp. H1585-H1596 ◽  
Author(s):  
B. P. Damiano ◽  
J. A. Mitchell ◽  
W. M. Cheung ◽  
R. Falotico
Keyword(s):  
Guinea Pig ◽  
Ventricular Function ◽  
Perfusion Pressure ◽  
Receptor Activation ◽  
Left Ventricular ◽  
Cardiac Response ◽  
Cell Surface Receptor ◽  
Thrombin Receptor ◽  
Coronary Perfusion ◽  
Thrombin Receptors

alpha-Thrombin alters vascular tone via a cell surface receptor. We used isolated guinea pig hearts perfused with buffer at constant flow to assess the effects of thrombin-receptor activation on coronary perfusion pressure, left ventricular function, and electrocardiogram. alpha-Thrombin produced concentration-dependent (0.03-1 U/ml), transient decreases in perfusion pressure followed by sustained increases. Concurrently, alpha-thrombin markedly reduced ventricular function. SFLLRN, a peptide that directly activates thrombin receptors, had qualitatively similar effects, except that it was less potent (0.1-30 microM). FSLLRN, a structurally similar peptide that does not activate thrombin receptors, had no effect. alpha-Thrombin and SFLLRN also changed S-T segment level and T-wave morphology. Previous alpha-thrombin exposure markedly inhibited the response to a alpha-thrombin but only moderately attenuated the response to SFLLRN. However, previous SFLLRN exposure did not alter subsequent response to alpha-thrombin or SFLLRN. Pretreatment with hirudin (3 U/ml), an inhibitor of thrombin's proteolytic action, prevented alpha-thrombin but not SFLLRN responses. Cromakalim (0.5 microM), a coronary vasodilator, reversed the effects of alpha-thrombin and SFLLRN on ventricular function, suggesting that depression of ventricular function resulted, in part, from vasoconstriction-induced myocardial perfusion deficit. Our results show that alpha-thrombin at physiologically relevant concentrations, has marked effects on coronary vascular resistance and ventricular function in isolated guinea pig hearts that are mediated by the proteolytically activated thrombin receptor.

Interaction Mode of the Phe-Phenyl Group of Thrombin Receptor-Tethered Ligand SFLLRNP in Receptor Activation

1998 ◽  
Vol 124 (2) ◽  
pp. 354-358 ◽  
Author(s):  
T. Nose ◽  
T. Fujita ◽  
M. Nakajima ◽  
Y. Inoue ◽  
T. Costa ◽  
...  
Keyword(s):  
Phenyl Group ◽  
Receptor Activation ◽  
Thrombin Receptor ◽  
Interaction Mode ◽  
Tethered Ligand

scholarly journals Mutations in the B.1.1.7 SARS-CoV-2 spike protein reduce receptor-binding affinity and induce a flexible link to the fusion peptide

2021 ◽  
Author(s):  
Eileen Socher ◽  
Marcus Conrad ◽  
Lukas Heger ◽  
Friedrich Paulsen ◽  
Heinrich Sticht ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Affinity ◽  
Receptor Binding ◽  
Fusion Peptide ◽  
Cell Surface Receptor ◽  
Spike Protein ◽  
Single Amino Acid ◽  
Wild Type Virus ◽  
Wild Type ◽  
Linear Interaction

AbstractThe B.1.1.7 variant of the SARS-CoV-2 virus shows enhanced infectiousness over the wild type virus, leading to increasing patient numbers in affected areas. A number of single amino acid exchanges and deletions within the trimeric viral spike protein characterize this new SARS-CoV-2 variant. Crucial for viral entry into the host cell is the interaction of the spike protein with the cell surface receptor angiotensin-converting enzyme 2 (ACE2) as well as integration of the viral fusion peptide into the host membrane. Respective amino acid exchanges within the SARS-CoV-2 variant B.1.1.7 affect inter-monomeric contact sites within the spike protein (A570D and D614G) as well as the ACE2-receptor interface region (N501Y), which comprises the receptor-binding domain (RBD) of the viral spike protein. However, the molecular consequences of mutations within B.1.1.7 on spike protein dynamics and stability, the fusion peptide, and ACE2 binding are largely unknown. Here, molecular dynamics simulations comparing SARS-CoV-2 wild type with the B.1.1.7 variant revealed inter-trimeric contact rearrangements, altering the structural flexibility within the spike protein trimer. In addition to reduced flexibility in the N-terminal domain of the spike protein, we found increased flexibility in direct spatial proximity of the fusion peptide. This increase in flexibility is due to salt bridge rearrangements induced by the D614G mutation in B.1.1.7 found in pre- and post-cleavage state at the S2’ site. Our results also imply a reduced binding affinity for B.1.1.7 with ACE2, as the N501Y mutation restructures the RBD-ACE2 interface, significantly decreasing the linear interaction energy between the RBD and ACE2.Our results demonstrate how mutations found within B.1.1.7 enlarge the flexibility around the fusion peptide and change the RBD-ACE2 interface, which, in combination, might explain the higher infectivity of B.1.1.7. We anticipate our findings to be starting points for in depth biochemical and cell biological analyses of B.1.1.7, but also other highly contagious SARS-CoV-2 variants, as many of them likewise exhibit a combination of the D614G and N501Y mutation.

Sign in / Sign up

Export Citation Format

Share Document