Agonist-Specific, High-Affinity Binding Epitopes Are Contributed by an Arginine in the N-Terminus of the Human Oxytocin Receptor†

Biochemistry ◽  
2002 ◽  
Vol 41 (16) ◽  
pp. 5086-5092 ◽  
Author(s):  
Victoria J. Wesley ◽  
Stuart R. Hawtin ◽  
Helen C. Howard ◽  
Mark Wheatley
Keyword(s):  
Oxytocin Receptor ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
N Terminus

Short polyglutamine peptide forms a high-affinity binding site for thioflavin-T at the N-terminus

2012 ◽  
Vol 10 (30) ◽  
pp. 5787 ◽  
Author(s):  
Shigeru Matsuoka ◽  
Motoki Murai ◽  
Toshio Yamazaki ◽  
Masayuki Inoue
Keyword(s):  
Binding Site ◽  
Thioflavin T ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
N Terminus ◽  
High Affinity Binding Site

Determinants of High-Affinity Binding and Receptor Activation in the N-Terminus of CCL-19 (MIP-3β)

Biochemistry ◽  
2004 ◽  
Vol 43 (12) ◽  
pp. 3670-3678 ◽  
Author(s):  
T. R. Ott ◽  
F. M. Lio ◽  
D. Olshefski ◽  
X.-J. Liu ◽  
R. S. Struthers ◽  
...  
Keyword(s):  
Receptor Activation ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
N Terminus

A Gly/Ala switch contributes to high affinity binding of benzoxazinone-based non-peptide oxytocin receptor antagonists

FEBS Letters ◽  
2004 ◽  
Vol 579 (2) ◽  
pp. 349-356 ◽  
Author(s):  
Stuart R. Hawtin ◽  
Sookhee N. Ha ◽  
Douglas J. Pettibone ◽  
Mark Wheatley
Keyword(s):  
Oxytocin Receptor ◽  
Affinity Binding ◽  
Receptor Antagonists ◽  
High Affinity Binding ◽  
High Affinity

Conformational Activation of Zymogen-Like Thrombin Variants by Tight Binding Ligands

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3070-3070
Author(s):  
Anuja Khan ◽  
Peter Panizzi ◽  
Heather K Kroh ◽  
Paul E. Bock ◽  
Sriram Krishnaswamy
Keyword(s):  
Active Site ◽  
Affinity Binding ◽  
Terminal Sequence ◽  
Binding Studies ◽  
High Affinity Binding ◽  
High Affinity ◽  
Fluorescence Measurements ◽  
N Terminus ◽  
Substrate Cleavage ◽  
Binding Cleft

Abstract A series of well-established structural transitions lead to proteinase formation following zymogen cleavage in the family of serine proteinases homologous to chymotrypsin. These structural changes are triggered by the formation of a salt bridge following the generation of a new N-terminus that inserts in a sequence-specific way into a binding cleft. Based on the established dependence of proteinase maturation on the N-terminal sequence, we prepared a series of recombinant variants of human thrombin (IIa) in which the I16-V17-E18 N-terminal sequence in wild type IIa (IIaWT) was replaced with I-G-E (IIaIGE), T-A-T (IIaTAT) or V-I-E (IIaVIE). In comparison to IIaWT, these variants exhibited greatly diminished ability to hydrolyze peptidyl substrates. Initial velocities measured with H-Dphenylalanyl- L-pipecolyl-L-arginine-p-nitroanilide (S2238) and L-pyroglutamyl-L-prolyl-L-arginyl-p-nitroanilide (S2366) established a graded reduction in activity ranging from ~0.4% (IIaIGE) to < 0.2% (IIaVIE and IIaTAT) that was also dependent on the concentration of the peptidyl substrate relative to Km. These findings are expected for variants that remain zymogen-like because of an impaired ability to drive proteinase formation. This interpretation was confirmed with studies using a fragment of staphylocoagulase (SC1–325). Structural studies have established SC1–325 to function as a conformational activator of the zymogen, prethrombin 2, that is accomplished by high affinity binding and the insertion of its N-terminus into the binding cleft. Saturating concentrations of SC1–325 had a small inhibitory effect on IIaWT but restored the rate of peptidyl substrate cleavage by IIaTAT, IIaVIE and IIaIGE to levels observed with IIaWT. While SC1–325 could bind to prothrombin and prethrombin 2 with high affinity and yield catalytic activity, a deletion mutant of SC lacking 13 residues (SC13–325) produced no detectable activity in the zymogen species. This result is consistent with the essential role played by the N-terminus of SC in conformational activation. SC13–325 was found to bind to the series of IIa variants with inferred equilibrium dissociation constants of ~10−10M. Surprisingly, SC13–325 restored peptidyl substrate cleavage by the zymogen-like variants of IIa to levels seen with IIaWT. It follows that rescue of the zymogen-like variants of IIa does not require N-terminal insertion by SC. These inferences with peptidyl substrates were further pursued with binding studies examining the ability of Nα-dansyl-(p-guanidino)-L-phenylalanine-piperidide (I-2581) to bind to the active site. Fluorescence measurements indicated that I-2581 bound with high affinity to IIaWT (Kd ~5 nM) but in an undetectably weak manner to IIaIGE. However, saturating concentrations of SC13–325 could completely restore high affinity binding of I-2581 to IIaIGE. Thus, rescue of peptidyl substrate cleavage by SC13–325 is matched by a restored ability of the zymogenlike variants to accommodate ligands at the active site. The findings raise the possibility that rescue of these variants by SC arises from ligand-dependent effects that stabilize proteinase structure and overcome the sub-optimal ability of I-G-E, T-A-T or V-I-E to insert properly into the binding cleft. This possibility was further investigated using the soluble extracellular domain of thrombomodulin (sTM) which represents one of the tightest binding natural ligands for IIa. As with SC13–325, high concentrations of sTM were also found to enhance peptidyl substrate cleavage by IIaIGE. Extent of rescue of IIaIGE by sTM was also dependent on the substrate and the concentration of substrate relative to Km. In complex with sTM, IIaIGE exhibited a partial restoration in protein C activation. While we have employed these N-terminal insertion variants as prototypic zymogen-like forms of IIa, rescue of these variants by strong ligands points to a generalized allosteric mechanism by which thrombin function may be regulated. Comparable or related phenomena may underlie the regulated action of thrombin on its range of biological substrates.

scholarly journals The N terminus of interleukin-8 (IL-8) receptor confers high affinity binding to human IL-8

1994 ◽  
Vol 269 (28) ◽  
pp. 18263-18266
Author(s):  
H. Suzuki ◽  
G.N. Prado ◽  
N. Wilkinson ◽  
J. Navarro
Keyword(s):  
Interleukin 8 ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
N Terminus

Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines

1990 ◽  
Vol 63 (02) ◽  
pp. 193-203 ◽  
Author(s):  
John R Shainoff ◽  
Deborah J Stearns ◽  
Patricia M DiBello ◽  
Youko Hishikawa-Itoh
Keyword(s):  
Peritoneal Macrophages ◽  
Affinity Binding ◽  
Macrophage Cell ◽  
High Affinity Binding ◽  
Amino Terminal ◽  
High Affinity ◽  
Terminal Domain ◽  
Weak Binding ◽  
Amino Terminal Domain

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells

1994 ◽  
Vol 72 (03) ◽  
pp. 465-474 ◽  
Author(s):  
Neelesh Bangalore ◽  
William N Drohan ◽  
Carolyn L Orthner
Keyword(s):  
Binding Sites ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Affinity Binding ◽  
Cell Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Gla Domain ◽  
Umbilical Vein Endothelial Cells

SummaryActivated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

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