scholarly journals Characterization of vascular permeability factor/vascular endothelial growth factor receptors on mononuclear phagocytes

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2767-2773 ◽  
Author(s):  
H Shen ◽  
M Clauss ◽  
J Ryan ◽  
AM Schmidt ◽  
P Tijburg ◽  
...  
Keyword(s):  
Binding Sites ◽  
Mononuclear Phagocytes ◽  
Affinity Binding ◽  
Vascular Endothelial ◽  
Vascular Permeability Factor ◽  
High Affinity Binding ◽  
Lower Affinity ◽  
High Affinity ◽  
Transduction Mechanisms ◽  
Endothelial Growth Factor

Abstract Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a polypeptide mediator, elaborated by certain tumors and other cell types, that exerts multiple effects on endothelium via interaction with a class of high-affinity binding sites. In this report, the interaction of VPF/VEGF with human mononuclear phagocytes (MPs) is characterized. Radioligand binding studies at 4 degrees C showed the presence of a single class of binding sites, kd approximately 300 to 500 pmol/L (approximately 20 times lower affinity than the high-affinity binding site on endothelial cells [ECs]), the occupancy of which correlated with VPF/VEGF-induced MP migration and expression of tissue factor. These binding results were paralleled by functional experiments which indicated that the same VPF/VEGF preparations were about an order of magnitude less effective in stimulating MP chemotaxis than in inducing EC proliferation. When MPs with surface-bound 125I-VPF/VEGF were warmed to 37 degrees C, endocytosis and degradation occurred. Occupancy of VPF/VEGF binding site resulted in subsequent activation of intracellular signal transduction mechanisms, as shown by an increase in MP intracellular calcium concentration. Cross-linking studies with 125I-VPF/VEGF showed a new high-molecular weight band (corresponding to putative 125I- VPF/VEGF-receptor complex), the appearance of which was blocked by excess unlabeled VPF/VEGF. Consistent with these results, immunoprecipitation of 32PO4-labeled MPs exposed to VPF/VEGF showed a single band of similar mobility, not seen in untreated controls. These results demonstrate that the interaction of VPF/VEGF with MPs, though of lower affinity than that observed with ECs, also results from interaction of the polypeptide with a specific cell-surface protein and leads to activation of intracellular transduction mechanisms.

scholarly journals Characterization of vascular permeability factor/vascular endothelial growth factor receptors on mononuclear phagocytes

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2767-2773 ◽  
Author(s):  
H Shen ◽  
M Clauss ◽  
J Ryan ◽  
AM Schmidt ◽  
P Tijburg ◽  
...  
Keyword(s):  
Binding Sites ◽  
Mononuclear Phagocytes ◽  
Affinity Binding ◽  
Vascular Endothelial ◽  
Vascular Permeability Factor ◽  
High Affinity Binding ◽  
Lower Affinity ◽  
High Affinity ◽  
Transduction Mechanisms ◽  
Endothelial Growth Factor

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a polypeptide mediator, elaborated by certain tumors and other cell types, that exerts multiple effects on endothelium via interaction with a class of high-affinity binding sites. In this report, the interaction of VPF/VEGF with human mononuclear phagocytes (MPs) is characterized. Radioligand binding studies at 4 degrees C showed the presence of a single class of binding sites, kd approximately 300 to 500 pmol/L (approximately 20 times lower affinity than the high-affinity binding site on endothelial cells [ECs]), the occupancy of which correlated with VPF/VEGF-induced MP migration and expression of tissue factor. These binding results were paralleled by functional experiments which indicated that the same VPF/VEGF preparations were about an order of magnitude less effective in stimulating MP chemotaxis than in inducing EC proliferation. When MPs with surface-bound 125I-VPF/VEGF were warmed to 37 degrees C, endocytosis and degradation occurred. Occupancy of VPF/VEGF binding site resulted in subsequent activation of intracellular signal transduction mechanisms, as shown by an increase in MP intracellular calcium concentration. Cross-linking studies with 125I-VPF/VEGF showed a new high-molecular weight band (corresponding to putative 125I- VPF/VEGF-receptor complex), the appearance of which was blocked by excess unlabeled VPF/VEGF. Consistent with these results, immunoprecipitation of 32PO4-labeled MPs exposed to VPF/VEGF showed a single band of similar mobility, not seen in untreated controls. These results demonstrate that the interaction of VPF/VEGF with MPs, though of lower affinity than that observed with ECs, also results from interaction of the polypeptide with a specific cell-surface protein and leads to activation of intracellular transduction mechanisms.

Oxytocin receptors in the oviduct during the oestrous cycle of the ewe

1990 ◽  
Vol 124 (3) ◽  
pp. 353-359 ◽  
Author(s):  
V. J. Ayad ◽  
S. A. McGoff ◽  
D. C. Wathes
Keyword(s):  
Binding Sites ◽  
Luteal Phase ◽  
Dissociation Constants ◽  
Relative Concentration ◽  
Oestrous Cycle ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
Lower Affinity ◽  
High Affinity ◽  
Oxytocin Receptors

ABSTRACT The presence of oxytocin receptors in ovine oviduct has been investigated. High-affinity binding sites for [3H]oxytocin were detected in crude membrane fractions prepared from the oviducts of ewes killed during the oestrous period. The dissociation constant calculated for these sites in competition studies was 1·7 nmol/l. Similar dissociation constants were calculated for [Arg8]-vasopressin and the oxytocin-specific agonists [Gly7]-oxytocin and [Thr4, Gly7]-oxytocin, indicating that these sites represent oxytocin receptors. At least one additional site of lower affinity and undetermined identity was present. The relative concentration of oxytocin-binding sites in preparations of oviduct membranes were estimated in ewes killed at different stages of the oestrous cycle using a single concentration of [3H]oxytocin. Binding was low during the luteal phase of the cycle but increased to a maximum at oestrus (77·7 fmol/mg protein). Binding fell after ovulation, reaching what appeared to be basal concentrations by the early luteal stage of the cycle. Binding to oviductal membranes from prepubertal, anoestrous and pregnant ewes was also low, but in anoestrous animals which had been treated with progesterone and oestrogen it was similar to values measured in ewes at oestrus. These results are consistent with the existence of oviductal oxytocin receptors which are regulated by ovarian steroids. We conclude that oxytocin receptors are present in the oviduct of the ewe around the time of ovulation. The significance of oxytocin to events taking place in the oviduct at this time remains to be determined. Journal of Endocrinology (1990) 124, 353–359

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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