10 research outputs found
Activation of PAR-2 Elicits NO-Dependent and CGRP-Independent Dilation of the Dural Artery
mRNA distribution of CGRP and its receptor components in the trigeminovascular system and other pain related structures in rat brain, and effect of intracerebroventricular administration of CGRP on Fos expression in the TNC
The metabolic impact of β-hydroxybutyrate on neurotransmission: Reduced glycolysis mediates changes in calcium responses and KATP channel receptor sensitivity
mRNA expression of 5-hydroxytryptamine 1B, 1D, and 1F receptors and their role in controlling the release of calcitonin gene-related peptide in the rat trigeminovascular system
Meal-stimulated glucagon release is associated with postprandial blood glucose level and does not interfere with glycemic control in children and adolescents with new-onset type 1 diabetes
Interactions of plasminogen activator inhibitor-1 with vitronectin involve an extensive binding surface and induce mutual conformational rearrangements
In order to explore early events during the association of plasminogen activator inhibitor-1 (PAI-1) with its cofactor vitronectin, we have applied a robust strategy that combines protein engineering, fluorescence spectroscopy, and rapid reaction kinetics. Fluorescence stopped-flow experiments designed to monitor the rapid association of PAI-1 with vitronectin indicate a fast, concentration-dependent, biphasic binding of PAI-1 to native vitronectin but only a monophasic association with the somatomedin B (SMB) domain, suggesting that multiple phases of the binding interaction occur only when full-length vitronectin is present. Nonetheless, in all cases, the initial fast interaction is followed by slower fluorescence changes attributed to a conformational change in PAI-1. Complementary experiments using an engineered, fluorescently silent PAI-1 with non-natural amino acids showed that concomitant structural changes occur as well in native vitronectin. Furthermore, we have measured the effect of vitronectin on the rate of insertion of the reactive center loop into β-sheet A of PAI-1 during reaction with target proteases. With a variety of PAI-1 variants, we observe that both full-length vitronectin and the SMB domain have protease-specific effects on the rate of loop insertion but that the two exhibit clearly different effects. These results support a model for PAI-1 binding to vitronectin in which the interaction surface extends beyond the region of PAI-1 occupied by the SMB domain. In support of this model are recent results that define a PAI-1-binding site on vitronectin that lies outside the somatomedin B domain (Schar, C. R., Blouse, G. E., Minor, K. H., and Peterson, C. B. (2008) J. Biol. Chem. 283, 10297-10309) and the complementary site on PAI-1 (Schar, C. R., Jensen, J. K., Christensen, A., Blouse, G. E., Andreasen, P. A., and Peterson, C. B. (2008) J. Biol. Chem. 283, 28487-28496). © Copyright 2009 by the American Chemical Society