LOCALIZATION OF VITRONECTIN BINDING DOMAIN IN PLASMINOGEN-ACTIVATOR INHIBITOR-1

Citation
Da. Lawrence et al., LOCALIZATION OF VITRONECTIN BINDING DOMAIN IN PLASMINOGEN-ACTIVATOR INHIBITOR-1, The Journal of biological chemistry, 269(21), 1994, pp. 15223-15228
Citations number
41
Categorie Soggetti
Biology
ISSN journal
00219258
Volume
269
Issue
21
Year of publication
1994
Pages
15223 - 15228
Database
ISI
SICI code
0021-9258(1994)269:21<15223:LOVBDI>2.0.ZU;2-U
Abstract
Plasminogen activator inhibitor type 1 (PAI-1) is the rapid physiologi c inhibitor of tissue-type plasminogen activator and urokinase-type pl asminogen activator (uPA). In plasma and the extracellular matrix, PAI -1 is associated with the adhesive glycoprotein vitronectin. In order to characterize the PAI-1 structural domain responsible for binding to vitronectin, the segment of the PAI-1 cDNA encoding amino acids 13-14 7 (nucleotides 248-650) was randomly mutagenized and subcloned into a bacterial expression vector containing the mature PAI-1 coding sequenc e. Recombinant PAI-1 mutants were expressed in Escherichia coli and ba cterial lysates assayed in duplicate for uPA inhibitory activity and v itronectin binding. Of 190 clones screened, six consistently demonstra ted decreased vitronectin binding relative to uPA inhibitory activity. DNA sequence analysis of four of these clones identified 10 unique mi ssense mutations, all located between base pairs 298 and 641, with eac h clone containing between one and four substitutions. Each substituti on was expressed independently by site-directed mutagenesis and again analyzed for uPA inhibitory activity and vitronectin binding. Five poi nt mutations that selectively disrupt vitronectin binding were identif ied. All 5 residues are located on the exterior of the PAI-1 structure . These findings appear to define a complex binding surface that bridg es alpha-helices C and E to beta-strand 1A and includes amino acids 55 , 109, 110, 116, and 123. These results suggest that vitronectin bindi ng may stabilize the active conformation of PAI-1 by restricting the m ovement of beta-sheet A and thereby preventing insertion of the reacti ve center loop.