Plasminogen Activator Inhibitor 1 Contains a Cryptic High Affinity Receptor Binding Site that Is Exposed upon Complex Formation with Tissue-type Plasminogen Activator ,

Abstract

SummaryThe low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic receptor, mediates the cellular internalization of tissue-type (t-PA) and urokinase-type (u-PA) plasminogen activator and their complexes with plasminogen activator inhibitor type 1 (PAI-1). LRP preferentially binds the complexed forms, exemplified by equilibrium dissociation constants (KD) that are at least an order of magnitude lower than those of the free components. To understand the molecular interactions, underlying the preference of the receptor for complexes rather than for the free components, we have performed a detailed analysis of the affinity and kinetics of the binding of PAI-1 and t-PA:PAI-1 complexes to the receptor, using surface plasmon resonance. To assess the involvement of the heparin-binding domain of PAI-1 for the interaction with LRP, we determined the equilibrium dissociation constants for the binding to LRP of a panel of PAI-1 mutants with single- and multiple amino-acid substitutions of the basic residues that constitute the heparin binding site of PAI-1 (K65, K69, R76, K80 and K88). The binding of these PAI-1 mutants was partially reduced with a 2 to 4 fold increase in KD values for single (K80, K88) and combined (K80, 88) substitution mutant proteins respectively. LRP binding of complexes, composed of t-PA with either wild type PAI-1 or any one of the single PAI-1 mutants indicated a major role of lysine 69 (K69) for the binding of t-PA:PAI-1 complexes to LRP (KD values of 6.1, 3.7, 75.4, 5.4, 12.5 and 8.1 nM for wild type, K65A, K69A, R76A, K80A and K88A complexes, respectively). Since the KD for the binding of free t-PA to LRP is 158 nM, we conclude that the PAI-1 moiety harbors the major determinant for t-PA:PAI-1 complex binding to LRP. The in vitro binding studies were extended by binding and clearance studies with COS-1 cells. Degradation of both 125I-t-PA:PAI-1 K69A and 125I-t-PA:PAI-1 K69A K80A K88A complexes after 2 h of incubation was reduced compared to the degradation of 125I-t-PA:PAI-1 complexes. We conclude that PAI-1 contains a cryptic binding site (lysine 69) for LRP, that is specifically expressed upon t-PA:PAI-1 complex formation.

LRP1 controls biosynthetic and endocytic trafficking of neuronal prion protein

“…Overall, however, converging lines of evidence argue that LRP1 is the key endocytic partner of PrP C on these neurons. This conclusion is perhaps not unexpected, given the role of LRP1 in mediating the endocytosis of the GPI-anchored plasminogen activator receptor (Horn et al, 1998). Furthermore, Taylor and Hooper (Taylor and Hooper, 2007) reported that siRNA LRP1 knockdown of LRP1 inhibits the 100 M Cu 2+ -dependent endocytosis of exogenous transfection-expressed PrP C on SH-SY5Y cells.…”

“…Binding through a basic motif to low-density lipoprotein receptorrelated protein 1 (LRP1) is the mechanism by which the GPIanchored urokinase plasminogen activator receptor is internalised by means of coated pits (Horn et al, 1998). This prompted us to investigate whether LRP1 is the endocytic partner for neuronal PrP C (Morris et al, 2006).…”

Differential Regulation of Extracellular Tissue Inhibitor of Metalloproteinases-3 Levels by Cell Membrane-bound and Shed Low Density Lipoprotein Receptor-related Protein 1

“…Interestingly, heparin has been shown to directly inhibit interaction of other ligands, including factor IXa (49), apolipoprotein A-V (50), C4b-binding protein (51) and PAI-1 (52), to LRP-1. In the case of PAI-1, the heparin binding and LRP-1 binding regions have been found to overlap (53,54). Binding of proteins to heparin is commonly mediated by clusters of positively charged residues (55).…”

Binding of Low Density Lipoprotein to Platelet Apolipoprotein E Receptor 2′ Results in Phosphorylation of p38MAPK

“…Regeneration of the sensor chip surface was performed by incubating with 10 mmol/liter taurodeoxycholic acid, 100 mmol/liter Tris (pH 9.0) for 2 min. Data were analyzed as described previously (34).…”