Horn I R, van den Berg B M, Moestrup S K, Pannekoek H, van Zonneveld A J
Department of Biochemistry, Academic Medical Center, University of Amsterdam, The Netherlands.
Thromb Haemost. 1998 Nov;80(5):822-8.
The low density lipoprotein receptor-related protein (LRP), a multi-functional 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.
低密度脂蛋白受体相关蛋白（LRP）是一种多功能内吞受体，介导组织型纤溶酶原激活剂（t-PA）和尿激酶型纤溶酶原激活剂（u-PA）及其与纤溶酶原激活剂抑制剂1型（PAI-1）复合物的细胞内化。LRP优先结合复合形式，以平衡解离常数（KD）为例，其至少比游离成分低一个数量级。为了解受体对复合物而非游离成分偏好背后的分子相互作用，我们使用表面等离子体共振对PAI-1和t-PA:PAI-1复合物与受体结合的亲和力和动力学进行了详细分析。为评估PAI-1的肝素结合域在与LRP相互作用中的作用，我们测定了一组PAI-1突变体与LRP结合的平衡解离常数，这些突变体对构成PAI-1肝素结合位点的碱性残基（K65、K69、R76、K80和K88）进行了单氨基酸和多氨基酸取代。这些PAI-1突变体的结合部分降低，单个（K80、K88）和组合（K80、88）取代突变体蛋白的KD值分别增加2至4倍。由t-PA与野生型PAI-1或任何一种PAI-1单突变体组成的复合物与LRP的结合表明，赖氨酸69（K69）在t-PA:PAI-1复合物与LRP的结合中起主要作用（野生型、K65A、K69A、R76A、K80A和K88A复合物与LRP结合的KD值分别为6.1、3.7、75.4、5.4、12.5和8.1 nM）。由于游离t-PA与LRP结合的KD为158 nM，我们得出结论，PAI-1部分包含t-PA:PAI-1复合物与LRP结合的主要决定因素。通过与COS-1细胞的结合和清除研究扩展了体外结合研究。与125I-t-PA:PAI-1复合物的降解相比，孵育2小时后125I-t-PA:PAI-1 K69A和125I-t-PA:PAI-1 K69A K80A K88A复合物的降解减少。我们得出结论，PAI-1含有一个针对LRP的隐蔽结合位点（赖氨酸69），该位点在t-PA:PAI-1复合物形成时特异性表达。
