Warshawsky I, Bu G, Schwartz A L
Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110.
Biochemistry. 1995 Mar 14;34(10):3404-15. doi: 10.1021/bi00010a032.
A 39-kDa protein copurifies with the low-density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) and inhibits the binding and/or cellular uptake of ligands by this receptor. We recently utilized glutathione S-transferase (GST)-39-kDa fusion protein constructs to demonstrate that constructs encoding amino-terminal residues 1-114 and carboxy-terminal residues 115-319 of the 39-kDa protein independently bind to purified LRP and to LRP on hepatoma cells with similar affinities as the full-length GST-39-kDa protein (Kd approximately 8-10 nM). These regions, however, inhibit ligand binding to LRP differently: GST/1-114 inhibits both tissue-type plasminogen activator (t-PA) and alpha 2-macroglobulin-methylamine (alpha 2M*) binding whereas GST/115-319 only potently inhibits t-PA binding. Four domains, containing residues 18-24 and 100-107 within amino-terminal constructs and residues 200-225 and 311-319 within carboxy-terminal constructs, are required for inhibition of ligand binding. In the present study, we generated additional 39-kDa protein constructs to precisely define residues within each domain required for inhibition of t-PA and alpha 2M* binding to LRP. The potential importance of these residues in mediating direct binding both to purified LRP and to LRP on hepatoma cells was examined. Within amino-terminal residues 1-114, alanine 103 and leucine 104 are required for inhibition of t-PA and alpha 2M* binding. These residues, however, are not required for binding either to purified LRP or to LRP on hepatoma cells. Within domain 18-24, arginine 21 is required for inhibition of t-PA and alpha 2M* binding as well as for the direct binding of amino-terminal constructs to LRP. Within carboxy-terminal domains 200-225 and 311-319, leucine 222 and leucine 319 are both required for inhibition of t-PA binding. Deletion of leucine 319 changes the ligand specificity from inhibition of t-PA binding to inhibition of alpha 2M* binding. Thus, leucine 319 is not required for direct binding to LRP whereas leucine 222 is required for high-affinity binding to LRP.
一种39 kDa的蛋白质与低密度脂蛋白受体相关蛋白/α2-巨球蛋白受体（LRP）共同纯化，并抑制该受体对配体的结合和/或细胞摄取。我们最近利用谷胱甘肽S-转移酶（GST）-39 kDa融合蛋白构建体证明，编码39 kDa蛋白氨基末端残基1-114和羧基末端残基115-319的构建体分别以与全长GST-39 kDa蛋白相似的亲和力（解离常数约为8-10 nM）与纯化的LRP以及肝癌细胞上的LRP结合。然而，这些区域对LRP配体结合的抑制作用不同：GST/1-114抑制组织型纤溶酶原激活剂（t-PA）和α2-巨球蛋白-甲胺（α2M*）的结合，而GST/115-319仅有效抑制t-PA的结合。氨基末端构建体中的18-24位残基和100-107位残基以及羧基末端构建体中的200-225位残基和311-319位残基是抑制配体结合所必需的。在本研究中，我们构建了更多的39 kDa蛋白构建体，以精确确定每个结构域中抑制t-PA和α2M与LRP结合所需的残基。研究了这些残基在介导与纯化的LRP以及肝癌细胞上的LRP直接结合中的潜在重要性。在氨基末端残基1-114中，丙氨酸103和亮氨酸104是抑制t-PA和α2M结合所必需的。然而，这些残基对于与纯化的LRP或肝癌细胞上的LRP结合并非必需。在18-24结构域中，精氨酸21是抑制t-PA和α2M结合以及氨基末端构建体与LRP直接结合所必需的。在羧基末端结构域200-225和311-319中，亮氨酸222和亮氨酸319都是抑制t-PA结合所必需的。缺失亮氨酸319会使配体特异性从抑制t-PA结合转变为抑制α2M结合。因此，亮氨酸319对于与LRP直接结合并非必需，而亮氨酸222是与LRP高亲和力结合所必需的。
