Wong A, Hwang S M, McDevitt P, McNulty D, Stadel J M, Johanson K
Department of Cellular Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.
Mol Pharmacol. 1996 Sep;50(3):529-37.
The vitronectin receptor (alpha v beta 3) is a member of the integrin superfamily that mediates cell attachment on arginine-glycine-aspartic acid (RGD)-containing adhesive proteins. A solid-phase microtiter assay was developed to investigate the binding properties of purified alpha v beta 3, using tritiated [3H]SK&F-107260 as the radiolabeled ligand. alpha v beta 3, purified from human platelets, human placenta, and chicken osteoclasts, bound [3H]SK&F-107260 saturably and specifically. Saturation binding studies using platelet alpha v beta 3 revealed a single class of high affinity binding sites, exhibiting a Kd of 1.44 nM and Bmax of 0.20 mol of [3H]SK&F-107260/mol of alpha v beta 3. [3H]SK&F-107260 binding was inhibited by a variety of RGD-containing peptides and by the snake venom protein echistatin, whereas an RGE-containing peptide and four nonpeptide fibrinogen receptor (alpha IIb beta 3) antagonists failed to do so. This study shows that alpha v beta 3 exhibits distinct ligand specificity from the structurally homologous fibrinogen receptor, alpha IIb beta 3. The relative potencies of the RGD-containing peptides in inhibiting [3H]SK&F-107260 binding to alpha v beta 3 were the same as their relative potencies in inhibiting biotinylated-fibrinogen binding to the receptor. alpha v beta 3 purified from chicken osteoclasts and human placenta bound [3H]SK&F-107260 with similar affinities and displayed the same pharmacological profile as the platelet vitronectin receptor. The alpha v beta 3 antagonists inhibited the attachment of MG63 human osteosarcoma cells or rat osteoclasts to recombinant rat osteopontin. The rank order of potency of the antagonists in the cell adhesion assays was similar to that of the receptor binding assay, suggesting that the purified alpha v beta 3-[3H]SK&F-107260 binding assay is a valid reflection of the ligand binding to alpha v beta 3 on cell systems.
玻连蛋白受体（αvβ3）是整合素超家族的成员，可介导细胞黏附于含精氨酸 - 甘氨酸 - 天冬氨酸（RGD）的黏附蛋白。我们开发了一种固相微量滴定分析法，以氚标记的[3H]SK&F - 107260作为放射性标记配体，研究纯化的αvβ3的结合特性。从人血小板、人胎盘和鸡破骨细胞中纯化得到的αvβ3可饱和且特异性地结合[3H]SK&F - 107260。使用血小板αvβ3进行的饱和结合研究显示存在一类单一的高亲和力结合位点，其解离常数（Kd）为1.44 nM，最大结合量（Bmax）为每摩尔αvβ3结合0.20摩尔的[3H]SK&F - 107260。多种含RGD的肽以及蛇毒蛋白echistatin可抑制[3H]SK&F - 107260的结合，而含RGE的肽和四种非肽类纤维蛋白原受体（αIIbβ3）拮抗剂则无此作用。本研究表明，αvβ3与结构同源的纤维蛋白原受体αIIbβ3具有不同的配体特异性。含RGD的肽在抑制[3H]SK&F - 107260与αvβ3结合方面的相对效力，与其在抑制生物素化纤维蛋白原与该受体结合方面的相对效力相同。从鸡破骨细胞和人胎盘中纯化得到的αvβ3以相似的亲和力结合[3H]SK&F - 107260，并表现出与血小板玻连蛋白受体相同的药理学特征。αvβ3拮抗剂可抑制MG63人骨肉瘤细胞或大鼠破骨细胞与重组大鼠骨桥蛋白的黏附。拮抗剂在细胞黏附试验中的效力排序与受体结合试验相似，这表明纯化的αvβ3 - [3H]SK&F - 107260结合试验能有效反映配体与细胞系统上αvβ3的结合情况。
