Hantgan R R, Rocco M, Nagaswami C, Weisel J W
Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.
Protein Sci. 2001 Aug;10(8):1614-26. doi: 10.1110/ps.3001.
The platelet integrin alphaIIbbeta3 is representative of a class of heterodimeric receptors that upon activation bind extracellular macromolecular ligands and form signaling clusters. This study examined how occupancy of alphaIIbbeta3's fibrinogen binding site affected the receptor's solution structure and stability. Eptifibatide, an integrin antagonist developed to treat cardiovascular disease, served as a high-affinity, monovalent model ligand with fibrinogen-like selectivity for alphaIIbbeta3. Eptifibatide binding promptly and reversibly perturbed the conformation of the alphaIIbbeta3 complex. Ligand-specific decreases in its diffusion and sedimentation coefficient were observed at near-stoichiometric eptifibatide concentrations, in contrast to the receptor-perturbing effects of RGD ligands that we previously observed only at a 70-fold molar excess. Eptifibatide promoted alphaIIbbeta3 dimerization 10-fold more effectively than less selective RGD ligands, as determined by sedimentation equilibrium. Eptifibatide-bound integrin receptors displayed an ectodomain separation and enhanced assembly of dimers and larger oligomers linked through their stalk regions, as seen by transmission electron microscopy. Ligation with eptifibatide protected alphaIIbbeta3 from SDS-induced subunit dissociation, an effect on electrophoretic mobility not seen with RGD ligands. Despite its distinct cleft, the open conformer resisted guanidine unfolding as effectively as the ligand-free integrin. Thus, we provide the first demonstration that binding a monovalent ligand to alphaIIbbeta3's extracellular fibrinogen-recognition site stabilizes the receptor's open conformation and enhances self-association through its distant transmembrane and/or cytoplasmic domains. By showing how eptifibatide and RGD peptides, ligands with distinct binding sites, each affects alphaIIbbeta3's conformation, our findings provide new mechanistic insights into ligand-linked integrin activation, clustering and signaling.
血小板整合素αIIbβ3是一类异二聚体受体的代表，这类受体在激活后可结合细胞外大分子配体并形成信号簇。本研究考察了αIIbβ3纤维蛋白原结合位点的占据如何影响受体的溶液结构和稳定性。依替巴肽是一种开发用于治疗心血管疾病的整合素拮抗剂，作为对αIIbβ3具有纤维蛋白原样选择性的高亲和力单价模型配体。依替巴肽的结合迅速且可逆地扰乱了αIIbβ3复合物的构象。在接近化学计量的依替巴肽浓度下，观察到其扩散系数和沉降系数出现配体特异性降低，这与我们之前仅在70倍摩尔过量时观察到的RGD配体对受体的扰动效应形成对比。通过沉降平衡测定，依替巴肽促进αIIbβ3二聚化的效率比选择性较低的RGD配体高10倍。如透射电子显微镜所见，结合依替巴肽的整合素受体显示出胞外结构域分离以及通过其柄区连接的二聚体和更大寡聚体的组装增强。用依替巴肽进行连接可保护αIIbβ3免受SDS诱导的亚基解离，这是RGD配体未观察到的对电泳迁移率的影响。尽管其裂隙不同，但开放构象与无配体的整合素一样有效地抵抗胍变性。因此，我们首次证明，将单价配体结合到αIIbβ3的细胞外纤维蛋白原识别位点可稳定受体的开放构象，并通过其远距离的跨膜和/或细胞质结构域增强自缔合。通过展示具有不同结合位点的配体依替巴肽和RGD肽如何各自影响αIIbβ3的构象，我们的发现为配体连接的整合素激活、聚集和信号传导提供了新的机制见解。
