Kashiwagi H, Tomiyama Y, Tadokoro S, Honda S, Shiraga M, Mizutani H, Handa M, Kurata Y, Matsuzawa Y, Shattil S J
The Second Department of Internal Medicine, Osaka University Medical School, and Department of Transfusion, Osaka University Hospital, Osaka, Japan.
Blood. 1999 Apr 15;93(8):2559-68.
Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, alphaIIbbeta3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of alphaIIbbeta3. One clone (AM-1) expressed constitutively active alphaIIbbeta3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antialphaIIbbeta3 antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of beta3(T562N) was responsible for receptor activation. In fact, T562N also activated alphaVbeta3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated alphaIIbbeta3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of beta3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to alphaIIbbeta3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125(FAK), indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of beta3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling.
由内向外的信号传导通过改变受体亲和力和/或亲合力来调节整合素的配体结合功能。例如，αIIbβ3在静息血小板中处于低亲和力/亲合力状态，血小板激动剂激活该受体可使纤维蛋白原结合。此外，整合素细胞质尾部的某些突变和截短与高亲和力/亲合力受体相关。为了进一步评估整合素激活的结构基础，对稳定的中国仓鼠卵巢（CHO）细胞转染子进行筛选，以寻找αIIbβ3的高亲和力/亲合力变体。一个克隆（AM-1）表达组成型激活的αIIbβ3，证据如下：（1）可溶性纤维蛋白原和PAC1（一种模拟配体的抗αIIbβ3抗体）的结合；以及（2）纤维蛋白原依赖性细胞聚集。293细胞中的序列分析和突变体表达证明，β3富含半胱氨酸的细胞外部分中的单个氨基酸取代（T562N）是受体激活的原因。事实上，T562N也激活了αVβ3，导致可溶性纤维蛋白原与293细胞自发结合。相比之下，T562A和T562Q均未激活αIIbβ3，这表明在第562位残基处获得天冬酰胺是相关变量。T562N还导致β3的异常糖基化，但这与受体激活无关。可溶性纤维蛋白原与αIIbβ3（T562N）的结合不足以触发pp125（FAK）的酪氨酸磷酸化，这表明在整合素信号传导过程中，激活该蛋白酪氨酸激酶还需要额外的配体结合后事件。这些研究在β3位于配体结合区域和细胞质尾部之间的区域中发现了一种新的功能获得性突变，并且表明该区域参与了由内向外信号传导过程中的整合素结构变化。
