Muscle Cell Signaling

许多肌营养不良症是由肌营养不良蛋白糖蛋白复合体(DGC)缺陷引起的。这个 DGC和整合素结合细胞外基质层粘连蛋白-2(O2BlYl)和层粘连蛋白-2或A7(31整合素)缺陷 导致其他的肌病。层粘连蛋白与DGC或整合素的结合导致细胞内 信号通路，可以影响细胞的生存和增殖，并与肌病和 肌肉萎缩。产生于层粘连蛋白/DGC/整合素的细胞信号已被认为是相当复杂的。我们会 确定蛋白酪氨酸激酶的特征，它可能是该信号转导中最早的事件之一，并定位 合成人营养素的磷酸化部位。这一信号的生理功能尚不清楚，但可能 机械接受的与机械接受或失眠相关的当肌肉收缩或拉伸时，纤维强度保持不变- ED，但在没有刺激的情况下，它会萎缩。层粘连蛋白/DGC/整合素信号可能是一种正常的生理信号 对这种机械运动做出反应，保持肌肉。失巢是细胞不丢失的过程-- 在正常的细胞环境中发生细胞凋亡。我们将通过以下方式测试这两个替代假设 确定层粘连蛋白/细胞附着和拉伸/收缩对起源于 DGC和整合素，并确定凋亡或增殖信号是否受到影响。使用抑制剂和 通过阻断抗体，我们将确定DGC或A7(31)整合素是否参与了受影响的信号转导。 其他通过p38、ERK1/2、AKT、JNK、FAK、Gs和c-src家族的信号通路都有 与细胞外基质与肌膜的结合有关。每一项的激活和位置 这些将被确定为肌细胞结合细胞外基质成分以及当肌细胞拉伸时， 悬浮保存，或允许附着在母体上。还将确定细胞凋亡的程度，并 总而言之，这些信号事件将清楚地说明生存能力如何受到这些信号事件的影响。 层粘连蛋白α亚单位(LG1-5)的五个球状结构域为整合素和DGC提供了结合位点。 我们已经表达并纯化了层粘连蛋白A1-LG4-5结构域，并表明，依赖于剂量，它 会导致细胞增殖或死亡。另一位合作者提供了Q2-LG4-5结构域蛋白。通过 比较这两种蛋白质对细胞活力和细胞信号的影响，我们将确定受体 负责增殖和死亡反应以及观察到的细胞死亡的性质。通过截断 突变，我们将进一步定位负责的层粘连蛋白-a序列。

Many muscular dystrophies are caused by defects in the dystrophin glycoprotein complex (DGC). The DGC and integrins bind extracellular matrix laminin-2 (o2BlYl) and defects in laminin-2 or a7(31 integrin cause other myopathies. Laminin-binding to either the DGC or integrins causes changes in the cell's signaling pathways, can effect cell survival and proliferation, and is germane to the myopathies and to muscle atrophy. Cell signaling arising at laminin/DGC/integrin is already known to be quite complex. We will characterize the protein tyrosine kinase which may be one of the earliest events in this signaling and localize the site of syntrophin phosphorylation. The physiological function of this signaling is not known but may be related to mechanoreception or anoikis. When muscle is contracted or stretched, fiber strength is maintain- ed, but when unstimulated it atrophies. The laminin/DGC/integrin signaling may be a normal physiological response to this mechanical motion, maintaining the muscle. Anoikis is the process by which cells not lo- cated in a normal cellular environment undergo apoptosis. We will test these two alternative hypotheses by determining the effect of laminin/cell attachment and stretching/contraction on the cell signaling originating at the DGC and integrins and determine if apoptosis or proliferative signaling is affected. Using inhibitors and blocking antibodies, we will determine whether the DGC or a7(31 integrin is involved in the affected signaling. Other signaling pathwaysthrough p38, ERK1/2, AKT, JNK, FAK, Gs, and c-src family kinases have all been linked to the binding of extracellular matrix to the sarcolemma. The activation and location of each of these will be determined as myocytes bind extracellular matrix components and as the myocytes are stretch, held in suspension, or allowed to attachto matrix. The extent of apoptosis will also be determined and the sum of these will give a clear picture of how viability is affected by these signaling events. The five globular domains of laminin's a-subunit (LG1-5) provide binding sites for integrins and the DGC. We have expressed and purified the laminin a1-LG4-5 domain and have shown that, depending on dose, it can cause cell proliferation or death. Another collaborator has provided the Q2-LG4-5domain protein. By comparing the effects of the two proteins on cell viability and cell signaling, we will determine the receptor responsible for proliferative and death responses and the nature of the cell death observed. By truncation mutation, we will further localize the laminin-a sequences responsible.