Muscle Cell Signaling

描述(申请人提供)：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。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的缺陷(31整合素导致其他肌病。层粘连蛋白与DGC或整合素的结合可引起细胞信号通路的改变，影响细胞的生存和增殖，并与肌肉疾病和肌肉萎缩密切相关。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。我们将确定蛋白酪氨酸激酶的特征，这可能是该信号中最早的事件之一，并定位合成营养素磷酸化的位置。这一信号的生理功能尚不清楚，但可能与机械感受或失巢有关。当肌肉收缩或拉伸时，纤维强度保持不变，但当不受刺激时，它会萎缩。层粘连蛋白/DGC/整合素信号可能是对这种机械运动的正常生理反应，维持肌肉。失巢凋亡是指非正常细胞环境中的细胞发生凋亡的过程。我们将通过确定层粘连蛋白/细胞附着和拉伸/收缩对起源于DGC和整合素的细胞信号的影响来检验这两种替代假设，并确定是否影响细胞凋亡或增殖信号。通过使用抑制剂和阻断抗体，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家族的信号通路都与细胞外基质与肌膜的结合有关。当肌细胞与细胞外基质成分结合时，以及当肌细胞被拉伸、悬浮或被允许附着于基质时，这些细胞的激活和位置将被确定。细胞凋亡的程度也将被确定，这些因素的总和将给出这些信号事件如何影响生存能力的清晰图景。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。我们已经表达和纯化了层粘连蛋白A1-LG4-5结构域，并表明它可以导致细胞增殖或死亡，这取决于剂量。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，我们将确定负责增殖和死亡反应的受体以及观察到的细胞死亡的性质。通过截断突变，我们将进一步定位相关的层粘连蛋白-a序列。

DESCRIPTION (provided by applicant): 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 pathways through 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 attach to 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-LG4-5 domain 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.