Transmembrane Regulation of Ectodomain Shedding

项目摘要 胞外结构域脱落，是一种完整膜蛋白的蛋白水解裂解， 细胞外结构域，影响多种生物学上重要的蛋白质，包括生长 因子前体、细胞因子受体、淀粉样前体蛋白和细胞粘附受体，和 蛋白聚糖因此胞外区脱落功能障碍常导致各种疾病的发生。很长的- 本研究的长期目标是阐明脱落调控的分子和结构基础 穿过细胞膜细胞内蛋白质可以通过直接与细胞膜相互作用来调节脱落。 脱落蛋白底物的胞质结构域。最典型的例子是钙调蛋白 (CaM)与L-选择素结合以抑制L-选择素的脱落。关于调控机制的一个线索是 有充分记录但无法解释的观察结果，即膜近端之间的距离 脱落的切割位点在L-选择素和细胞膜上，而不是切割位点处的序列， 是减少活动的关键缩短距离可消除脱落。近膜区 L-选择素胞质结构域与CaM相互作用，但它只有12个残基长，比CaM短得多。 典型的钙调素结合序列我们最近研究了钙调素与L-选择素衍生的 肽表明，L-选择素中的CaM结合区也可能包括L-选择素的一部分， 跨膜结构域我们假设钙调素与L-选择素的相互作用影响其跨膜功能， 结构域的膜，这反过来又改变构象和/或可及性的脱落 切割位点在膜的另一侧。L-选择素跨膜结构域的一部分可以， 在CaM缔合后，分配成CaM，从而移动整个TM结构域并脱落切割 使脱落分裂位点与细胞质之间的距离缩短， 膜，并有效抑制L-选择素的脱落。在具体目标1中， 钙调素与L-选择素衍生的水溶性肽相互作用的基础将进一步 表征了在特异性目的2中，在膜模拟中CaM与L-选择素片段的相互作用 环境的特征将与NMR和荧光光谱。该片段包含 脱落切割位点，L-选择素的跨膜和胞质结构域。重点将是 检测L-选择素跨膜结构域中的任何构象和/或位置变化， CaM协会。在特异性目的3中，L-选择素片段中脱落切割位点的变化 由CaM联合诱导的，包括脱落切割位点之间距离的任何变化 和膜双层的特征。CaM与L- 选择素肽将有助于阐明CaM调节L-选择素脱落的潜在机制， 并提供了一般的脱落调节机制的见解。

PROJECT SUMMARY Ectodomain shedding, the proteolytic cleavage of an integral membrane protein to release the extracellular domain from the host cell, affects a variety of biologically important proteins including growth factor precursors, cytokine receptors, amyloid precursor proteins and cell adhesion receptors, and proteoglycans. Therefore malfunction of ectodomain shedding often leads to various diseases. The long- term objective of this project is to elucidate the molecular and structural basis for shedding regulation across the cell membrane. Intracellular proteins can regulate shedding by interacting directly with the cytoplasmic domain of the shedding protein substrate. The best characterized example is the calmodulin (CaM) association with L-selectin to inhibit shedding of L-selectin. One clue to the regulation mechanism is the well documented but unexplained observation that the distance between the membrane-proximal shedding cleavage site in L-selectin and the cell membrane, rather than the sequence at the cleavage site, is critical to shedding activity. Shortening the distance abolishes shedding. The membrane-proximal region of the L-selectin cytoplasmic domain interacts with CaM, but it is only 12 residues long, much shorter than a typical CaM-binding sequence. Our recent study on the interaction of CaM with L-selectin-derived peptides suggested that the CaM-binding region in L-selectin may also include a portion of the L-selectin transmembrane domain. We hypothesize that CaM interaction with L-selectin affects its transmembrane domain in the membrane, which in turn changes the conformation and/or accessibility of the shedding cleavage site on the other side of the membrane. A portion of the L-selectin transmembrane domain may, upon CaM association, partition into CaM, thereby moving the entire TM domain and shedding cleavage site toward the cytoplasm and shortening the distance between the shedding cleavage site and the membrane, and effectively inhibit shedding of L-selectin. In Specific Aim 1, the energetic and structural basis for the interaction of CaM with water-soluble peptides derived from L-selectin will be further characterized. In Specific Aim 2, the interaction of CaM with a L-selectin fragment in membrane-mimicking environments will be characterized with NMR and fluorescence spectroscopy. The fragment contains the shedding cleavage site, the transmembrane and cytoplasmic domains of L-selectin. The focus will be to detect any conformational and/or positional changes in the L-selectin transmembrane domain induced by CaM association. In Specific Aim 3, changes at the shedding cleavage site in the L-selectin fragment induced by CaM association, including any changes in the distance between the shedding cleavage site and the membrane bilayer, will be characterized. A careful study of the complexes of CaM with the L- selectin peptides will help to elucidate the mechanism underlying CaM regulation of L-selectin shedding, and provide insights into shedding regulation mechanisms in general.