INTEGRIN LIGANDS IN SYNAPTIC PLASTICITY

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。A.具体目标具体目标的范围没有改变。 B。研究和结果这项工作的一个关键方法是使用一种结合整合素的合成肽。该肽被设计为含有两个可被市售抗体识别的序列。一个抗原位点是六组氨酸序列（6xHis），第二个是Myc序列。肽已经制成，我们已经将其连接到名为APDP的光敏交联剂上。使用RGD-His-Myc-APDP交联剂（下文称为RGD-连接体），一组实验测试了RGD-连接体是否与海马体结合以及交联活性是否由光诱导。使用不同的光测试三种激活范例。确定最佳激活光源为365 nm，该光源来自放置在离海马切片约6 cm处的灯。我们的第一次免疫印迹分析表明交联反应起作用了。用抗His抗体和抗Myc抗体检测到多条带。这些实验的总体解释是，RGD-接头如预测的那样起作用，靶向整联蛋白，以便将交联活性定位在整联蛋白本身。将进行下一组实验以测试在拉播实验中是否看到相同的条带，然后进行一组实验以测试相同的分子是否在CA 1中交联。更长范围的实验是通过质谱鉴定从CA 1分离的交联分子，然后敲低它们在器官型培养切片中的表达。发展器官型培养的工作已经开始。海马切片已被放置在培养箱中，以测试其可维持的时间。 补编：尚未提交补编。 C.目前的意义是RGD肽将交联剂递送至整联蛋白的潜力。初步结果表明，该方法非常干净和具体。这意味着在组织中几乎没有RGD-接头的非特异性结合，并且可以代表显示RGD肽作为在其细胞表面上表达整联蛋白的细胞的优异靶向分子的数据。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A. Specific Aims The scopes of the Specific Aims have not changed. B. Studies and ResultsA key approach of the work is the use of a synthetic peptide that binds integrins. The peptide was designed to contain two sequences that are recognized by commercially available antibodies. One antigen site is a six-histidine sequence (6xHis) and the second is a Myc sequence. The peptide has been made and we have linked it to a photoactive cross-linker named APDP. Using the RGD-His-Myc-APDP cross-linker (hereafter referred to as RGD-linker) a set of experiments tested whether the RGD-linker binds to hippocampus and whether the cross-linking activity was induced by light. Three activation paradigms were tested using different lights. The best source of activating light was determined to be 365 nm from a lamp that was placed approximately 6 cm from the hippocampal slice. Our first analysis of immunoblots indicate the cross-linking reaction worked. Using anti-His antibody and anti-Myc antibody several bands were detected. The overall interpretation of these experiments is that the RGD-linker functions as predicted, targeting to integrins in order to position the cross-linking activity at the integrin itself. The next set of experiments will be conducted to test whether the same bands are seen in pull-sown experiments, then a set of experiments to test whether the same molecules are cross-linked in CA1. The longer-range experiments are to identify by mass spectrometry isolated cross-linked molecules from CA1 and then knockdown their expression in organotypic cultured slice. Work on the development of organotypic culture has begun. Hippocampal slice has been placed in an incubator to test for the time it can be maintained. Supplements: Supplements have not yet been submitted. C. Significance The current significance is the potential of RGD peptide to deliver cross-linker to integrins. The preliminary results indicate that the approach is exceptionally clean and specific. This means there is little non-specific binding of RGD-linker in the tissue and could represent data showing that RGD peptide serves as an excellent targeting molecule to cells expressing integrins on their cell surface.