INTEGRIN LIGANDS IN SYNAPTIC PLASTICITY

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。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结合整合素的合成肽。该肽被设计成包含两个可被市售抗体识别的序列。一个抗原位点是6组氨酸序列（6xHis），另一个抗原位点是Myc序列。这种肽已经合成了，我们把它和一种叫做APDP的光活性交联剂连接起来。利用rgd - hs - myc - apdp交联剂（以下简称rgd -link），我们进行了一组实验，测试了rgd -link是否与海马结合，以及其交联活性是否受光诱导。用不同的光测试了三种激活范式。最佳的激活光源被确定为365纳米，距离海马体切片约6厘米。我们对免疫印迹的第一次分析表明交联反应起作用了。用抗his抗体和抗myc抗体检测出多个条带。这些实验的总体解释是，rgd连接子的功能与预期一致，以整合素为目标，以便将交联活性定位在整合素本身。下一组实验将测试在拉播种实验中是否看到相同的条带，然后一组实验将测试在CA1中是否有相同的交联分子。更远距离的实验是通过质谱法鉴定分离的CA1交联分子，然后敲低其在器官型培养切片中的表达。器官型培养的开发工作已经开始。海马体切片放置在培养箱中，以测试它能维持多久。补编：尚未提交补编。当前的意义在于RGD肽具有向整合素传递交联剂的潜力。初步结果表明，该方法是非常明确和具体的。这意味着RGD-linker在组织中几乎没有非特异性结合，并且可以代表数据表明RGD肽作为细胞表面表达整合素的细胞的极好靶向分子。

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.