INTEGRIN LIGANDS IN SYNAPTIC PLASTICITY

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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.