Dergai M. Identification and chracterization of isoforms of adaptor protein intersectin 1

Object of the study is regulation of functional diversity of adaptor proteins' isoforms. Subject of the study - transcriptional and protein isoforms of adaptor protein ITSN1. Methods of the study - cloning of cDNA fragments, affinity chromatography, RT-PCR on isolated cells, transient transfection of eukariotyc cells, Western-mlot and Southern-blot analyses, immunoprecipitation, precipitation of protein complexes by GST-fused proteins, site-directed mutagenesis, modeling of protein ternary structure, confocal microscopy. Results and novelty - in the study group of transcriptional isoforms containing novel alternative exon 22a was identified. Structure and expression of these transcripts were studied. Protein product of ITSN1-22a was shown. This isoform colocalizes with major isoform ITSN1-s and forms with it protein complex. Isoform ITSN1-22a has specific intra- and intermolecular interactions. In the study transcripts with exon 20 are characterized. Inclusion of this exon is neuron-specific. The transcripts with exon 20 are typical of mammals only. Insertion encoded by exon 20 changes specificity of SH3A domain binding. It was shown that these changes were caused by rearrangements in third pocket in the interaction interface of the domain. Similar mechanism was shown for neuron-specific form of Src1 kinase.