Alternative splicing affecting the SH3A domain controls the binding properties of intersectin 1 in neurons

Institute of Molecular Biology and Genetics, 150 Zabolotnogo Street, Kyiv 03143, Ukraine.
Biochemical and Biophysical Research Communications (Impact Factor: 2.28). 09/2008; 372(4):929-34. DOI: 10.1016/j.bbrc.2008.05.156
Source: PubMed

ABSTRACT Intersectin 1 (ITSN1) is a conserved adaptor protein implicated in endocytosis, regulation of actin cytoskeleton rearrangements and mitogenic signaling. Its expression is characterized by multiple alternative splicing. Here we show neuron-specific expression of ITSN1 isoforms containing exon 20, which encodes five amino acid residues in the first SH3 domain (SH3A). In vitro binding experiments demonstrated that inclusion of exon 20 changes the binding properties of the SH3A domain. Endocytic proteins dynamin 1 and synaptojanin 1 as well as GTPase-activating protein CdGAP bound the neuron-specific variant of the SH3A domain with higher affinity than ubiquitously expressed SH3A. In contrast, SOS1, a guanine nucleotide exchange factor for Ras, and the ubiquitin ligase Cbl mainly interact with the ubiquitously expressed isoform. These results demonstrate that alternative splicing leads to the formation of two pools of ITSN1 with potentially different properties in neurons, affecting ITSN1 function as adaptor protein.

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    ABSTRACT: Adaptor/scaffold proteins of the intersectin (ITSN) family are important components of endocytic and signalling complexes. They coordinate trafficking events with actin cytoskeleton rearrangements and modulate the activity of a variety of signalling pathways. In this review, we present our results as a part of recent findings on the func-tion of ITSNs, the role of alternative splicing in the generation of ITSN1 diversity and the potential relevance of ITSNs for neurodegenerative diseases and cancer. Introduction. Adaptor/scaffold proteins are important components of many cellular processes and signalling systems. Classical scaffolds typically do not posses any enzymatic activity. They function as platforms for the assembly of multiprotein complexes and can help to lo-calize signalling molecules to a specific compartment of the cell or/and regulate the efficiency of a signalling pathway [1, 2]. Scaffold proteins may both facilitate or inhibit sig-nal transduction depending on their concentration in cer-tain compartments, regulating the strength, specificity and duration of signal propagation. Adaptor/scaffold proteins of intersectin (ITSN) fa-mily are characterized by the presence of multiple do-mains that mediate protein-protein interactions. By bin-ding to numerous proteins, ITSNs assemble multimeric complexes implicated in clathrin-and caveolin-media-ted endocytosis, actin cytoskeleton rearrangements, cell signalling and survival (reviewed in [3, 4]). Abnorma-lities of expression of the ITSN1 gene, which is located on chromosome 21, were associated with the endocytic anomalies reported in patients with Down syndrome and Alzheimer's disease [5–7]. In this review, we present our results and summa-rize recent findings of other laboratories concerning the role of ITSN family members in the formation of clath-rin-coated vesicles and regulation of signal transduc-tion and actin cytoskeleton rearrangements. We also describe the impact of alternative processing on the ge-neration of diversity of the ITSN family and regulation of the ITSN genes expression. Role of ITSN in endocytosis and signalling. ITSN family consists of proteins encoded by two genes, ITSN1 and ITSN2, located on human chromosomes 21 and 2, respectively [8, 9]. ITSN1 and ITSN2 are evolutiona-rily conserved proteins and have the same domain orga-nization. The short isoform (ITSN-S) consists of two Eps15 homology domains (EH1 and EH2), a coiled-coil region (CCR) and five Src homology 3 domains (SH3A–E). The long isoform (ITSN-L) contains an ex-tended C-terminus consisting of a Dbl homology (DH), a Pleckstrin homology (PH) and a C2 domain.
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    ABSTRACT: Alternative splicing (AS) generates vast transcriptomic complexity in the vertebrate nervous system. However, the extent to which trans-acting splicing regulators and their target AS regulatory networks contribute to nervous system development is not well understood. To address these questions, we generated mice lacking the vertebrate- and neural-specific Ser/Arg repeat-related protein of 100 kDa (nSR100/SRRM4). Loss of nSR100 impairs development of the central and peripheral nervous systems in part by disrupting neurite outgrowth, cortical layering in the forebrain, and axon guidance in the corpus callosum. Accompanying these developmental defects are widespread changes in AS that primarily result in shifts to nonneural patterns for different classes of splicing events. The main component of the altered AS program comprises 3- to 27-nucleotide (nt) neural microexons, an emerging class of highly conserved AS events associated with the regulation of protein interaction networks in developing neurons and neurological disorders. Remarkably, inclusion of a 6-nt, nSR100-activated microexon in Unc13b transcripts is sufficient to rescue a neuritogenesis defect in nSR100 mutant primary neurons. These results thus reveal critical in vivo neurodevelopmental functions of nSR100 and further link these functions to a conserved program of neuronal microexon splicing.
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    ABSTRACT: Intersectin 1 (ITSN1) is a multifunctional adaptor protein which is involved in endocytosis, exocytosis and cellular signaling and it is also associated with such pathologies as Down syndrome and Alzheimer’s disease. The aim of this study was to identify new ITSN1 protein partners which are implicated in membrane trafficking. Methods. In silico analysis by Scansite online resource had identified a GTPase activating protein oligophrenin 1 (OPHN1) as a potential partner of ITSN1 SH3A domain. GST pull-down and immunoprecipitation were used to prove complex formation between ITSN1 and OPHN1. Subcellular protein localization was determined by immunofluorescence and confocal microscopy. Results. We have shown that brain-specific and ubiquitously expressed SH3A domain isoforms of ITSN1 interact with OPHN1. ITSN1 and OPHN1 form complexes in both resting and stimulated to exocytosis PC12 cell line. Conclusions. GTPase activating protein OPHN1 and adaptor protein ITSN1 interact in PC12 cell line independently of exocytosis stimulation.
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