Endocytic adaptor protein intersectin 1 forms a complex with microtubule stabilizer STOP in neurons

Intersectin 1 (ITSN1) is a multidomain adaptor protein that functions in clathrin-mediated endocytosis and signal transduction. This protein is highly abundant in neurons and is implicated in Down syndrome, Alzheimer's disease and, possibly, other neurodegenerative disorders. Here we used an in vitro binding assay combined with MALDI-TOF mass spectrometry to identify novel binding partners of ITSN1. We found that the neuron-specific isoform of the stable tubule-only polypeptide (STOP) interacts with SH3A domain of ITSN1. STOP and ITSN1 were shown to form a complex in vivo and to partially co-localize in rat primary hippocampal neurons. As STOP is a microtubule-stabilizing protein that is required for several forms of synaptic plasticity in the hippocampus, identification of this interaction raises the possibility of ITSN1 participation in this process.     

Intersectin 1 forms complexes with SGIP1 and Reps1 in clathrin-coated pits

Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor protein involved in clathrin-mediated endocytosis, cellular signaling and cytoskeleton rearrangement. ITSN1 gene is located on human chromosome 21 in Down syndrome critical region. Several studies confirmed role of ITSN1 in Down syndrome phenotype. Here we report the identification of novel interconnections in the interaction network of this endocytic adaptor. We show that the membrane-deforming protein SGIP1 (Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1) and the signaling adaptor Reps1 (RalBP associated Eps15-homology domain protein) interact with ITSN1 in vivo. Both interactions are mediated by the SH3 domains of ITSN1 and proline-rich motifs of protein partners. Moreover complexes comprising SGIP1, Reps1 and ITSN1 have been identified. We also identified new interactions between SGIP1, Reps1 and the BAR (Bin/amphiphysin/Rvs) domain-containing protein amphiphysin 1. Immunofluorescent data have demonstrated colocalization of ITSN1 with the newly identified protein partners in clathrin-coated pits. These findings expand the role of ITSN1 as a scaffolding molecule bringing together components of endocytic complexes.     

Intersectin 1 forms a complex with adaptor protein Ruk/CIN85 in vivo independently of epidermal growth factor stimulation

Intersectin 1 (ITSN1) is an adaptor protein involved in clathrin-mediated endocytosis, apoptosis, signal transduction and cytoskeleton organization. Here, we show that ITSN1 forms a complex with adaptor protein Ruk/CIN85, implicated in downregulation of receptor tyrosine kinases. The interaction is mediated by the SH3A domain of ITSN1 and the third or fourth proline-rich blocks of Ruk/CIN85, and does not depend on epidermal growth factor stimulation, suggesting a constitutive association of ITSN1 with Ruk/CIN85. Moreover, both proteins colocalize in MCF-7 cells with their common binding partner, the ubiquitin ligase c-Cbl. The possible biological role of the interaction between ITSN1 and Ruk/CIN85 is discussed.     

Autoregulation of Fox protein expression to produce dominant negative splicing factors

The Fox proteins are a family of regulators that control the alternative splicing of many exons in neurons, muscle, and other tissues. Each of the three mammalian paralogs, Fox-1 (A2BP1), Fox-2 (RBM9), and Fox-3 (HRNBP3), produces proteins with a single RNA-binding domain (RRM) flanked by N- and C-terminal domains that are highly diversified through the use of alternative promoters and alternative splicing patterns. These genes also express protein isoforms lacking the second half of the RRM (FoxΔRRM), due to the skipping of a highly conserved 93-nt exon. Fox binding elements overlap the splice sites of these exons in Fox-1 and Fox-2, and the Fox proteins themselves inhibit exon inclusion. Unlike other cases of splicing autoregulation by RNA-binding proteins, skipping the RRM exon creates an in-frame deletion in the mRNA to produce a stable protein. These FoxΔRRM isoforms expressed from cDNA exhibit highly reduced binding to RNA in vivo. However, we show that they can act as repressors of Fox-dependent splicing, presumably by competing with full-length Fox isoforms for interaction with other splicing factors. Interestingly, the Drosophila Fox homolog contains a nearly identical exon in its RRM domain that also has flanking Fox-binding sites. Thus, rather than autoregulation of splicing controlling the abundance of the regulator, the Fox proteins use a highly conserved mechanism of splicing autoregulation to control production of a dominant negative isoform.     

The LMP2A protein of Epstein–Barr virus regulates phosphorylation of ITSN1 and Shb adaptors by tyrosine kinases

Latent Membrane Protein 2A (LMP2A) is an Epstein–Barr virus-encoded protein that is important for the maintenance of latent infection. Its activity affects cellular differentiation, migration, proliferation and B cell survival. LMP2A resembles a constitutively activated B cell antigen receptor and exploits host kinases to activate a set of downstream signaling pathways. In the current study we demonstrate the interaction of LMP2A with intersectin 1 (ITSN1), a key endocytic adaptor protein. This interaction occurs via both the N- and C-tails of LMP2A and is mediated by the SH3 domains of ITSN1. Additionally, we identified the Shb adaptor and the Syk kinase as novel binding ligands of ITSN1. The Shb adaptor interacts simultaneously with the phosphorylated tyrosines of LMP2A and the SH3 domains of ITSN1 and mediates indirect interaction of ITSN1 to LMP2A. Syk kinase promotes phosphorylation of both ITSN1 and Shb adaptors in LMP2A-expressing cells. In contrast to ITSN1, Shb phosphorylation depends additionally on Lyn kinase activity.Considering that Shb and ITSN1 are implicated in various receptor tyrosine kinase signaling, our results indicate that LMP2A can affect a number of signaling pathways by regulating the phosphorylation of the ITSN1 and Shb adaptors.     

Nova autoregulation reveals dual functions in neuronal splicing

The Nova family of neuron-specific RNA-binding proteins were originally identified as targets in an autoimmune neurologic disease characterized by failure of motor inhibition. Nova-1 regulates alternative splicing of pre-mRNAs encoding the inhibitory neurotransmitter receptor subunits GABA(A)Rgamma2 and GlyRalpha2 by directly binding intronic elements, resulting in enhancement of exon inclusion. Here we identify exon E4 in the Nova-1 pre-mRNA itself, encoding a phosphorylated protein domain, as an additional target of Nova-dependent splicing regulation in the mouse spinal cord. Nova binding to E4 is necessary and sufficient for Nova-dependent exon exclusion. E4 harbors five repeats of the known Nova-binding tetranucleotide YCAY and mutation of these elements destroys Nova-dependent regulation. Furthermore, swapping of the sites from Nova-1 and GABA(A)Rgamma2 indicates that the ability of Nova to enhance or repress alternative exon inclusion is dependent on the position of the Nova-binding element within the pre-mRNA. These studies demonstrate that in addition to its previously described role as a splicing activator, Nova autoregulates its own expression by acting as a splicing repressor.     

TIA1 prevents skipping of a critical exon associated with spinal muscular atrophy

Prevention of skipping of exon 7 during pre-mRNA splicing of Survival Motor Neuron 2 (SMN2) holds the promise for cure of spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. Here, we report T-cell-restricted intracellular antigen 1 (TIA1) and TIA1-related (TIAR) proteins as intron-associated positive regulators of SMN2 exon 7 splicing. We show that TIA1/TIAR stimulate exon recognition in an entirely novel context in which intronic U-rich motifs are separated from the 5' splice site by overlapping inhibitory elements. TIA1 and TIAR are modular proteins with three N-terminal RNA recognition motifs (RRMs) and a C-terminal glutamine-rich (Q-rich) domain. Our results reveal that any one RRM in combination with a Q domain is necessary and sufficient for TIA1-associated regulation of SMN2 exon 7 splicing in vivo. We also show that increased expression of TIA1 counteracts the inhibitory effect of polypyrimidine tract binding protein, a ubiquitously expressed factor recently implicated in regulation of SMN exon 7 splicing. Our findings expand the scope of TIA1/TIAR in genome-wide regulation of alternative splicing under normal and pathological conditions.     

Autoinhibition of GEF activity in intersectin 1 is mediated by the short SH3-DH domain linker

Intersectin 1L (ITSN1L) acts as a specific guanine nucleotide exchange factor (GEF) for the small guanine nucleotide binding protein Cdc42 via its C‐terminal DH domain. Interestingly, constructs of ITSN1L that comprise additional domains, for instance the five SH3 domains amino‐terminal of the DH domain, were shown to be inhibited in their exchange factor activity. Here, we investigate the inhibitory mechanism of ITSN1L in detail and identify a novel short amino acid motif which mediates autoinhibition. We found this motif to be located in the linker region between the SH3 domains and the DH domain, and we show that within this motif W1221 acts as key residue in establishing the inhibitory interaction. This assigns ITSN1L to a growing class of GEFs that are regulated by a short amino acid motif inhibiting GEF activity by an intramolecular interaction. Moreover, we quantify the interaction between the ITSN1L SH3 domains and the Cdc42 effector N‐WASP using fluorescence anisotropy binding experiments. As the SH3 domains are not involved in autoinhibition, binding of N‐WASP does not release inhibition of nucleotide exchange activity in kinetic experiments, in contrast to earlier observations.     

Intersectin 2, a new multimodular protein involved in clathrin-mediated endocytosis

Intersectin 1 (ITSN1) is a binding partner of dynamin that has been shown to participate in clathrin-mediated endocytosis. Here we report the characterization of a new human gene, ITSN2, highly similar to ITSN1. Alternative splicing of ITSN2 generates a short isoform with two EH domains, a coiled-coil region and five SH3 domains, and a longer isoform containing extra carboxy domains (DH, PH and C2 domains), suggesting that it could act as a guanine nucleotide exchange factor for Rho-like GTPases. ITSN2 expression analysis indicates that it is widely expressed in human tissues. Intersectin 2 isoforms show a subcellular distribution similar to other components of the endocytic machinery and co-localize with Eps15. Moreover, their overexpression, as well as the corresponding ITSN1 protein forms, inhibits transferrin internalization.     

Position-dependent effects of hnRNP A1/A2 in SMN1/2 exon7 splicing

Heterogeneous nuclear ribonucleoprotein A1 and A2 (hnRNP A1/2) is a ubiquitously expressed RNA binding protein known to bind intronic or exonic splicing silencer. Binding of hnRNP A1/2 to survival of motor neuron gene (SMN1/2) exon 7 and flanking sequences strongly inhibits the inclusion of exon 7, which causes spinal muscular atrophy, a common genetic disorder. However, the role of hnRNP A1/2 on the side away from exon 7 is unclear. Here using antisense oligonucleotides, we fished an intronic splicing enhancer (ISE) near the 3′-splice site (SS) of intron 7 of SMN1/2. Mutagenesis identified the efficient motif of the ISE as “UAGUAGG”, coupled with RNA pull down and protein overexpression, we proved that hnRNP A1/2 binding to the ISE promotes the inclusion of SMN1/2 exon 7. Using MS2-tethering array and “UAGGGU” motif walking, we further uncovered that effects of hnRNP A1/2 on SMN1/2 exon 7 splicing are position-dependent: exon 7 inclusion is inhibited when hnRNP A1/2 binds proximal to the 5′SS of intron 7, promoted when its binds proximal to the 3′SS. These data provide new insights into the splicing regulatory mechanism of SMN1/2.     

EHSH1/intersectin, a protein that contains EH and SH3 domains and binds to dynamin and SNAP-25. A protein connection between exocytosis and endocytosis?

In yeast two-hybrid screens for proteins that bind to SNAP-25 and may be involved in exocytosis, we isolated a protein called EHSH1 (for EH domain/SH3 domain-containing protein). Cloning of full-length cDNAs revealed that EHSH1 is composed of an N-terminal region with two EH domains, a central region that is enriched in lysine, leucine, glutamate, arginine, and glutamine (KLERQ domain), and a C-terminal region comprised of five SH3 domains. The third SH3 domain is alternatively spliced. Data bank searches demonstrated that EHSH1 is very similar to Xenopus and human intersectins and to human SH3P17. In addition, we identified expressed sequence tags that encode a second isoform of EHSH1, called EHSH2. EHSH1 is abundantly expressed in brain and at lower levels in all other tissues tested. In binding studies, we found that the central KLERQ domain of EHSH1 binds to recombinant or native brain SNAP-25 and SNAP-23. The C-terminal SH3 domains, by contrast, quantitatively interact with dynamin, a protein involved in endocytosis. Dynamin strongly binds to the alternatively spliced central SH3 domain (SH3C) and the two C-terminal SH3 domains (SH3D and SH3E) but not to the N-terminal SH3 domains (SH3A and SH3B). Immunoprecipitations confirmed that both dynamin and SNAP-25 are complexed to EHSH1 in brain. Our data suggest that EHSH1/intersectin may be a novel adaptor protein that couples endocytic membrane traffic to exocytosis. The ability of multiple SH3 domains in EHSH1 to bind to dynamin suggests that EHSH1 can cluster several dynamin molecules in a manner that is regulated by alternative splicing.     

Adaptor Proteins Intersectin 1 and 2 Bind Similar Proline-Rich Ligands but Are Differentially Recognized by SH2 Domain-Containing Proteins

Background

Scaffolding proteins of the intersectin (ITSN) family, ITSN1 and ITSN2, are crucial for the initiation stage of clathrin-mediated endocytosis. These proteins are closely related but have implications in distinct pathologies. To determine how these proteins could be separated in certain cell pathways we performed a comparative study of ITSNs.

Methodology/Principal Findings

We have shown that endogenous ITSN1 and ITSN2 colocalize and form a complex in cells. A structural comparison of five SH3 domains, which mediated most ITSNs protein-protein interactions, demonstrated a similarity of their ligand-binding sites. We showed that the SH3 domains of ITSN2 bound well-established interactors of ITSN1 as well as newly identified ITSNs protein partners. A search for a novel interacting interface revealed multiple tyrosines that could be phosphorylated in ITSN2. Phosphorylation of ITSN2 isoforms but not ITSN1 short isoform was observed in various cell lines. EGF stimulation of HeLa cells enhanced tyrosine phosphorylation of ITSN2 isoforms and enabled their recognition by the SH2 domains of the Fyn, Fgr and Abl1 kinases, the regulatory subunit of PI3K, the adaptor proteins Grb2 and Crk, and phospholipase C gamma. The SH2 domains mentioned were unable to bind ITSN1 short isoform.

Conclusions/Significance

Our results indicate that during evolution of vertebrates ITSN2 acquired a novel protein-interaction interface that allows its specific recognition by the SH2 domains of signaling proteins. We propose that these data could be important to understand the functional diversity of paralogous ITSN proteins.