Modern complexity of the eukaryotic membrane transport system is considered the result of paralogous gene expansion. In mammals clathrin-mediated endocytosis (CME) is mediated by a machinery of proteins represented mostly by at least two paralogues. 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. So far it remains obscure to what extent the roles of ITSNs are common in tissues where they are coexpressed. Thus, the aim of this work was to characterize ITSN2 and perform a comparative analysis with ITSN1. Given differential expression of ITSN2 long isoform in human embryonic tissues we studied its involvement in early embryonic development using Xenopus laevis as a model. We show that transcripts of ITSN2-L can be detected in Xenopus embryos from the first cleavage onwards. Overexpression of functional domains of ITSN2-L in embryos resulted in aberrant phenotypes. The strongest phenotype was produced by the C-terminal extension of ITSN2-L. Embryos displayed hyperpigmentation and gastrulation failure that were incompatible with survival. The C-terminus of ITSN2-L includes the DH-PH tandem, a nucleotide exchange factor for the small GTPase Cdc42 and the C2 domain. Further investigations revealed that the DH-PH tandem was responsible for the development of the phenotype affecting the actin cytoskeleton in embryos. Observed developmental defects depended on Cdc42. The effect of expression of the constitutively active GTPase strongly resembled that of the DH-PH tandem. The dominant negative Cdc42 partially rescued developmental defects induced by the expression of the DH-PH tandem. Thus, our data indicate that the ITSN2 exchange factor regulates the activity of Cdc42 during embryo development affecting actin cytoskeleton in Xenopus embryos. The next step of this work was functional comparison of intersectins. So far these proteins were investigated independently except for a recent study of Henne et al. However, a comparison of ITSN1 and ITSN2 is of current importance to understand to what extent they could substitute for each other under normal and pathological conditions. The data about ITSN2 and its functional role are limited. It was shown that many paralogous proteins involved in membrane-trafficking processes perform similar functions in distinct cellular compartments. We now provide evidence that ITSN1 and its paralogue ITSN2 are components of common protein complexes and have a predominantly common intracellular distribution in the cell line studied. Previously, ITSN1 was suggested to be constitutively associated with the endocytic adaptor Eps15 in a protein complex. Presumably, ITSN2 is also engaged in this scaffolding complex crucial for progression of clathrin-mediated endocytosis. Regarding the fact that ITSNs have the same domain organization, a similar subcellular distribution and are components of common complexes, we investigated to what extent pools of their binding partners could be different. Most known ITSN1 interactors are ligands of its five SH3 domains. A comparison of the SH3 domain structures of ITSNs showed a high similarity of their ligand-binding sites. Amino acid mismatches within respective SH3 domain pairs were predominantly located beyond the ligand-binding sites. The results from in vitro binding experiments demonstrate the ability of the SH3 domains of ITSN2 to pull down all the protein partners of ITSN1 investigated here. Moreover, novel SH3 ligands, the endocytic adaptors POB1 and Reps1 as well as the signaling proteins Sema6A and SPRY2, were common to both ITSNs. Data from the comparison of ligand-binding sites and in vitro binding experiments suggest that the SH3 domains of ITSNs bind predominantly similar ligands. Certainly, the possibility of specific partners for the certain SH3 domains can not be excluded. There are several possible explanations for this phenomenon. First, selection kept the ligand binding sites of the ITSNs SH3 domains similar whereas amino acid residues beyond the sites varied to a greater extent. Another explanation concerns noncanonical functions of the SH3 domains and the n existence of additional interaction interfaces. Given identified similarities between the ITSNs, we searched for novel binding interfaces that could provide specific protein-protein interactions. Sequence analysis revealed that ITSNs differ in the number of tyrosine residues and their location within the molecule. We intended to search for a putative novel protein-interaction interface that could be distinct between ITSN molecules. A comparison of the primary sequences of the ITSNs demonstrated that ITSN2-S contains 60% more tyrosine residues than ITSN1-S. Accumulation of tyrosines in ITSN2 isoforms was clearly observed in the range of its vertebrate orthologues from fish to primates. All the conserved tyrosine residues of ITSN1-S were located within protein domains whereas additional tyrosine residues in ITSN2-S were found in interdomain regions. Tyrosine phosphorylation of ITSN2 isoforms but not ITSN1-S was detected in various cell lines. This is in good correspondence with phosphoproteomic data about this modification of ITSNs. Acquisition by ITSN2 of additional tyrosine residues and their posttranslational modification lead us to assume that tyrosinebased linear motifs emerged during evolution to regulate ITSN2. Evolution of linear motifs is considered to be the fastest and major mechanism involved in changing protein interaction networks. One could expect that phosphorylation of the linear motif allows recognition of the motif by proteins bearing phosphotyrosine-binding domains. We have demonstrated that the SH2 domains of the kinases Fyn, Fgr and Abl1, the regulatory subunit of PI3K, the adaptor proteins Grb2 and Crk, and phospholipase C gamma could mediate binding to ITSN2. In spite of phosphorylation of ITSN2 isoforms in growing cells, interactions with the SH2 domains were detected only in EGF-stimulated cells. It could be suggested that EGF treatment induces specific phosphorylation of motifs recognized by certain SH2 domains. Our results indicate that during evolution of vertebrates ITSN2 acquired novel protein-interaction interface that allows its specific recognition by the SH2 domains of signaling proteins. We propose that these data could be important for understanding the functional diversity of paralogous ITSN proteins and establishing their roles in diseases. Keywords: intersectin, early embryonic development, protein-protein interactions, SH3 domains, tyrosine phosphorylation.
