RTK signaling regulator SPRY2 associates with endocytic adaptor ITSN1 in vivo

Abstract

Mitogenic signaling from RTK (receptor tyrosine kinase) is tightly linked to endocytosis that regulates the fidelity of signal transduction. Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor implicated in the earliest stages of clathrin-mediated endocytosis and involved in malignant transformation when overexpressed. The aim of this study was to find proteins that could connect ITSN1 to RTK signaling. Methods. In silico prediction of interaction motifs, expression of proteins in bacterial system and mammalian cell culture, immunoprecipitation, GST pull-down analysis, immunofluorescent studies. Results. Using «Scansite» service a binding of ITSN1 SH3A domain to signaling protein SPRY2 was predicted. For experimental verification of this interaction the GST fu- sion SH3 domains of ITSN1 were produced and purified from Escherichia coli. GST pull-down analysis showed that SH3A, N-SH3A (neuron-specific variant), SH3C and SH3E domains of ITSN1 were able to pull-down Flag- SPRY2 from cell lysate. Immunofluorescent analysis together with coimmunoprecipitation indicated that Omni- ITSN1 and Flag-SPRY2 were partially colocalized and formed a complex in vivo. Conclusions. Our data showed that endocytic adaptor ITSN1 and signaling protein SPRY2 are associated in vivo presumably via PRD/SH3 interaction

ITSN protein family: regulation of diversity, role in signalling and pathology

“…We and other laboratories have demonstrated that ITSN1 through its SH3 domains forms complexes with E3-ubiquitin ligases of Cbl family [30][31][32] as well as with Sprouty 2, a negative regulator of Cbl [33,34] (Fig. We and other laboratories have demonstrated that ITSN1 through its SH3 domains forms complexes with E3-ubiquitin ligases of Cbl family [30][31][32] as well as with Sprouty 2, a negative regulator of Cbl [33,34] (Fig.…”

Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells

“…ITSN1 is a multidomain and multifunctional adaptor protein which is involved in clathrin-and caveolin-dependent endocytosis [1,2], Ca 2+ -regulated exocytosis [3] and synaptic vesicles retrieval [4]. It is also implicated in cellular signaling [5][6][7] and neuron survival [8]. Abnormalities of ITSN1 expression are associated with the endocytic anomalies reported in Down syndrome brains and early stages of Alzheimer's disease as well as with neurodegeneration in Huntington's disease [9,10].…”