Structural diversity and differential expression of novel human intersectin 1 isoforms

Abstract

Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor protein that functions in clathrin-mediated endocytosis, cell signalling and cytoskeleton rearrangements. The ITSN1 gene encodes two main isoforms: a short form (ITSN1-s), which is ubiquitously expressed and consists of two Eps15 homology (EH) domains and five Src homology 3 (SH3) domains, and a long form (ITSN1-l), which is predominantly expressed in the brain and contains three additional domains, a Dbl homology (DH) domain, a Pleckstrin homology (PH) domain and a C2 domain. Using computational analysis of the EST database and 3' RACE we determined the length of the 3' untranslated region of ITSN1-l and demonstrated that the polyadenylation site is located 11,559 nt downstream of the stop codon of the ITSN1-l mRNA. Recently, additional splicing events affecting ITSN1 transcripts were reported, but full-length transcriptional isoforms with different combinations of alternatively spliced exons remained unknown. Here we report the identification of fifteen novel transcriptional isoforms of the human ITSN1 gene with full-length coding sequences that are the result of different combinations of the alternatively spliced exons 5, 6/6', 20, 23, 25, 26, 26a and 35. The isoforms identified differ in domain organization and expression level in different tissues and more likely contribute to the modulation of many complex protein interactions in which ITSN1 participates.

Modulation of Intersectin-1s Lung Expression Induces Obliterative Remodeling and Severe Plexiform Arteriopathy in the Murine Pulmonary Vascular Bed

“…A potential limitation of our murine model is that pulmonary ECs were transduced only with the NH 2 -terminal protein fragment produced by granzyme B cleavage of ITSN; the potential contribution of the C-terminal fragment to this mouse phenotype was not considered. However, as previously reported, in late-stage PAH, when pulmonary artery ECs display a stable hyperproliferative phenotype, ITSN and EH ITSN expression could be regulated at the mRNA levels by alternative mRNA splicing, a process highly characteristic to ITSN 51 ; it is likely that ITSN's cleavage by granzyme B with generation of the EH ITSN is just the trigger for excessive EC proliferation and formation of vascular lesions and that the expression of alternatively spliced EH ITSN transcript may be responsible for PAH progression and severe arteriopathy.…”

A Novel p38 Mitogen-activated Protein Kinase/Elk-1 Transcription Factor-dependent Molecular Mechanism Underlying Abnormal Endothelial Cell Proliferation in Plexogenic Pulmonary Arterial Hypertension

“…Real time PCR as described (4,28) confirmed the extent of ITSN-1s mRNA down-regulation in EC PAH compared with EC Ctrl (not shown). These results strongly indicate a stable EC phenotype and a possible regulation of ITSN-1s and EH ITSN expression in late stage PAH at mRNA levels and by alternative mRNA splicing, a process highly characteristic to ITSNs (41). Altogether, the findings demonstrate that ITSN-1s is down-regulated in lung EC PAH .…”

Intersectin multidomain adaptor proteins: Regulation of functional diversity

“…In contrast to the widely expressed ITSN2-L isoform, ITSN1-L is expressed predominantly in neurons (Pucharcos et al, 2000(Pucharcos et al, , 2001Guipponi et al, 1998). However, low levels of ITSN1-L expression were detected in adult kidney, liver and placenta (Pucharcos et al, 2001), as well as in fetal liver, lung, kidney and muscle (Tsyba et al, 2004;Kropyvko et al, 2010a). Western blot analysis of neuron lysates and lysates of glial cells from rat brain did not reveal ITSN1-S isoforms in neurons (Yu et al, 2008).…”

“…Many splicing events cause frameshifts in ITSNs mRNA and introduce premature termination codons that lie more than 50 nucleotides upstream of an exon-exon junction (Tsyba et al, 2004;Kropyvko et al, 2010a;Pucharcos et al, 2001;Seifert et al, 2007). Such mRNAs are expected to be degraded via the nonsense-mediated mRNA decay (NMD) pathway (McGlincy and Smith, 2008).…”