Regulation of dynamin-2 assembly-disassembly and function through the SH3A domain of intersectin-1s

Abstract

Intersectin-1s (ITSN-1s), a five Src homology 3 (SH3) domain-containing protein, is critically required for caveolae and clathrin-mediated endocytosis (CME), due to its interactions with dynamin (dyn). Of the five SH3A-E domains, SH3A is unique because of its high affinity for dyn and potent inhibition of CME. However, the molecular mechanism by which SH3A integrates in the overall function of ITSN-1s to regulate the endocytic process is not understood. Using biochemical and functional approaches as well as high-resolution electron microscopy, we show that SH3A exogenously expressed in human lung endothelial cells caused abnormal endocytic structures, distorted caveolae clusters, frequent staining-dense rings around the caveolar necks and 60% inhibition of caveolae internalization. In vitro studies further revealed that SH3A, similar to full-length ITSN-1s stimulates dyn2 oligomerization and guanosine triphosphatase (GTP)ase activity, effects not detected when other SH3 domains of ITSN-1s were used as controls. Strikingly, in the presence of SH3A, dyn2–dyn2 interactions are stabilized and despite continuous GTP hydrolysis, dyn2 oligomers cannot disassemble. SH3A may hold up caveolae release from the plasma membrane and formation of free-transport vesicles, by prolonging the lifetime of assembled dyn2. Altogether, our results indicate that ITSN-1s, via its SH3A has the unique ability to regulate dyn2 assembly–disassembly and function during endocytosis.

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

“…After the resulting cDNA vectors were verified by DNA sequencing to ensure sequence integrity, they were transformed into E. coli BL-21(DE3) pLysS (Invitrogen). The GST fusion proteins were then purified as described previously (29).…”

Intersectin‐1s: An Important Regulator of Cellular and Molecular Pathways in Lung Injury

“…157,158 As caveolae biogenesis is an emerging field, the findings reported in the KD ITSN mouse, demonstrating that the caveolae density in endothelia is reduced, most probably as a consequence of impaired dynamin-2 recruitment to the endocytic site and deficient caveolar fission, are relevant. 159 Caveolae are associated with a plethora of cel-lular processes (i.e., endocytosis, cell proliferation, differentiation, and apoptosis, as well as cell migration) recently linked to the pathogenesis of idiopathic pulmonary fibrosis, lung cancer, inflammation, and vascular dysfunction. [160][161][162][163][164] Thus, our observations not only enhance the understanding of caveolar function but also link ITSN-1s to caveolae release from endothelial plasma membrane and thus to controlling their number.…”

Emerging Roles for Intersectin (ITSN) in Regulating Signaling and Disease Pathways

“…The expression of SH3A stabilized dynamin-2 oligomers and impaired dynamin-2 function such that caveolae could not detach from the plasma membrane. This in vivo effect is similar to that seen with SH3A overexpression in EC’s in cell culture [39]. Furthermore, acute depletion of ITSN1-S resulted in increased endothelial permeability and impaired interendothelial junctions [40].…”

“…How the SH3A domain interferes with dynamin-2-mediated endocytosis is not completely understood. Knezevic and colleagues overexpressed the SH3A domain in cultured human lung endothelial cells (EC’s) to examine its effects on endocytosis [39]. SH3A overexpression disrupted caveolae internalization in EC’s.…”