Molecular cloning, functional expression and characterization of scinderin, a Ca(2+)-dependent actin-filament severing protein present in secretory tissues.
Creators: name: Marcu, Monica Gianina.
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 1997
Subjects: subject: Biology, Molecular.

description: Chromaffin cells and platelets store amines in dense core vesicles whose contents are released by exocytosis. In chromaffin cells and probably in platelets, the cortical filamentous (F)-actin cytoskeleton represents a negative control for secretion, therefore it has to be locally disassembled (depolymerized) to allow vesicles movement toward the plasma membrane and exocytosis. Search for factors which might be involved in the regulation of F-actin networks resulted in the discovery in our laboratory of scinderin (Sc), a Ca$\sp{2+}$-dependent F-actin severing protein present in tissues with high secretory activity. Sc is mainly cortically localized, it has two Ca$\sp{2+}$-binding sites $\rm (K\sb{d}\ 5.9 \times 10\sp{-7}M,\ B\sb{max}$ 0.8 mol Ca$\sp{2+}$/mol protein; $\rm K\sb{d}\ 2.8 \times 10\sp{-6}M,$ B$\rm\sb{max}$ 1.9 mol Ca$\sp{2+}$/mol protein), and interacts with actin and PIP$\sb2$. Its activity is regulated by Ca$\sp{2+}$, pH and PIP$\sb2$. Here we report the molecular cloning and sequence elucidation of Sc, identification and localization of two PIP$\sb2$ and three actin binding sites. Nucleotide and amino acid sequence analysis indicates that Sc has 6 domains, each containing 3 internal sequence motifs, 63% and 53% homology with gelsolin and villin, respectively, two other F-actin severing proteins. Sc has three actin binding sites, two of each in domains 1 and 2, two PIP$\sb2$ binding sites with the same distribution. These two domains of Sc are of great importance for Ca$\sp{2+}$-dependent F-actin severing activity and thus for regulation of exocytosis. A truncated form of Sc (Sc$\sb{3-6})$ devoid of the putative actin binding sites had no effect on Ca$\sp{2+}$-induced exocytosis but nevertheless, it could bind to actin in a Ca$\sp{2+}$-independent manner. This fact lead to the discovery of another important actin binding site in the NH$\sb2$ terminal of domain 5 of Sc. This actin binding site is involved in nucleating actin polymerization and does not contribute directly to the severing activity of scinderin, though, its presence in the molecule is necessary for proper positioning of the active severing sites of the domains 1 and 2 of scinderin when binding actin. Functional studies involving recombinant Sc indicated that this protein potentiates Ca$\sp{2+}$-induced exocytosis in both, digitonin-permeabilized chromaffin cells and platelets. Recombinant Sc$\sb{1-715}$ potentiated Ca$\sp{2+}$-induced F-actin disassembly and exocytosis in permeabilized chromaffin cells and platelets, an effect blocked in the presence of peptides Sc-ABP$\sb1$ and Sc-ABP$\sb2$ (with sequences corresponding to the first two actin binding sites of Sc), exogenous $\gamma$-actin or the addition of PIP$\sb2$. The inhibitory effect of PIP$\sb2$ was blocked by peptide Sc-PIP$\sb2$BP (with the sequence corresponding to a PIP$\sb2$-binding site present in domain 1 of Sc). The results suggest that Sc-evoked cortical F-actin disassembly is required for secretion and that Sc is an important component of the exocytotic machinery.