Neurabin-II/Spinophilin

Abstract

In a preceding paper, we reported a novel actin filament (F-actin)-binding protein, named neurabin, which was specifically expressed in neural tissue and implicated in neurite formation. We purified from rat brain another F-actin-binding protein, which had a domain organization similar to that of neurabin but was ubiquitously expressed, and named it neurabin-II. The original neurabin, renamed neurabin-I, had 1095 amino acids and a calculated M r of 122,729, whereas neurabin-II had 817 amino acids and a calculated M r of 89,642. Both neurabin-I and -II had one F-actin-binding domain at the N-terminal region, one PDZ domain at the middle region, a domain known to interact with transmembrane proteins, and domains predicted to form coiledcoil structures at the C-terminal region. Both neurabin-I and -II bound along the sides of F-actin and showed F-actin-cross-linking activity. The subcellular distribution analysis indicated that neurabin-II was enriched at the postsynaptic density fraction in rat brain and the adherens junction fraction in rat liver. Immunofluorescence microscopic analysis revealed that neurabin-II was highly concentrated at the synapse in primary cultured rat hippocampal neurons and at the cadherinbased cell-cell adhesion sites in Madin-Darby canine kidney cells. Neurabin-II turned out to be the same as a recently reported protein phosphatase 1-binding protein named spinophilin. These results suggest that neurabin-II/spinophilin plays an important role in linking the actin cytoskeleton to the plasma membrane.Specialized membrane domains formed with transmembrane proteins, such as cell adhesion molecules, receptors, and channels, are often associated with the actin cytoskeleton (for reviews, see Refs. 1-4). The linkage between the actin cytoskeleton and the plasma membrane plays crucial roles in various cellular events, such as cell adhesion, cell motility, and cell shape determination, and the proteins linking the actin cytoskeleton to the transmembrane proteins have been identified (1-4). However, the molecular basis of this linkage is not fully understood.In a preceding paper, we purified from rat brain a novel F-actin-binding protein, named neurabin, which was specifically expressed in neural tissue and implicated in neurite formation (5). Neurabin had one F-actin-binding domain, one PDZ domain, and four domains predicted to form coiled-coil structures. The PDZ domain is found in many proteins, some of which are localized at cell-cell junctions (for review, see Ref. 6), such as PSD-95/SAP90 at synaptic junction, Dlg at septate junction, and ZO-1 and ZO-2 at tight junction. The PDZ domain binds to the unique C-terminal motifs of target proteins found in many transmembrane proteins, such as N-methyl-D-aspartate receptors and Shaker-type K ϩ channels (6). Neurabin is likely to serve as a linker between the actin cytoskeleton and a transmembrane protein(s) at synapse, although we have not yet identified its interacting transmembrane protein.During the purification of neurabin, we detected another F-...

Different behavior of l-Afadin and Neurabin-II during the formation and destruction of cell – cell adherens junction

“…This result, together with our previous result (Satoh et al, 1998), indicate that neurabin-II is localized at cadherin-based cell ± cell AJ both in epithelial and non-epithelial cells. In addition to their localization, neurabin-II and Ecadherin show the similar behavior in response to Rac activity, Ca 2+ concentrations and PMA.…”

“…Western blot analysis indicated that the protein level of l-afadin in MDCK cells expressing Rac1DA or Rac1DN was similar to that in wild-type cells (data not shown). We con®rmed that the accumulation of neurabin-II, but not ZO-1, was a ected by Rac activity as described (Takaishi et al, 1997;Satoh et al, 1998) (data not shown). These results suggest that there are at least two groups of proteins in cell ± cell AJ in MDCK cells: one group, including E-cadherin, b-catenin, neurabin-II, and lafadin, is sensitive to Rac activity; and the other group, including ZO-1, is insensitive to Rac activity.…”

“…Rac-regulated accumulation of l-Afadin and Neurabin-II, but not ZO-1, at cell ± cell AJ We have previously shown that E-cadherin, b-catenin, neurabin-II and F-actin densely accumulate at cell ± cell AJ in MDCK cells expressing a dominant active mutant of Rac1 (V12Rac1, Rac1DA), whereas they less densely accumulate in MDCK cells expressing a dominant negative mutant of Rac1 (N17Rac1, Rac1DN), in comparison with those in wild-type MDCK cells (Takaishi et al, 1997;Satoh et al, 1998). We next examined whether the accumulation of l-afadin and ZO-1 were a ected by Rac activity.…”

“…Both neurabin-I and -II have a similar domain structure, one F-actin-binding domain at the N-terminal region, one PDZ domain at the middle region, a domain known to interact with integral membrane proteins and domains predicted to form coiled-coil structures at the C-terminal region. Both the isoforms bind along the sides of F-actin and show Factin-crosslinking activity Satoh et al, 1998). Immuno¯uorescence microscopic analysis indicates that neurabin-I and -II are highly concentrated at the synapse in neuron and that neurabin-II is concentrated at cadherin-based cell ± cell AJ in MDCK cells.…”

“…We have recently isolated two novel F-actin-binding proteins which are localized at cell ± cell AJ, neurabin Satoh et al, 1998) and afadin . Neurabin has two isoforms: neurabin-I and -II.…”