Solution Structure of the Dimeric Cytoplasmic Domain of Syndecan-4^,

Abstract

The syndecans, transmembrane proteoglycans which are involved in the organization of cytoskeleton and/or actin microfilaments, have important roles as cell surface receptors during cell-cell and/or cell-matrix interactions. Since previous studies indicate that the function of the syndecan-4 cytoplasmic domain is dependent on its oligomeric status, the conformation of the syndecan-4 cytoplasmic domain itself is important in the understanding of its biological roles. Gel filtration results show that the syndecan-4 cytoplasmic domain (4L) itself forms a dimer stabilized by ionic interactions between peptides at physiological pH. Commensurately, the NMR structures demonstrate that syndecan-4L is a compact intertwined dimer with a symmetric clamp shape in the central variable V region with a root-mean-square deviation between backbone atom coordinates of 0.95 A for residues Leu(186)-Ala(195). The molecular surface of the 4L dimer is highly positively charged. In addition, no intersubunit NOEs in membrane proximal amino acid resides (C1 region) have been observed, demonstrating that the C1 region is mostly unstructured in the syndecan-4L dimer. Interestingly, two parallel strands of 4L form a cavity in the center of the dimeric twist similar to our previously reported 4V structure. The overall topology of the central variable region within the 4L structure is very similar to that of 4V complexed with the phosphatidylinositol 4,5-bisphosphate; however, the intersubunit interaction mode is affected by the presence of C1 and C2 regions. Therefore, we propose that although the 4V region in the full cytoplasmic domain has a tendency for strong peptide--peptide interaction, it may not be enough to overcome the repulsion of the C1 regions of syndecan-4L.

Structural and Cell Adhesion Properties of Zebrafish Syndecan-4 Are Shared with Higher Vertebrates

“…This prompted us to question whether the structure of the zebrafish syndecan-4 cytoplasmic domain, and therefore its function, would be affected by these changes. However, the solution structure of the dimeric cytoplasmic domain of the zebrafish syndecan-4 revealed a twisted clamp topology, which is very similar to that reported for the rat homologue (12,39). Most intermolecular NOE interactions were detected in residues from Ser 188 to Lys 199 , consistent with a symmetric dimer of the twisted clamp shape (Fig.…”

Regulation of Inositol Phospholipid Binding and Signaling through Syndecan-4

“…Syndecan-4 is alone among the family of cell surface heparan sulfate proteoglycans in having a cationic motif in its cytoplasmic V region that can bind inositol phospholipids and PKC␣ (18,19,(21)(22)(23)(24). We have shown previously by NMR spectroscopy that a dimer of V region is stabilized by one molecule of PtdIns(4,5)P 2 , and the cytoplasmic domain forms a unique "twisted clamp" structure that may be relevant to its function (21).…”

“…Size Exclusion Chromatography-Gel filtration procedures were as previously (22). Synthetic peptides or a mixture of synthetic peptide and phosphoinositide were loaded onto a Sephadex G-50 gel filtration column (0.7 ϫ 50 cm) equilibrated with 50 mM HEPES (pH 7.3), 0.1% CHAPS, and 150 mM NaCl.…”

“…Published data suggest that the C2 region of syndecan-4 cytoplasmic domain is flexible and may not participate in interactions with inositides (22). This region, and particularly its terminal FYA motif, does interact with PDZ domain proteins (28).…”

“…The two pairs of lysine residues appear to be critical for this activity, and the motif KKXXXKK is known to bind inositol polyphosphate based on previous studies, for example of synaptotagmin II (20). The functional consequences of inositol phospholipid binding at this site appears to be the stabilization of a dimer of syndecan-4 cytoplasmic domain in an unusual twisted clamp motif, determined by nuclear magnetic resonance (NMR) spectroscopy (21,22). Dimers, or more probably, higher order oligomers (23), then can bind protein kinase C␣ (PKC␣), and cause it to be strongly activated (18,23,24).…”