The Conserved Phenylalanine in the Transmembrane Domain Enhances Heteromeric Interactions of Syndecans

Abstract

The transmembrane domain (TMD) of the syndecans, a family of transmembrane heparin sulfate proteoglycans, is involved in forming homo-and heterodimers and oligomers that transmit signaling events. Recently, we reported that the unique phenylalanine in TMD positively regulates intramolecular interactions of syndecan-2. Besides the unique phenylalanine, syndecan-2 contains a conserved phenylalanine (SDC2-Phe-169) that is present in all syndecan TMDs, but its function has not been determined. We therefore investigated the structural role of SDC2-Phe-169 in syndecan TMDs. Replacement of SDC2-Phe-169 by tyrosine (S2F169Y) did not affect SDS-resistant homodimer formation but significantly reduced SDS-resistant heterodimer formation between syndecan-2 and -4, suggesting that SDC2-Phe-169 is involved in the heterodimerization/ oligomerization of syndecans. Similarly, in an in vitro binding assay, a syndecan-2 mutant (S2(F169Y)) showed a significantly reduced interaction with syndecan-4. FRET assays showed that heteromolecular interactions between syndecan-2 and -4 were reduced in HEK293T cells transfected with S2(F169Y) compared with syndecan-2. Moreover, S2(F169Y) reduced downstream reactions mediated by the heterodimerization of syndecan-2 and -4, including Rac activity, cell migration, membrane localization of PKC␣, and focal adhesion formation. The conserved phenylalanine in syndecan-1 and -3 also showed heterodimeric interaction with syndecan-2 and -4. Taken together, these findings suggest that the conserved phenylalanine in the TMD of syndecans is crucial in regulating heteromeric interactions of syndecans.Integral membrane receptors consist of an extracellular domain that binds specific ligands, a transmembrane domain (TMD) 2 that transmits signals in response to ligand binding, and a cytoplasmic domain to which the signals are transmitted by the TMD and that is thereby activated, resulting in a conformational change that causes binding or induction of enzymatic activity inside the cell (1, 2). The TMD is therefore critically important in transmitting signals from the external environment to the inside of the cell, with many recent investigations exploring the mechanism by which TMD interactions regulate cell signaling (2-6). TMDs of single-pass membrane receptors have been shown to cluster, resulting in homotypic and/or heterotypic interactions, with TMDs often forming not only homo-and/or heterodimers but higher-order oligomers in cell membranes (7-10). Most investigations of TMD interactions have analyzed homo-oligomerization in vitro and in vivo, with fewer to date assessing heterotypic TMD associations.One of the most investigated examples of hetero-oligomerization in biology involves the family of EGF receptors, also called the ErbB family (11-13). Although only four ErbB family members have been identified to date, ErbB1 (EGF receptor), ErbB2 (HER2, Neu), ErbB3 (HER3), and ErbB4 (HER4), they have been shown to form 28 homo-and heterodimers (12). These combinations are thought to result in diverse cellul...

Emerging roles of syndecan 2 in epithelial and mesenchymal cancer progression

“…The presence of a unique phenylalanine residue Phe (167) has been shown to bestow to the SDC molecule increased ability to oligomerize and to regulate specific cell functions, including cell migration . In addition, the hetero‐oligomerization of SDC2 and SDC4 is heavily dependent on the conserved phenylalanine Phe (169), as the generated SDC2 (F169Y) mutant exhibits attenuation of downstream activities including cell migration and focal adhesion formation . This may be important in cancer cell behaviour, where formation of hetero‐dimers with SDC4 significantly affects SDC2‐mediated functions, as shown in migration and growth of colon cancer cells .…”

Secondary structure and topology of the transmembrane domain of Syndecan‐2 in detergent micelles

“…1B). Backbone resonances including 15 N, 13 Ca, and 13 C' of residues from syndecan-2 excluding the artificial amino acids at the termini were fully assigned. Most Ha resonances were assigned based on a 3D-HBHACONH experiment.…”

“…5). The conserved residue F159 which was shown to be critical for dimerization [13] favored protein dimerization as its side chain could face to the same direction as the motif (Fig. 5).…”

“…Detergent-resistant TMD dimerization of syndecan-3 was observed [12]. Studies showed that the GXXXG (X is any amino acid) motif and the conserved phenylalanine residue in the TMD (F159 in syndecan-2) are critical for dimerization [13,14]. TMD may have different roles in various syndecans as it is shown that the TMD of syndecan-1 dimerizes weakly, the TMDs of syndecan-3 and syndecan-4 dimerizes strongly, and the TMD of syndecan-2 dimerizes very strongly [14].…”

Syndecans as Cell Surface Receptors in Cancer Biology. A Focus on their Interaction with PDZ Domain Proteins

“…Recent studies have revealed the potential of syndecan-2 and -4 to form hetero-oligomers, reducing each syndecan activity ( Choi et al, 2015 ). This hetero-oligomerization capacity may offer insight into an underlying modulating mechanism ( Kwon et al, 2015 ).…”