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Abstract
Syndecan-4 is a transmembrane heparan sulfate proteoglycan that can regulate cell-matrix interactions and is enriched in focal adhesions. Its cytoplasmic domain contains a central region unlike that of any other vertebrate or invertebrate syndecan core protein with a cationic motif that binds inositol phospholipids. In turn, lipid binding stabilizes the syndecan in oligomeric form, with subsequent binding and activation of protein kinase C. The specificity of phospholipid binding and its potential regulation are investigated here. Highest affinity of the syndecan-4 cytoplasmic domain was seen with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5P)(2)) and phosphatidylinositol 4-phosphate, and both promoted syndecan-4 oligomerization. Affinity was much reduced for 3-phosphorylated inositides while no binding of diacylglycerol was detected. Syndecan-2 cytoplasmic domain had negligible affinity for any lipid examined. Inositol hexakisphosphate, but not inositol tetrakisphosphate, also had high affinity for the syndecan-4 cytoplasmic domain and could compete effectively with PtdIns(4,5)P(2). Since inositol hexaphosphate binding to syndecan-4 does not promote oligomer formation, it is a potential down-regulator of syndecan-4 signaling. Similarly, phosphorylation of serine 183 in syndecan-4 cytoplasmic domain reduced PtdIns(4,5)P(2) binding affinity by over 100-fold, although interaction could still be detected by nuclear magnetic resonance spectroscopy. Only protein kinase Calpha was up-regulated in activity by the combination of syndecan-4 and PtdIns(4,5)P(2), with all other isoforms tested showing minimal response. This is consistent with the codistribution of syndecan-4 with the alpha isoform of protein kinase C in focal adhesions.
Highlights
Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)1 has multiple roles in cell signaling and the regulation of cell adhesion, morphology, and trafficking (1– 4)
The KKXXXKK motif within syndecan-4 V region provides a potential site for InsP6 binding (31), we showed previously that this inositol phosphate neither promotes syndecan-4 cytoplasmic domain oligomerization nor activates protein kinase C (PKC)␣ (18)
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, 21–24)
Summary
Materials—Syndecan-2 and Ϫ4 peptides corresponding to the entire cytoplasmic domains of human syndecan-4 (4L) and syndecan-2 (2L) were synthesized by SynPep (Dublin, CA), and their sequences confirmed by mass spectroscopy. These sequences were (C)RMKKKDEGSYDLGKKPIYKKAPTNEFYA and RMRKKDEGSYDLGERKPSSAAYQKAPTKEFYA, respectively. Peptides were incubated on ice for 30 min with the indicated concentrations of lipid in 10 mM Tris-HCl (pH 7.5) in a reaction volume of 100 l. Peptides were first incubated with differing concentrations of inositol phosphate or sulfate. Lipid affinities (M) for cytoplasmic domains of wild-type syndecan-4 (4L), phosphorylated syndecan-4 (p-4L), syndecan-4 with a truncation of the C-terminal last three amino acids (4⌬E), or wild-type syndecan-2 (2L).
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