Abstract
BackgroundHeparan sulfate proteoglycans (HSPGs) are one of the basic constituents of plasma membranes. Specific molecular interactions between HSPGs and a number of extracellular ligands have been reported. Mechanisms involved in controlling the localization and abundance of HSPG on specific domains on the cell surface, such as membrane rafts, could play important regulatory roles in signal transduction.Methodology/Principal FindingsUsing metabolic radiolabeling and sucrose-density gradient ultracentrifugation techniques, we identified [35S]sulfate-labeled macromolecules associated with detergent-resistant membranes (DRMs) isolated from a rat parathyroid cell line. DRM fractions showed high specific radioactivity ([35S]sulfate/mg protein), implying the specific recruitment of HSPGs to the membrane rafts. Identity of DRM-associated [35S]sulfate-labeled molecules as HSPGs was confirmed by Western blotting with antibodies that recognize heparan sulfate (HS)-derived epitope. Analyses of core proteins by SDS-PAGE revealed bands with an apparent MW of syndecan-4 (30–33 kDa) and syndecan-1 (70 kDa) suggesting the presence of rafts with various HSPG species. DRM fractions enriched with HSPGs were characterized by high sphingomyelin content and found to only partially overlap with the fractions enriched in ganglioside GM1. HSPGs could be also detected in DRMs even after prior treatment of cells with heparitinase.Conclusions/SignificanceBoth syndecan-1 and syndecan-4 have been found to specifically associate with membrane rafts and their association seemed independent of intact HS chains. Membrane rafts in which HSPGs reside were also enriched with sphingomyelin, suggesting their possible involvement in FGF signaling. Further studies, involving proteomic characterization of membrane domains containing HSPGs might improve our knowledge on the nature of HSPG-ligand interactions and their role in different signaling platforms.
Highlights
Heparan sulfate proteoglycans (HSPGs) are ubiquitous molecules among animal cells and are one of the basic constituents of plasma membranes
We have examined whether HSPGs in PTr cells are localized to membrane rafts
Quantitative analysis showed that 60–70% of [35S]sulfate-labeled material detected in low-density fractions could be removed by the trypsin treatment described in the Materials and Method section, suggesting that the majority of detergent-resistant membranes (DRMs)-associated proteoglycans resided in trypsin-accessible compartment and originated from the cell surface, which is in a good agreement with the detailed report by Takeuchi et al [18]
Summary
Heparan sulfate proteoglycans (HSPGs) are ubiquitous molecules among animal cells and are one of the basic constituents of plasma membranes. HSPGs show specific molecular interactions with a number of heparin-binding growth factors, cytokines, plasma membrane proteins (involved in cell-cell or cell-extracellular matrix interactions), and pathogens (such as viruses and plasmodium) [1,2]. HSPGs intercalated into cell membrane through their core proteins (e.g., syndecan family) or linked by glycosylphosphatidylinositol (GPI)-anchor (e.g., glypican family) bind to a number of extracellular ligands, intercept and regulate biological signals coming into the cells. The localization of HSPGs to specific domains on the cell surface likely plays an important regulatory role. Mechanisms involved in controlling the localization and abundance of HSPG on specific domains on the cell surface, such as membrane rafts, could play important regulatory roles in signal transduction
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