title: Syndecans reside in sphingomyelin-enriched low-density fractions of the plasma membrane isolated from a parathyroid cell line.
abstract: BACKGROUND
  Heparan 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.
  
  
  RESULTS
  Using metabolic radiolabeling and sucrose-density gradient ultracentrifugation techniques, we identified [(35)S]sulfate-labeled macromolecules associated with detergent-resistant membranes (DRMs) isolated from a rat parathyroid cell line. DRM fractions showed high specific radioactivity ([(35)S]sulfate/mg protein), implying the specific recruitment of HSPGs to the membrane rafts. Identity of DRM-associated [(35)S]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
  Both 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.
authors.affiliation: Section of Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan. kapobch@tmd.ac.jp
authors.forename: Katarzyna A
authors.initials: KA
authors.lastname: Podyma-Inoue
authors.forename: Miki
authors.initials: M
authors.lastname: Hara-Yokoyama
authors.forename: Tamayuki
authors.initials: T
authors.lastname: Shinomura
authors.forename: Tomoko
authors.initials: T
authors.lastname: Kimura
authors.forename: Masaki
authors.initials: M
authors.lastname: Yanagishita
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry; D019812:Heparan Sulfate Proteoglycans / Q000737:chemistry; D008024:Ligands; D021962:Membrane Microdomains / Q000378:metabolism; D008954:Models, Biological; D010280:Parathyroid Glands / Q000166:cytology; D011133:Polysaccharide-Lyases / Q000737:chemistry; D017434:Protein Structure, Tertiary; D051381:Rats; D015398:Signal Transduction; D013109:Sphingomyelins / Q000378:metabolism*; D053668:Syndecan-1 / Q000378:metabolism; D053671:Syndecan-4 / Q000378:metabolism; D053667:Syndecans / Q000502:physiology*; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D003902:Detergents; D000939:Epitopes; D019812:Heparan Sulfate Proteoglycans; D008024:Ligands; D013109:Sphingomyelins; D053668:Syndecan-1; D053671:Syndecan-4; D053667:Syndecans; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D011133:Polysaccharide-Lyases; C021349:heparitinsulfate lyase