SELF-ASSOCIATION OF N-SYNDECAN (SYNDECAN-3) CORE PROTEIN IS MEDIATED BY A NOVEL STRUCTURAL MOTIF IN THE TRANSMEMBRANE DOMAIN AND ECTODOMAINPLANKING REGION

We expressed domains of the core protein of the transmembrane heparan sulfate proteoglycan N-syndecan (syndecan-3) either individually or as maltose-binding protein fusion proteins. Biochemical characterization of the purified proteins revealed that some of them were capable of s elf-association and formed stable, noncovalent multimeric complexes. T he formation of N-syndecan core protein complexes was also demonstrate d in mammalian cells by in situ cross-linking. Identification of struc tural motifs in the core protein of N-syndecan responsible for the for mation of these complexes was accomplished by analyzing a series of co nstructs comprising different regions of the protein as well as site-d irected mutants. Self-association was assayed by SDS-polyacrylamide ge l electrophoresis, glutaraldehyde cross-linking, and size-exclusion hi gh pressure liquid chromatography. Our results indicated that (i) the transmembrane domain of the N-syndecan core protein was required but n ot sufficient for the formation of stable complexes; (ii) the minimal amino acid sequence that conferred the ability of the N-syndecan core protein to form multimeric complexes included the last four amino acid s (ERKE) of the extracellular domain plus the transmembrane domain; (i ii) point mutations that changed the basic residues in this sequence t o alanine residues either partially or completely abolished the abilit y of the N-syndecan core protein to form complexes; and (iv) replaceme nt of conserved glycine residues in the transmembrane domain with leuc ines abolished complex formation. This property is similar to the olig omerization activity of other transmembrane receptors and suggests tha t regulated self-association may be important for the biological activ ity of transmembrane proteoglycans.