Domain associations and ligand interactions of the mannose 6-phosphate/insulin-like growth factor II receptor

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) is involved in binding and internalization of insulin-like growth factor II (IGF-II) and M6P-bearing ligands as well as lysosomal biogenesis, growth regulation, and tumor suppression. Reports of interaction between the urokinase-type plasminogen activator receptor (uPAR) and the M6P/IGF2R provided a new perspective on the M6P/IGF2R as a tumor suppressor. Our goal was to map the uPAR binding site on the M6P/IGF2R using a panel of minireceptors containing different regions of the M6P/IGF2R extracytoplasmic (EC) domain. We found that soluble forms of uPAR bind the M6P/IGF2R at two sites in this region by a M6P-dependent mechanism, differing from interactions between full-length uPAR and the receptor, in which the binding was M6P-independent, both with low abundance and affinity. This receptor has recently been shown to function as a dimer at the cell surface, but neither the mechanism of inter-receptor interaction nor the dimerization domain has yet been identified. Investigating the domain associations of the M6P/IGF2R would therefore provide insight into the receptor's structural composition as it pertains to functional roles such as dimer formation, ligand binding, and growth suppression. We hypothesized that areas near the receptor's ligand binding domains would contribute preferentially to oligomerization. Two panels of minireceptors were synthesized that involved truncations of either the N- or C-terminal regions of the M6P/IGF2R encompassing deletions of various ligand binding domains. Immunoprecipitation assays showed that all of the minireceptors tested were able to associate with a full-length, epitope-tagged M6P/IGF2, and the presence of mutations in the ligand binding sites of the minireceptors had no effect on their ability to associate. All of the minireceptors were reciprocally able to bind to a ligand binding mutant receptor that is unable to bind M6P-bearing ligands or IGF-II. Thus, association within the heterodimers was not dependent on the presence of functional ligand binding domains. Collectively, these studies reveal that no specific dimerization domain exists, but rather that interactions between dimeric partners occur all along the receptor's EC region and that formation of heterologous receptor complexes leads to interference with normal receptor oligomerization and functional properties such as ligand binding.