Dimerization of the Insulin-like Growth Factor II/Mannose 6-Phosphate Receptor

Abstract

The insulin-like growth factor II/mannose 6-phosphate receptor (IGF2R) interacts with lysosomal enzymes through two binding domains in its extracytoplasmic domain. We report in the accompanying article (Byrd, J. C., and MacDonald, R. G. (2000) J. Biol. Chem. 275, 18638 -18646) that only one of the two extracytoplasmic mannose 6-phosphate (Man-6-P) binding domains is necessary for high affinity Man-6-P ligand binding, suggesting that, like the cation-dependent Man-6-P receptor, oligomerization of the IGF2R contributes to high affinity interaction with lysosomal enzymes. In the present study, we have directly characterized both naturally occurring and engineered forms of the IGF2R for their ability to form oligomeric structures. Whereas gel filtration chromatography suggested that purified bovine IGF2R species exist in a monomeric form, native gel electrophoresis allowed for the separation of dimeric and monomeric forms of the receptors with distinct phosphomannosyl ligand binding characteristics. The ability of the IGF2R to form oligomeric complexes was confirmed and localized to the extracytoplasmic domain through the use of epitope-tagged soluble IGF2R constructs bearing deletions of the transmembrane and cytoplasmic domains. Finally, chimeric receptors were engineered containing the extracytoplasmic and transmembrane domains of the IGF2R fused to the cytoplasmic domain of the epidermal growth factor receptor with which dimerization of the chimeras could be monitored by measuring autophosphorylation. Collectively, these results show that the IGF2R is capable of forming oligomeric complexes, most likely dimers, in the absence of Man-6-P ligands.

Mechanisms for High Affinity Mannose 6-Phosphate Ligand Binding to the Insulin-like Growth Factor II/Mannose 6-Phosphate Receptor

“…First, only one of the two extracytoplasmic Man-6-P binding domains is required for the formation of high affinity, strongly supporting the hypothesis that receptor oligomerization is responsible for the ability of multivalent ligands to interact with the receptor with high affinity. In addition, our laboratory has recently found that the IGF2R is capable of forming dimers (49). Second, regions of the IGF2R outside of the minimal Man-6-P binding domain play a role in the formation of high affinity sites.…”

Real Time Kinetics of Insulin-like Growth Factor II (IGF-II) Interaction with the IGF-II/Mannose 6-Phosphate Receptor

“…Approximately 90% of membrane-bound IGF-IIR is normally found within the cell. The remaining protein is present at the cell surface, where its extracellular domains can exist as monomers or dimers (7,8). A 23-amino acid transmembrane domain and a cytoplasmic tail containing recognition sequences ensure rapid internalization at the plasma membrane (9).…”

Binding of Urokinase-type Plasminogen Activator Receptor (uPAR) to the Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor

“…Transient expression of the minireceptors by calcium phosphatemediated transfection into 293T human embryonic kidney cells and immunoblot analysis of cell lysates to measure expression of each construct were also performed as described (37,43).…”

“…Radiolabeled pentamannose phosphate-bovine serum albumin (PMP-BSA) and unlabeled PMP-BSA were prepared as described previously (22,37). PMP-Sepharose was prepared as previously described (5).…”

“…Preparation, Expression, and Analysis of Epitope-tagged Soluble Minireceptors-The truncated Man-6-P/IGF2R minireceptors 1-3F, 1-8F, 1-15F, 7-9F, and 7-15F were tagged with the eight-residue FLAG epitope (DYKDDDDK) at the carboxyl terminus and cloned into the pCMV5 vector as previously described (37). The construct 1-15F, formerly called 15F (22), will be identified as 1-15F throughout this paper to clarify that this construct contains all 15 extracytoplasmic repeats but lacks the transmembrane and cytoplasmic domains.…”