Structure of uPAR, plasminogen, and sugar-binding sites of the 300 kDa mannose 6-phosphate receptor

Abstract

The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) mediates the intracellular transport of newly synthesized lysosomal enzymes containing mannose 6-phosphate on their N-linked oligosaccharides. In addition to its role in lysosome biogenesis, the CI-MPR interacts with a number of different extracellular ligands at the cell surface, including latent transforming growth factor-b, insulin-like growth factor-II, plasminogen, and urokinase-type plasminogen activator receptor (uPAR), to regulate cell growth and motility. We have solved the crystal structure of the N-terminal 432 residues of the CI-MPR at 1.8 Å resolution, which encompass three out of the 15 repetitive domains of its extracytoplasmic region. The three domains, which exhibit similar topology to each other and to the 46 kDa cation-dependent mannose 6-phosphate receptor, assemble into a compact structure with the uPAR/plasminogen and the carbohydrate-binding sites situated on opposite faces of the molecule. Knowledge of the arrangement of these three domains has allowed us to propose a model of the entire extracytoplasmic region of the CI-MPR that provides a context with which to envision the numerous binding interactions carried out by this multi-faceted receptor.

Identification of a Low Affinity Mannose 6-Phosphate-binding Site in Domain 5 of the Cation-independent Mannose 6-Phosphate Receptor

“…domains 3 and 9) of all CI-MPRs sequenced to date, and substitution of Gln-66, Arg-111, Glu-133, or Tyr-143 of the CD-MPR (31) or their corresponding residues in domains 3 and 9 of the CI-MPR (21) results in a decrease in the affinity of the receptor for a lysosomal enzyme by Ͼ1000-fold. The crystal structures of the CD-MPR (13,27) and domains 1-3 of the CI-MPR (28,29) confirm the importance of these residues by demonstrating that their location is within hydrogen bonding distance of the hydroxyl groups of the mannose ring. To evaluate whether equivalent Gln, Arg, Glu, and Tyr are present in other regions of the CI-MPR, we performed a structure-based sequence align- FIG.…”

“…Our crystal structures of the MPRs (13,(27)(28)(29)44) have provided insight into the mechanism by which these receptors recognize Man-6-P with high affinity. The core structure (Fig.…”

“…Interestingly, this study also revealed that domain 5 exhibits a comparable level of sequence identity to the CD-MPR as observed for domains 3 and 9. Our crystal structures of the CD-MPR (13,27) and domains 1-3 of the CI-MPR (28,29) plus mutagenesis studies (21,30,31) have identified conserved residues that are essential for Man-6-P binding. To evaluate the possibility that domain 5 binds carbohydrate, we performed a structure-based sequence alignment to compare domain 5 with the CD-MPR and domains 3 and 9 of the CI-MPR (see Fig.…”

“…The alignment reveals that domain 5 contains the four key residues (corresponding to Gln-66, Arg-111, Glu-133, and Tyr-143) that are conserved in the CD-MPR and in domains 3 and 9 of all CIMPRs sequenced to date and that have been demonstrated to be essential for high affinity Man-6-P binding. However, this sequence alignment reveals that domain 5 lacks two cysteine residues that, based on the structures of the CD-MPR (13,27) and domains 1-3 of the CI-MPR (28,29), are predicted to form a disulfide bond that is critical for the formation of a Man-6-P-binding pocket. Thus, it is unclear from the alignment analysis whether domain 5 harbors a Man-6-P-binding site.…”

A set of phosphatase-inert “molecular rulers” to probe for bivalent mannose 6-phosphate ligand–receptor interactions

“…16 Using this approach, they estimated the intramolecular distance of closest approach between the domain 3 and domain 9 M6P binding sites to be ~45 Å. In contrast, they estimated the interphosphate distance between the M6P caps of a bisphosphorylated oligosaccharide to be ~30 Å.…”