Two distinct mannose 6-phosphate (Man-6-P) receptors (MPRs), the cation-dependent MPR (CD-MPR) and the insulin-like growth factor II/MPR (IGF-II/MPR), recognize a diverse population of Man-6-P-containing ligands. The IGF-II/MPR is a type I transmembrane glycoprotein with a large extracytoplasmic region composed of 15 repeating domains that display sequence identity to each other and to the single extracytoplasmic domain of the CD-MPR. A structure-based sequence alignment of the two distinct Man-6-P-binding sites of the IGF-II/MPR with the CD-MPR implicates several residues of IGF-II/ MPR domains 3 and 9 as essential for Man-6-P binding. To test this hypothesis single amino acid substitutions were made in constructs encoding either the N-or the C-terminal Man-6-P-binding sites of the bovine IGF-II/ MPR. The mutant IGF-II/MPRs secreted from COS-1 cells were analyzed by pentamannosyl phosphateagarose affinity chromatography, identifying four residues (Gln-392, Ser-431, Glu-460, and Tyr-465) in domain 3 and four residues (Gln-1292, His-1329, Glu-1354, and Tyr-1360) in domain 9 as essential for Man-6-P recognition. Binding affinity studies using the lysosomal enzyme, ␤-glucuronidase, confirmed these results. Together these analyses provide strong evidence that the two Man-6-P-binding sites of the IGF-II/MPR are structurally similar to each other and to the CD-MPR and utilize a similar carbohydrate recognition mechanism.

The 46 kDa cation-dependent mannose 6-phosphate receptor (CD-MPR) plays an essential role in the biogenesis of lysosomes by diverting newly synthesized mannose 6-phosphate (Man-6-P)-containing lysosomal enzymes from the secretory pathway to acidified endosomes. Previous crystallographic studies of the CD-MPR have identified 11 amino acids within its carbohydrate binding pocket. These residues were evaluated quantitatively by assaying the binding affinity of mutant receptors containing a single amino acid substitution toward a lysosomal enzyme. The results show that substitution of Gln-66, Arg-111, Glu-133, or Tyr-143 results in a >800-fold decrease in affinity, demonstrating these four amino acids are essential for carbohydrate recognition by the CD-MPR. Solution binding and surface plasmon resonance analyses demonstrated that the presence of Mn2+ enhanced the affinity of the CD-MPR for a lysosomal enzyme by 2- to 4-fold and increased the stoichiometry of the interaction between a heterogeneous population of a lysosomal enzyme and the receptor by approximately 3-fold. In contrast, substitution of Asp-103 results in a protein that no longer exhibits enhanced binding affinities or altered stoichiometry in the presence of cations, and electron spin resonance demonstrated that the D103S mutant exhibits a 6-fold lower affinity for Mn2+ than the wild-type receptor (Kd = 3.7 6 1.4 mM versus 0.6 6 0.1 mM). Chemical cross-linking revealed that Mn2+ influences the stoichiometry of interaction between the CD-MPR and lysosomal enzymes by increasing the oligomeric state of the receptor from dimer to higher order oligomers. Taken together, these studies provide the molecular basis for high affinity carbohydrate recognition by the CD-MPR. Furthermore, Asp-103 has been identified as the key residue which mediates the effects of divalent cations on the binding properties of the CD-MPR.

The 46 kDa cation-dependent MPR (CD-MPR) plays a key role in the delivery of lysosomal enzymes to the lysosome by binding newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases and diverting them from the secretory pathway. Previous studies on a truncated form of the receptor comprised of only the soluble extracellular region (sCD-MPR, residues 1-154) have shown that the CD-MPR exists as a homodimer and exhibits two distinct conformations in the ligandbound versus ligand-unbound states, involving changes in quaternary structure and positioning of loop D, the residues of which form a side of the binding pocket in the presence of ligand. To determine the role of inter-monomer contacts in the functioning of the sCD-MPR, site-directed mutagenesis was used to generate a construct lacking a salt bridge (Glu19-Lys137) that tethers the N-terminal α-helix of one subunit to loop D of the other subunit in the ligand-bound form. Here we show by surface plasmon resonance analyses and NMR spectroscopy that the elimination of this inter-monomer salt bridge significantly decreases binding affinity of the mutant receptor (E19Q/K137M) towards lysosomal enzymes and Man-6-P. Analyses of the E19Q/K137M mutant receptor crystallized under various conditions revealed an altered quaternary structure that is intermediate between those observed in the ligand-bound and ligand-unbound states. Taken together, the results demonstrate a key role for inter-monomer interactions in the structure and functioning of the CD-MPR.The biogenesis of lysosomes requires the correct sorting of >60 acid hydrolases from their site of synthesis in the endoplasmic reticulum to their final destination in lysosomes (1). This targeting event is mediated by the 46 kDa cation-dependent mannose 6-phosphate receptor (CD-MPR) and the 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR), the only two members of the P-type family of lectins (2-4). The two MPRs recognize phosphomannosyl residues on high mannose-type N-glycans that serve to mark newly synthesized soluble acid hydrolases for delivery to endosomal/lysosomal compartments. The MPRs transport their cargo from the trans Golgi network (TGN) to endosomes where the acidic pH of the endosomal compartment facilitates disassembly of the complex. The released lysosomal enzymes are packaged into lysosomes while the receptors either return to the Golgi/ TGN via a retromer-assisted transport system (5) to repeat the process or move to the plasma membrane where the CI-MPR, but not the CD-MPR, functions to internalize exogenous ligands at the cell surface (6,7). Consistent with its role in intracellular trafficking, biochemical studies † This work was supported by National Institutes of Health Grant R01DK42667 (to N.M.D. and J.-J.P.K.) 1To whom correspondence should be addressed: Nancy M. Dahms have confirmed that the CD-MPR displays strict pH dependence with respect to ligand binding, with optimal binding occurring at the pH of the TGN (~pH 6.5) and little or no binding occurring at...