Ligand binding specificities of the two mannose 6-phosphate receptors.
Two mannose 6-phosphate (Man-6-P) receptors (MPRs) direct the vesicular transport of newly synthesized lysosomal enzymes that contain Man-6-P from the Golgi to a prelysosomal compartment. In order to understand the respective roles of the Mr = 46,000 cation-dependent (CD-) MPR and the Mr = 300,000 cation-independent (CI-) MPR in lysosomal targeting, an assay has been developed that simultaneously measures the relative affinity of each MPR for multiple ligands. Glycoproteins containing Man-6-P were affinity-purified from the metabolically labeled secretions of mutant mouse fibroblasts lacking both MPRs. They were incubated with purified MPRs, and the resulting receptor-ligand complexes were immunoprecipitated by anti-MPR monoclonal antibodies coupled to agarose beads. Ligands were eluted with Man-6-P and then quantified following SDS-polyacrylamide gel electrophoresis. Saturating concentrations of CI-MPR resulted in the complete recovery of each Man-6-P glycoprotein in receptor-ligand complexes. Apparent affinity constants ranged between 1 and 5 nM for the individual species. Ligands precipitated by the CD-MPR appeared identical to those bound by the CI-MPR, with apparent affinity constants ranging between 7 and 28 nM. The binding affinities of the two receptors for different ligands were not correlated, indicating that the two MPRs preferentially recognize different subsets of lysosomal enzymes. In addition, saturating levels of CD-MPR resulted in the precipitation of only 50% of the total input ligands, suggesting that the CD-MPR binds a subpopulation of the Man-6-P glycoproteins bound by the CI-MPR. These results provide a biochemical mechanism, which, in part, may explain the interaction of the two MPRs with overlapping yet distinct subsets of ligands in vivo.
Sleat
D E
DE
Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854, USA.
Lobel
P
P
D000818:Animals; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000201:enzymology; D008360:Mannosephosphates / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D010766:Phosphorylation; D017527:Receptor, IGF Type 2 / Q000378:metabolism*; D013379:Substrate Specificity
D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; C027693:mannose-6-phosphate