title: Mutational analysis of the binding site residues of the bovine cation-dependent mannose 6-phosphate receptor.
abstract: Mannose 6-phosphate receptors (MPRs) deliver soluble acid hydrolases to the lysosome in higher eukaryotic cells. The two MPRs, the cation-dependent MPR (CD-MPR) and the insulin-like growth factor II/cation-independent MPR, carry out this process by binding with high affinity to mannose 6-phosphate residues found on the N-linked oligosaccharides of their ligands. To elucidate the key amino acids involved in conveying this carbohydrate specificity, site-directed mutagenesis studies were conducted on the extracytoplasmic domain of the bovine CD-MPR. Single amino acid substitutions of the residues that form the binding pocket were generated, and the mutant constructs were expressed in transiently transfected COS-1 cells. Following metabolic labeling, mutant CD-MPRs were tested for their ability to bind pentamannosyl phosphate-containing affinity columns. Of the eight amino acids mutated, four (Gln-66, Arg-111, Glu-133, and Tyr-143) were found to be essential for ligand binding. In addition, mutation of the single histidine residue, His-105, within the binding site diminished the binding of the receptor to ligand, but did not eliminate the ability of the CD-MPR to release ligand under acidic conditions.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
authors.forename: L J
authors.initials: LJ
authors.lastname: Olson
authors.forename: M K
authors.initials: MK
authors.lastname: Hancock
authors.forename: D
authors.initials: D
authors.lastname: Dix
authors.forename: J J
authors.initials: JJ
authors.lastname: Kim
authors.forename: N M
authors.initials: NM
authors.lastname: Dahms
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017527:Receptor, IGF Type 2 / Q000737:chemistry*; D013329:Structure-Activity Relationship
chemical_list: D017527:Receptor, IGF Type 2