The two mannose 6-phosphate binding sites of the insulin-like growth factor-II/mannose 6-phosphate receptor display different ligand binding properties. J Biol Chem 1998 Aug 28;273(35):22358-66
Date
08/26/1998Pubmed ID
9712856DOI
10.1074/jbc.273.35.22358Scopus ID
2-s2.0-0032575546 (requires institutional sign-in at Scopus site) 27 CitationsAbstract
The two mannose 6-phosphate (Man-6-P) binding sites of the insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MPR) have been localized to domains 1-3 and 7-9, and studies have shown that Arg435 in domain 3 and Arg 1334 in domain 9 are essential for Man-6-P binding. To determine whether the IGF-II/MPR containing a single Man-6-P binding site is functional, clonal mouse L cell lines stably transfected with either mutant bovine IGF-II/MPR cDNA, containing substitutions at position 435 and/or 1334, or the wild type receptor cDNA were assayed for their ability to sort lysosomal enzymes to the lysosome. Mutant receptors containing a single Man-6-P binding site were approximately 50% less efficient than the wild type receptor in the overall targeting of lysosomal enzymes to the lysosome. Mutant receptors containing a substitution at Arg1334 (Dom9(Ala)), in contrast to those containing a substitution at Arg435 (Dom3(Ala)), were unable to target cathepsin D and beta-hexosaminidase to the lysosome. Equilibrium binding assays using 125I-labeled beta-glucuronidase demonstrated that Dom3(Ala) and Dom9(Ala) had a Kd of 2.0 and 4.3 nM, respectively. In addition, Dom3(Ala), unlike Dom9(Ala), was unable to completely dissociate from ligand under acidic pH conditions. These data indicate that the two Man-6-P binding sites of the IGF-II/MPR are not functionally equivalent.
Author List
Marron-Terada PG, Brzycki-Wessell MA, Dahms NMAuthor
Nancy M. Dahms PhD Professor in the Biochemistry department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsBinding Sites
Cathepsin D
Cattle
Cell Line
Chromatography, Affinity
Glucuronidase
Humans
Insulin-Like Growth Factor II
Lysosomes
Mannosephosphates
Mice
Protein Binding
Receptor, IGF Type 2
Recombinant Proteins
Substrate Specificity
Transfection