The two mannose 6-phosphate binding sites of the insulin-like growth factor-II/mannose 6-phosphate receptor display different ligand binding properties.
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.
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
P G
PG
Marron-Terada
M A
MA
Brzycki-Wessell
N M
NM
Dahms
D000818:Animals; D001665:Binding Sites; D002402:Cathepsin D / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002846:Chromatography, Affinity; D005966:Glucuronidase / Q000378:metabolism*; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology; D008360:Mannosephosphates / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D017527:Receptor, IGF Type 2 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D011994:Recombinant Proteins; C027693:mannose-6-phosphate; D007335:Insulin-Like Growth Factor II; D005966:Glucuronidase; D002402:Cathepsin D