title: Localization of the carbohydrate recognition sites of the insulin-like growth factor II/mannose 6-phosphate receptor to domains 3 and 9 of the extracytoplasmic region.
abstract: The insulin-like growth factor II/mannose 6-phosphate receptor is a multifunctional receptor that binds to a diverse array of mannose 6-phosphate (Man-6-P) modified proteins as well as nonglycosylated ligands. Previous studies have mapped its two Man-6-P binding sites to a minimum of three domains, 1-3 and 7-9, within its 15-domain extracytoplasmic region. Since the primary amino acid determinants of carbohydrate recognition by the insulin-like growth factor II/mannose 6-phosphate receptor are predicted by sequence alignment to the cation-dependent mannose 6-phosphate receptor to reside within domains 3 and 9, constructs encoding either domain 3 alone or domain 9 alone were expressed in a Pichia pastoris expression system and tested for their ability to bind several carbohydrate ligands, including Man-6-P, pentamannosyl phosphate, the lysosomal enzyme, beta-glucuronidase, and the carbohydrate modifications (mannose 6-sulfate and Man-6-P methyl ester) found on Dictyostelium discoideum lysosomal enzymes. Although both constructs were functional in ligand binding and dissociation, these studies demonstrate the ability of domain 9 alone to fold into a high affinity (K(d) = 0.3 +/- 0.1 nm) carbohydrate-recognition domain whereas the domain 3 alone construct is capable of only low affinity binding (K(d) approximately 500 nm) toward beta-glucuronidase, suggesting that residues in adjacent domains (domains 1 and/or 2) are important, either directly or indirectly, for optimal binding by domain 3.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
authors.forename: Michael K
authors.initials: MK
authors.lastname: Hancock
authors.forename: Rama D
authors.initials: RD
authors.lastname: Yammani
authors.forename: Nancy M
authors.initials: NM
authors.lastname: Dahms
mesh_terms: D000818:Animals; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry*; D002846:Chromatography, Affinity; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004023:Dictyostelium / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008247:Lysosomes / Q000201:enzymology / Q000378:metabolism; D010843:Pichia / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017527:Receptor, IGF Type 2 / Q000378:metabolism*
chemical_list: D002241:Carbohydrates; D018076:DNA, Complementary; D008024:Ligands; D017527:Receptor, IGF Type 2; D007335:Insulin-Like Growth Factor II; D006596:Hexosaminidases