RESUMO
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.
Assuntos
Catepsina D/metabolismo , Glucuronidase/metabolismo , Manosefosfatos/metabolismo , Receptor IGF Tipo 2/metabolismo , Animais , Sítios de Ligação , Bovinos , Linhagem Celular , Cromatografia de Afinidade , Humanos , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Lisossomos/enzimologia , Camundongos , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , TransfecçãoRESUMO
A recombinant baculovirus containing the cDNA for the full-length bovine cation-dependent mannose 6-phosphate receptor (CD-MPR) was generated by homologous recombination. Spodoptera frugiperda (Sf9) and Trichoplusia ni 5B1-4 (High Five) insect cells, which do not contain endogenous MPRs as determined by Western blot analyses and pentamannosyl phosphate-agarose affinity chromatography, were infected with recombinant baculovirus. The infected cells expressed 26-, 29-, 32-, 35-, and 39-kDa species that were immunologically reactive with antisera raised against the native bovine CD-MPR and quantitative Western analysis demonstrated 1-2 x 10(6) CD-MPR molecules/cell, a level which is comparable to that expressed normally in a variety of cell lines and tissues. Digestion with endo-beta-N-acetylglucosaminidase H indicated that these multiple species were due to the presence of either 0, 1, 2, 3, or 4 N-linked oligosaccharide chains, respectively, and that the majority (87%) of these carbohydrates were of the high-mannose type. The receptor produced was biologically active since it bound specifically to a pentamannosyl phosphate-agarose column and exhibited acid-dependent ligand dissociation. In addition, studies using a homobifunctional cross-linking agent on the purified CD-MPR suggested that, like the receptor expressed in mammalian cells, the insect-produced CD-MPR existed as a dimer in the membrane.
Assuntos
Baculoviridae/genética , Cátions , Expressão Gênica , Receptor IGF Tipo 2/genética , Spodoptera/metabolismo , Acetilglucosaminidase/metabolismo , Animais , Western Blotting , Configuração de Carboidratos , Bovinos , Cromatografia de Afinidade , Vetores Genéticos , Glicosilação , Substâncias Macromoleculares , Oligossacarídeos/análise , Receptor IGF Tipo 2/química , Receptor IGF Tipo 2/metabolismo , Proteínas Recombinantes/química , TransfecçãoRESUMO
The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) binds two distinct ligands, mannose 6-phosphate (Man-6-P) and insulin-like growth factor II (IGF-II). The extracytoplasmic region of the receptor is composed of 15 homologous repeating domains and domains 1-3 and 7-9 have been shown to contain the two Man-6-P binding sites. To determine the location of the single IGF-II binding site, truncated forms of the M6P/IGF-II receptor were expressed transiently in COS-1 cells and assayed for their ability to bind iodinated human recombinant IGF-II. The binding of [125I]IGF-II to the receptors in the presence or absence of excess unlabeled IGF-II, IGF-I, or insulin was determined by incubation with homobifunctional cross-linking agents followed by SDS-polyacrylamide gel electrophoresis. These binding studies demonstrated that a construct encoding domains 5-11 bound 0.9 mol of IGF-II/mol of receptor, whereas a construct encoding domains 5-10 exhibited no detectable binding to IGF-II. These results indicate that the IGF-II binding site of the M6P/IGF-II receptor is contained within domains 5-11 and that residues in domain 11 play an important role in IGF-II binding.
Assuntos
Fator de Crescimento Insulin-Like II/metabolismo , Receptor IGF Tipo 2/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Bovinos , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Humanos , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Manosefosfatos/metabolismo , Dados de Sequência Molecular , Peso Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Receptor IGF Tipo 2/biossíntese , Receptor IGF Tipo 2/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Deleção de SequênciaRESUMO
Bovine, human, and rat cation-independent mannose 6-phosphate receptors (CI-MPRs) are capable of binding both mannose 6-phosphate and insulin-like growth factor-II (IGF-II). However, the receptor isolated from either chicken or frog lacks the high affinity IGF-II-binding site. To determine whether CI-MPRs isolated from a species that is closely related to placental mammals can bind IGF-II, the MPRs were purified from a marsupial, the American opossum (Didelphis virginiana), by phosphomannan-Sepharose affinity chromatography and then tested for their ability to bind IGF-II. Opossum liver expressed both the CI-MPR and the cation-dependent MPR (CD-MPR). Both receptors contained Asn-linked oligosaccharides. In contrast to CD-MPRs isolated from other species, the opossum CD-MPR displayed heterogeneity with respect to the number of Asn-linked oligosaccharide chains it contains. The CI-MPR isolated from opossum liver, like the CI-MPR from bovine liver, bound iodinated human recombinant IGF-II. However, Scatchard analysis revealed that the opossum CI-MPR bound IGF-II with a lower affinity (Kd = 14.5 nM) than the bovine receptor (Kd = 0.2 nM). The addition of excess IGF-II, but not IGF-I or insulin, inhibited binding to [125I]IGF-II, indicating that the opossum CI-MPR exhibits specificity for IGF-II. These results suggest that the emergence of a high affinity IGF-II-binding site in the CI-MPR occurred in evolution before the divergence of marsupials and placental mammals from their last common ancestor.