title: An insulin-like growth factor II (IGF-II) affinity-enhancing domain localized within extracytoplasmic repeat 13 of the IGF-II/mannose 6-phosphate receptor.
abstract: Insulin-like growth factor II (IGF-II) and phosphomannosylated glycoproteins bind to distinct sites on the same receptor, the IGF-II/mannose 6-phosphate receptor (IGF2R). Analysis of truncated receptors (minireceptors) has been used to map the IGF-II binding site within the receptor's extracytoplasmic domain, which consists of 15 homologous repeats. A minireceptor consisting of repeat 11 contained the minimal elements for binding IGF-II, but with 5- to 10-fold lower relative binding affinity than the full-length receptor. We hypothesized that the complete, high-affinity IGF-II binding site is formed by interaction between the primary site in repeat 11 and a putative affinity-enhancing domain. To determine the minimum portion of the IGF2R's extracytoplasmic domain needed for expression of high-affinity IGF-II binding, a nested set of FLAG epitope-tagged minireceptors encompassing repeats 11 through 15 was prepared and transiently expressed in 293T cells. Minireceptors containing repeats 11-13 or 11-15 exhibited high affinity, comparable to the full-length receptor (IC50 = 1-2 nM), whereas constructs containing repeat 11 only or repeats 11-12 did not (IC50 = 10-20 nM). These data suggested that the affinity-enhancing domain is located within repeat 13, which contains a unique 43-residue insert that has approximately 50% sequence identity to the type II repeat of fibronectin. Although a repeat 13 minireceptor did not bind IGF-II on its own, an 11-13 minireceptor containing a deletion of the 43-residue insert exhibited low IGF-II binding affinity (IC50 = 10-20 nM). Expression of mutant receptors from a full-length IGF2R construct bearing a deletion of the 43-residue insert was very low relative to wild type. Depletion assays using IGF-II-Sepharose showed that the mutant receptor had lower affinity for IGF-II than the wild-type receptor. This study reveals that two independent receptor domains are involved in the formation of a high-affinity binding site for IGF-II, and that a complete repeat 13 is required for high-affinity IGF-II binding.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha 68198-4525, USA.
authors.forename: G R
authors.initials: GR
authors.lastname: Devi
authors.forename: J C
authors.initials: JC
authors.lastname: Byrd
authors.forename: D H
authors.initials: DH
authors.lastname: Slentz
authors.forename: R G
authors.initials: RG
authors.lastname: MacDonald
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D005813:Genes, Synthetic; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017527:Receptor, IGF Type 2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D017527:Receptor, IGF Type 2; D011993:Recombinant Fusion Proteins; D007335:Insulin-Like Growth Factor II