AN INSULIN-LIKE-GROWTH-FACTOR-II (IGF-II) AFFINITY-ENHANCING DOMAIN LOCALIZED WITHIN EXTRACYTOPLASMIC REPEAT-13 OF THE IGF-II MANNOSE-6-PHOSPHATE RECEPTOR

Insulin-like growth factor II (IGF-II) and phosphomannosylated glycopr oteins bind to distinct sites on the same receptor, the IGF-II/mannose 6-phosphate receptor (IGF2R). Analysis of truncated receptors (minire ceptors) has been used to map the IGF-II binding site within the recep tor's extracytoplasmic domain, which consists of 15 homologous repeats . A minireceptor consisting of repeat 11 contained the minimal element s for binding IGF-II, but with 5- to 10-fold lower relative binding af finity than the full-length receptor. We hypothesized that the complet e, 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 dom ain needed for expression of high-affinity IGF-II binding, a nested se t of FLAG epitope-tagged minireceptors encompassing repeats 11 through 15 was prepared and transiently expressed in 293T cells. Minireceptor s containing repeats 11-13 or 11-15 exhibited high affinity, comparabl e to the full-length receptor (IC50 = 1-2 nM), whereas constructs cont aining repeat 11 only or repeats 11-12 did not (IC50 = 10-20 nM). Thes e data suggested that the affinity-enhancing domain is located within repeat 13, which contains a unique 43-residue insert that has similar to 50% sequence identity to the type II repeat of fibronectin. Althoug h a repeat 13 minireceptor did not bind IGF-II on its own, an 11-13 mi nireceptor containing a deletion of the 43-residue insert exhibited lo w IGF-II binding affinity (IC,, = 10-20 nM). Expression of mutant rece ptors from a full-length IGF2R construct bearing a deletion of the 43- residue insert was very low relative to wild type. Depletion assays us ing IGF-ll-Sepharose showed that the mutant receptor had lower affinit y for IGF-II than the wild-type receptor. This study reveals that two independent receptor domains are involved in the formation of a high-a ffinity binding site for IGF-II, and that a complete repeat 13 is requ ired for high-affinity IGF-II binding.