Localization of the Insulin-like Growth Factor II Binding Site to Amino Acids 1508–1566 in Repeat 11 of the Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor

Abstract

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) binds insulin-like growth factor II (IGF-II) with high affinity. To localize the IGF-II binding site within the 15 repeating units that form the extracytoplasmic domain of the receptor, purified human M6P/IGF-II receptor was digested with thermolysin, and the fragments were analyzed for their ability to bind 125I-IGF-II in a cross-linking assay. Two IGF-II-binding receptor fragments of 23 and 37 kDa were purified. Sequence analysis revealed that the fragments consist of disulfide connected peptides comprising amino acids 1331-1566 and 1331-1697 of the receptor repeats 9-12. In a second approach we expressed truncated forms of the M6P/IGF-II receptor fused to the C terminus of the extracytoplasmic domain of the 46-kDa mannose 6-phosphate receptor. Fusion proteins containing M6P/IGF-II receptor repeats 10-15, 10-11, or 11-15 bound IGF-II, whereas a fusion protein containing the single repeat 10 failed to bind. This result indicates that repeat 11 (amino acids 1508-1650) is sufficient for binding of IGF-II. Residues 1508-1566, which are shared by the 23-kDa IGF-II-binding fragment and repeat 11, are proposed to form the IGF-II binding site of the M6P/IGF-II receptor.

M6P/IGF2R is mutated in squamous cell carcinoma of the lung

“…The minimal binding region for IGF2 in the human receptor is located in the N-terminal portion of repeat 11 (Dahms et al, 1994;Garmroudi et al, 1996;Schmidt et al, 1995), and repeat 13 further enhances the IGF2 binding a nity of the M6P/IGF2R (Devi et al, 1998). The mannose 6-phosphate binding sites and the IGF2 binding and enhancing regions of the M6P/ IGF2R gene were screened for mutations in the tumors with LOH.…”

“…A G:C?C:G transversion was identi®ed at a non CpG site in exon 33 that results in the substitution of Arg for Gly1564 (Figure 2c). This amino acid alteration occurs within the identi®ed minimal IGF2 binding site (Dahms et al, 1994;Garmroudi et al, 1996;Schmidt et al, 1995), and is predicted from the 3-D structure of the CDM6PR to be in a putative loop region between beta strands 2 and 3 (Roberts et al, 1998). The substitution of Arg, a large charged amino acid, for Gly1564, a small neutral amino acid, would be expected to signi®cantly alter receptor tertiary structure and function.…”

Real Time Kinetics of Insulin-like Growth Factor II (IGF-II) Interaction with the IGF-II/Mannose 6-Phosphate Receptor

“…Domains 1-15 had the highest affinity for IGF-II, suggesting that other unknown IGF-II binding enhancing domains may also exist. Previously reported affinity values of IGF-II for full-length receptor have ranged from 0.2 to 5 nM for bovine IGF-IIR (47, 48) and 15 nM for placental purified human IGF-IIR (6). Differences may be due to experimental approach, since measurements have been determined both by affinity blots and surface plasmon resonance, with variations due to either the quality of purified proteins or stability of recombinant proteins.…”

“…The glycosylated protein (270 kDa) has 15 extracytosolic repeat domains containing distinct binding sites for phosphomannosyl residues and IGF-II in mammals and marsupials. Mannosylated proteins bind to domains 1-3 and 7-9 (4, 5), and IGF-II binds to domain 11 (6). Approximately 90% of membrane-bound IGF-IIR is normally found within the cell.…”

Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice

“…We hypothesized that a portion of IGF-II (hereafter referred to as the ''GILT tag'') retaining the ability to bind to the IGF-II͞CI-MPR would serve as an effective targeting moiety when fused to lysosomal enzymes. Such a GILT-tagged protein would target the identical receptor targeted by Man6-P (albeit to a distinct binding site), thereby sharing the identical endocytic pathway for lysosomal targeting with Man6-Pcontaining proteins (22)(23)(24)(25)(26)(27).…”