Abstract
The mammalian cation-independent mannose 6-phosphate receptor (CI-MPR) binds mannose 6-phosphate-bearing glycoproteins and insulin-like growth factor (IGF)-II. However, the CI-MPR from the opossum has been reported to bind bovine IGF-II with low affinity (Dahms, N. M., Brzycki-Wessell, M. A., Ramanujam, K. S., and Seetharam, B. (1993) Endocrinology 133, 440-446). This may reflect the use of a heterologous ligand, or it may represent the intrinsic binding affinity of this receptor. To examine the binding of IGF-II to a marsupial CI-MPR in a homologous system, we have previously purified kangaroo IGF-II (Yandell, C. A., Francis, G. L., Wheldrake, J. F., and Upton, Z. (1998) J. Endocrinol. 156, 195-204), and we now report the purification and characterization of the CI-MPR from kangaroo liver. The interaction of the kangaroo CI-MPR with IGF-II has been examined by ligand blotting, radioreceptor assay, and real-time biomolecular interaction analysis. Using both a heterologous and homologous approach, we have demonstrated that the kangaroo CI-MPR has a lower binding affinity for IGF-II than its eutherian (placental mammal) counterparts. Furthermore, real-time biomolecular interaction analysis revealed that the kangaroo CI-MPR has a higher affinity for kangaroo IGF-II than for human IGF-II. The cDNA sequence of the kangaroo CI-MPR indicates that there is considerable divergence in the area corresponding to the IGF-II binding site of the eutherian receptor. Thus, the acquisition of a high-affinity binding site for regulating IGF-II appears to be a recent event specific to the eutherian lineage.
Highlights
The cation-independent mannose 6-phosphate receptor (CIMPR)1 is a multifunctional protein that binds proteins bearing mannose 6-phosphate moieties, as well as insulin-like growth factor (IGF)-II
The Triton X-100 solubilized liver membranes were passed over the phosphomannan-Sepharose column, and the protein retained after washing with Buffer D was IGF-II Binding to the Kangaroo cation-independent mannose 6-phosphate receptor (CI-MPR)
The opossum study was performed using a heterologous ligand, and it was not known whether amino acid differences between opossum and bovine IGF-II or amino acid differences in the IGF-II binding region of the receptor itself accounted for this difference in affinity
Summary
The cation-independent mannose 6-phosphate receptor (CIMPR)1 is a multifunctional protein that binds proteins bearing mannose 6-phosphate moieties, as well as insulin-like growth factor (IGF)-II. We have cloned the kangaroo CI-MPR cDNA sequence for the region proposed to be the IGF-II binding site on the mammalian receptor and have compared the sequences. Both the kangaroo and bovine receptors bound 125I-labeled IGF-II, and this binding could be displaced by the addition of excess unlabeled rhIGF-II (Fig. 2, a and b), but not IGF-I (data not shown).
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