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Altered Ligand Binding by Insulin-like Growth Factor II/Mannose 6-Phosphate Receptors Bearing Missense Mutations in Human Cancers¹

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198-4525 [G. R. D., J. C. B., R. G. M.]; Department of Safety of Medicines, Zeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire, SK10 4TG, United Kingdom [A. T. D. S.]; and Department of Radiation Oncology, Duke University Medical Center, Durham, North Carolina 27710 [R. L. J.]

The M6P/IGF2R gene, encoding the insulin-like growth factor II (IGF-II)/mannose 6-phosphate receptor (IGF2R), is frequently inactivated during carcinogenesis. M6P/IGF2R is postulated to be a tumor suppressor gene due to its ability to bind and degrade the mitogen IGF-II, promote activation of the growth inhibitor transforming growth factor ß, and regulate the targeting of lysosomal enzymes. In this study, we determined the effects of four M6P/IGF2R missense mutations associated with loss of heterozygosity in hepatocellular and breast cancers on the ligand binding properties of full-length membrane-bound receptors. Site-directed mutagenesis was used to prepare COOH-terminal, c-myc epitope-tagged human IGF2R cDNA expression constructs bearing point mutations that lead to the substitutions I1572T, G1464E, G1449V, and Q1445H, all of which are located in the receptor’s extracytoplasmic domain. Ligand binding was measured in plasma membranes from 293T cells expressing full-length receptors. No binding of ¹²⁵I-IGF-II to I1572T mutant receptors was observed. Binding to G1449V mutant receptors was decreased by 50% relative to wild-type (WT). However, IGF-II binding to the G1464E and Q1445H mutant receptors was equivalent to WT when plasma membranes were assayed immediately after preparation. The phosphomannosylated pseudoglycoprotein pentamannose 6-phosphate-BSA (PMP-BSA) was synthesized as a ligand for the M6P binding site. Binding of ¹²⁵I-PMP-BSA was equivalent to WT for the I1572T, G1464E, and Q1445H mutations, but there was a 60% reduction in PMP-BSA binding to the G1449V mutant receptor. Thus, several missense mutations in M6P/IGF2R disrupt the ligand binding functions of the intact IGF2R, lending further support to the hypothesis that the M6P/IGF2R is a tumor suppressor gene.

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