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J. Biol. Chem., Vol. 280, Issue 22, 21067-21077, June 3, 2005

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Domain Interactions of the Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor^*

Jodi L. Kreiling, James C. Byrd, and Richard G. MacDonald¶

From the Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198-5870

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) forms oligomeric structures important for optimal function in binding and internalization of Man-6-P-bearing extracellular ligands as well as lysosomal biogenesis and growth regulation. However, neither the mechanism of inter-receptor interaction nor the dimerization domain has yet been identified. We hypothesized that areas near the ligand binding domains of the receptor would contribute preferentially to oligomerization. Two panels of minireceptors were constructed that involved truncations of either the N- or C-terminal regions of the M6P/IGF2R encompassing deletions of various ligand binding domains. -FLAG or -Myc-based immunoprecipitation assays showed that all of the minireceptors tested were able to associate with a full-length, Myc-tagged M6P/IGF2R (WT-M). In the -FLAG but not -Myc immunoprecipitation assays, the degree of association of a series of C-terminally truncated minireceptors with WT-M showed a positive trend with length of the minireceptor. In contrast, length did not seem to affect the association of the N-terminally truncated minireceptors with WT-M, except that the 12th extracytoplasmic repeat appeared exceptionally important in dimerization in the -FLAG assays. The presence of mutations in the ligand-binding sites of the minireceptors had no effect on their ability to associate with WT-M. Thus, association within the heterodimers was not dependent on the presence of functional ligand binding domains. Heterodimers formed between WT-M and the minireceptors demonstrated high affinity IGF-II and Man-6-P-ligand binding, suggesting a functional association. We conclude that there is no finite M6P/IGF2R dimerization domain, but rather that interactions between dimer partners occur all along the extracytoplasmic region of the receptor.

Received for publication, November 16, 2004 , and in revised form, February 24, 2005.

^* This work was supported in part by National Institutes of Health Grant 5R01CA91885 (to R. G. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of pre-doctoral stipend support provided by Graduate Studies; Bukey, McDonald, Emley, and Widaman fellowships; the Dr. Fred W. Upson grant-in-aid award through the University of Nebraska Medical Center, and the NASA Space Grant Scholarship.

Present address: Washington University School of Medicine, Rheumatology Division, 4921 Parkview Place, Campus Box 8045, St. Louis, MO 63110.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, NE 68198-5870. Tel.: 402-559-7824; Fax: 402-559-6650; E-mail: rgmacdon{at}unmc.edu.

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