Tài liệu Báo cáo khoa học: High-affinity ligand binding by wild-type/mutant heteromeric complexes of

High-affinity ligand binding by wild-type/mutant

heteromeric complexes of the mannose

6-phosphate/insulin-like growth factor II receptor

Michelle A. Hartman1

, Jodi L. Kreiling2

, James C. Byrd1 and Richard G. MacDonald1

1 Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA

2 Department of Chemistry, University of Nebraska, Omaha, NE, USA

The mannose 6-phosphate ⁄ insulin-like growth factor II

receptor (M6P⁄IGF2R) is a 300 kDa transmembrane

glycoprotein that has diverse ligand-binding properties

contributing to several important cellular functions

[1,2]. Insulin-like growth factor II (IGF-II) binding to

the M6P⁄IGF2R leads to uptake into the cell and deg￾radation of the growth factor in lysosomes [3–6]. This

activity reduces IGF-II availability in the pericellular

Keywords

insulin-like growth factor II; ligand binding;

mannose 6-phosphate; mannose

6-phosphate ⁄ insulin-like growth factor II

receptor; oligomerization

Correspondence

R. G. MacDonald, Department of

Biochemistry and Molecular Biology,

University of Nebraska Medical Center,

985870 Nebraska MED CTR, Omaha, NE

68198 5870, USA

Fax: +1 402 559 6650

Tel:+1 402 559 7824

E-mail: [email protected]

(Received 17 October 2008, revised 19

December 2008, accepted 21 January 2009)

doi:10.1111/j.1742-4658.2009.06917.x

The mannose 6-phosphate ⁄ insulin-like growth factor II receptor has diverse

ligand-binding properties contributing to its roles in lysosome biogenesis

and growth suppression. Optimal receptor binding and internalization of

mannose 6-phosphate (Man-6-P)-bearing ligands requires a dimeric struc￾ture leading to bivalent high-affinity binding, presumably mediated by

cooperation between sites on both subunits. Insulin-like growth factor II

(IGF-II) binds to a single site on each monomer. It is hypothesized that

IGF-II binding to cognate sites on each monomer occurs independently,

but bivalent Man-6-P ligand binding requires cooperative contributions

from sites on both monomers. To test this hypothesis, we co-immunopre￾cipitated differentially epitope-tagged soluble mini-receptors and assessed

ligand binding. Pairing of wild-type and point-mutated IGF-II binding sites

between two dimerized mini-receptors had no effect on the function of the

contralateral binding site, indicating IGF-II binding to each side of the

dimer is independent and manifests no intersubunit effects. As expected,

heterodimeric receptors composed of a wild-type monomer and a mutant

bearing two Man-6-P-binding knockout mutations form functional IGF-II

binding sites. By contrast to prediction, such heterodimeric receptors also

bind Man-6-P-based ligands with high affinity, and the amount of binding

can be attributed entirely to the immunoprecipitated wild-type receptors.

Anchoring of both C-terminal ends of the heterodimer produces optimal

binding of both IGF-II and Man-6-P ligands. Thus, IGF-II binds indepen￾dently to both subunits of the dimeric mannose 6-phosphate ⁄ insulin-like

growth factor II receptor. Although wild-type ⁄ mutant hetero-oligomers

form readily when mixed, it appears that multivalent Man-6-P ligands bind

preferentially to wild-type sites, possibly by cross-bridging receptors within

clusters of immobilized receptors.

Abbreviations

Glc-6-P, glucose 6-phosphate; HA, hemagglutinin; HBS, Hepes-buffered saline; HBST, HBS containing 0.05% Triton X-100; IGF-II, insulin-like

growth factor II; M6P ⁄ IGF2R, mannose 6-phosphate ⁄ insulin-like growth factor II receptor; Man-6-P, mannose 6-phosphate; pBSKII+,

pBluescript SK II+; PMP-BSA, pentamannosyl 6-phosphate-BSA.

FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS 1915