High affinity insulin binding by soluble insulin receptor extracellular domain fused to a leucine zipper

Insulin receptors (IRs) that are truncated at the end of the ectodomain form dimers that bind insulin with different characteristics to wild type receptors. These soluble IRs have lowered affinity for insulin compared with full-length IR, and exhibit linear Scatchard plots in contrast to the curvili...

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Bibliographic Details
Published in:FEBS letters 2000-08, Vol.479 (1), p.15-18
Main Authors: Hoyne, Peter A., Cosgrove, Leah J., McKern, Neil M., Bentley, John D., Ivancic, Neva, Elleman, Thomas C., Ward, Colin W.
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Cell Line
CHO Cells
Cricetinae
Dimerization
DNA Primers - genetics
Ectodomain chimera
Expression
High affinity binding
Humans
In Vitro Techniques
Insulin - metabolism
Insulin receptor
Kinetics
Leucine zipper
Leucine Zippers - genetics
Protein Structure, Quaternary
Protein Structure, Tertiary
Receptor, Insulin - chemistry
Receptor, Insulin - genetics
Receptor, Insulin - metabolism
Recombinant Fusion Proteins
Saccharomyces cerevisiae - genetics
Solubility
Transfection
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Summary:Insulin receptors (IRs) that are truncated at the end of the ectodomain form dimers that bind insulin with different characteristics to wild type receptors. These soluble IRs have lowered affinity for insulin compared with full-length IR, and exhibit linear Scatchard plots in contrast to the curvilinear plots obtained with full-length IR, IR truncated at the C-terminus of the transmembrane region and IR ectodomains fused to the self-associating constant domains from Fc or λ immunoglobulins. In this report, we have fused the IR ectodomain to the 33 residue leucine zipper from the transcriptional activator GCN4 of Saccharomyces cerevisiae. This fusion protein binds insulin with high affinity in a manner comparable to native receptor. The respective dissociation constants were K d1 8.2×10 −11 M and K d2 1.6×10 −8 M for hIRedZip and K d1 5.7×10 −11 M and K d2 6.3×10 −9 M for membrane-anchored, native receptor.
ISSN:0014-5793
1873-3468
DOI:10.1016/S0014-5793(00)01872-X