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Related Experiment Videos

The insulin receptor. Structural basis for high affinity ligand binding.

M Böni-Schnetzler, W Scott, S M Waugh

    The Journal of Biological Chemistry
    |June 15, 1987
    PubMed
    Summary

    Reducing insulin receptors with dithiothreitol breaks disulfide bonds, affecting insulin binding affinity. Optimal pH conditions minimize binding loss, revealing that receptor half association is crucial for high-affinity insulin binding.

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    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Cellular Signaling

    Background:

    • The insulin receptor (IR) is a tetrameric glycoprotein crucial for glucose homeostasis.
    • Understanding IR structure-function relationships, particularly ligand binding, is vital for metabolic research.

    Purpose of the Study:

    • To investigate the role of disulfide bonds in insulin receptor structure and insulin binding affinity.
    • To determine the impact of reduction conditions on receptor conformation and ligand interaction.

    Main Methods:

    • Treatment of soluble and membrane-bound human placental insulin receptors with dithiothreitol (DTT) at varying pH.
    • Kinetic analysis of disulfide bond reduction and insulin binding.
    • Scatchard analysis and sucrose density gradient centrifugation to assess receptor dissociation and binding characteristics.

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    Main Results:

    • DTT treatment at pH 8.5 reduces inter-half disulfide bonds, causing dissociation into alpha beta dimers with lower insulin affinity.
    • Intra-chain disulfides in the alpha subunit, part of the ligand-binding domain, are susceptible to reduction at pH 7.6, leading to significant binding loss.
    • Reduction in membranes at pH 8.5 preserves affinity, but subsequent solubilization causes dissociation and a 10-fold decrease in binding affinity (Kd).

    Conclusions:

    • The association of two alpha beta receptor halves (four ligand-binding domains) is essential for high-affinity insulin binding.
    • Specific intra-chain disulfides within the alpha subunit directly modulate ligand binding.
    • pH-dependent reduction strategies can differentiate between structural changes affecting receptor dissociation and those impacting ligand affinity.