High-affinity insulin binding: insulin interacts with two receptor ligand binding sites

Area of Science:

• Biochemistry
• Molecular Biology
• Endocrinology

Background:

• Insulin receptor binding is complex, involving high- and low-affinity sites or negative cooperativity.
• Receptor dimeric structure and insulin's bivalent nature are key to high-affinity interactions.
• Previous studies implicated the L1 domain and fibronectin repeats in insulin binding.

Purpose of the Study:

• To identify specific amino acid residues in the insulin receptor involved in ligand binding.
• To elucidate the structural determinants of high-affinity insulin-receptor interactions.

Main Methods:

• Structure-directed alanine scanning mutagenesis of the insulin receptor holo-receptor.
• Transient expression of receptor mutants in 293 cells.
• Determination of insulin binding affinities to expressed receptor mutants.

Main Results:

• Mutations of Lys(484), Leu(552), Asp(591), Ile(602), Lys(616), Asp(620), and Pro(621) reduced insulin binding affinity 2-5 fold.
• Most mutations affected holo-receptor binding but not secreted receptor binding, indicating direct involvement in the binding site.
• These residues constitute a novel ligand binding site on the insulin receptor.

Conclusions:

• Specific residues within the insulin receptor are critical for high-affinity insulin binding.
• The identified residues form a novel ligand binding site, contributing to the complexity of insulin signaling.
• Insulin interacts in trans with both receptor binding sites to achieve high-affinity binding.