High-Affinity Insulin Binding: Insulin Interacts with Two Receptor Ligand Binding Sites

Citations Over TimeTop 10% of 2008 papers

Abstract

The interaction of insulin with its receptor is complex. Kinetic and equilibrium binding studies suggest coexistence of high- and low-affinity binding sites or negative cooperativity. These phenomena and high-affinity interactions are dependent on the dimeric structure of the receptor. Structure-function studies of insulin analogs suggest insulin has two receptor binding sites, implying a bivalent interaction with the receptor. Alanine scanning studies of the secreted recombinant receptor implicate the L1 domain and a C-terminal peptide of the receptor alpha subunit as components of one ligand binding site. Functional studies suggest that the first and second type III fibronectin repeats of the receptor contain a second ligand binding site. We have used structure-directed alanine scanning mutagenesis to identify determinants in these domains involved in ligand interactions. cDNAs encoding alanine mutants of the holo-receptor were transiently expressed in 293 cells, and the binding properties of the expressed receptor were determined. Alanine mutations of Lys(484), Leu(552), Asp(591), Ile(602), Lys(616), Asp(620), and Pro(621) compromised affinities for insulin 2-5-fold. With the exception of Asp(620), none of these mutations compromised the affinity of the recombinant secreted receptor for insulin, indicating that the perturbation of the interaction is at the site of mutation and not an indirect effect on the interaction with the binding site of the secreted receptor. These residues thus form part of a novel ligand binding site of the insulin receptor. Complementation experiments demonstrate that insulin interacts in trans with both receptor binding sites to generate high-affinity interactions.

Related Papers

• → The Insulin Receptor and Its Cellular Targets1(2001)226 cited
• → Grb10 Inhibits Insulin-stimulated Insulin Receptor Substrate (IRS)-Phosphatidylinositol 3-Kinase/Akt Signaling Pathway by Disrupting the Association of IRS-1/IRS-2 with the Insulin Receptor(2003)121 cited
• → Interaction between the Grb10 SH2 Domain and the Insulin Receptor Carboxyl Terminus(1996)104 cited
• → Identification of the Rat Adapter Grb14 as an Inhibitor of Insulin Actions(1998)102 cited
• → Human growth factor receptor bound 14 binds the activated insulin receptor and alters the insulin-stimulated tyrosine phosphorylation levels of multiple proteins(2001)27 cited