Characterization of the Functional Insulin Binding Epitopes of the Full-length Insulin Receptor

Abstract

Mutational analyses of the secreted recombinant insulin receptor extracellular domain have identified a ligand binding site composed of residues located in the L1 domain (amino acids 1-470) and at the C terminus of the ␣ subunit (amino acids [705][706][707][708][709][710][711][712][713][714][715] , and Phe 89 on insulin-induced receptor autophosphorylation. They had no effect on the maximal response to insulin but produced an increase in the EC 50 commensurate with their effect on the affinity of the receptor for insulin.The initiating event in the insulin-signaling cascade is the binding of insulin to a specific plasma membrane receptor. This interaction has been studied extensively and was found to be extremely complex (see Ref. 1 for review). Scatchard plots (2) of equilibrium binding data are concave and curvilinear, suggesting heterogeneity of ligand binding sites, negatively cooperative site-site interactions, or a combination of both (1). These properties and high affinity interactions with insulin are dependent on the dimeric structure of the receptor; insulin binds non-cooperatively to a single population of binding sites in the monomeric receptor (3-5). The stoichiometry of binding to the native receptor appears to be one insulin molecule to one receptor dimer (6). The secreted recombinant extracellular domain of the receptor exhibits properties similar to those of the receptor monomer and has a stoichiometry of two insulin molecules to one receptor dimer (6, 7).A number of hypothetical models of insulin-receptor interactions have been proposed to explain these findings (7-9). The model that best explains the experimental findings is that of De Meyts (1). This proposes that insulin has two topographically distinct receptor binding sites and that the receptor has two topographically distinct insulin binding sites/monomer. In this model, insulin binds asymmetrically to the insulin dimer, with each of its binding sites contacting its cognate receptor site on a separate monomer. Although the detailed structural basis of this model has yet to be elucidated, the structure-function relationships of the insulin molecule and the receptor have been studied extensively and provide support for the essentials of the model.Insulin (1, 10). However, hagfish insulin, in which these residues and the tertiary structure of the molecule are conserved (11), displays anomalous receptor binding behavior (11,12), suggesting that other residues outside this region might also be involved in receptor interactions. This is supported by the finding that two recombinant insulin analogues with substitutions in residues located on the opposite side of the molecule, Leu A13 to Ser and Leu B17 to Gln, exhibited similar receptor binding behavior to hagfish insulin (7,8).The structure and structure-function relationships of the insulin receptor are not as well documented. It is a dimeric transmembrane protein (see Ref. 1 for review). Each monomer consists of disulfide-linked ␣ and ␤ subunits. The ␣ subunits are wholly extracellular and c...

Protective hinge in insulin opens to enable its receptor engagement

“…This observation rationalizes (i) the high activity of an Ala B26 analog (36), (ii) the dispensability of Tyr B26 in truncated analogs (37), and (iii) that whereas holoreceptor substitution Arg14Ala (near both Tyr B26 and the B25 main chain) impairs insulin binding >10 3 -fold, Ala substitution of Asp12 (in contact only with Tyr B26 ) impairs hormone binding by only 6-fold (33). We suggest that the conservation of Tyr B26 (2) arises not from its role in receptor binding but instead from its contribution to proinsulin folding (38) and insulin self-assembly (2,39).…”

“…Lined by Pro716 and Pro718 in IR-A (Pro716 and Lys718 in IR-B) and multiple main-chain atoms, this open surface presumably contributes to the rigid requirement for an aromatic side chain at B25 (2,4,35). In the holoreceptor, substitution of Val715 by Ala eliminates detectable hormone binding (33). It is unclear, however, why substitution of Phe B25 by Ala, deleterious in full-length insulin (13), is by contrast well tolerated in truncated analog des-pentapeptide[B26-B30]-insulin-amide (see below) (35).…”

“…Relative activities of insulin analogs were evaluated in an in vitro assay of hormone-stimulated receptor autophosphorylation (33).…”

Design of an Insulin Analog with Enhanced Receptor Binding Selectivity

“…Receptor Binding Assays-Binding assays employ a FLAG epitope-tagged holoreceptor (either IR isoform B or IGFR) transiently expressed in human 293 PEAK cells (CRL-2828, American Tissue Culture Collection) (35,36). The receptors were partially purified by wheat germ agglutinin (WGA) chromatography and further fractionated on binding to polystyrene 96-well plates (Nunc Maxisorb) coated with an anti-FLAG monoclonal antibody (FLAG M2 immunoglobulin G; Sigma).…”

Structure of the insulin receptor–insulin complex by single-particle cryo-EM analysis

“…Each insulin is located between the L1 domain of one IR-monomer, the FnIII-1 domain of the other IR-monomer, and the α-CT helix (Figure 1c). Residues of the L1 subdomain and the α-CT helix, previously identified as essential for insulin binding by a variety of biochemical approaches 13 , have been proposed to represent the IR S1 site 18 . Structures of “S1 microreceptors” (containing L1, CR and a portion of the α-CT) in complex with insulin and insulin mimetics 7 provided the first atomic details of some IR-insulin key interactions (Extended Data Table 1).…”