Kinetic Evidence for the Sequential Association of Insulin Binding Sites 1 and 2 to the Insulin Receptor and the Influence of Receptor Isoform,

Abstract

Through binding to and signaling via the insulin receptor (IR), insulin is involved in multiple effects on growth and metabolism. The current model for the insulin-IR binding process is one of a biphasic reaction. It is thought that the insulin peptide possesses two binding interfaces (sites 1 and 2), which allow it to bridge the two alpha-subunits of the insulin receptor during the biphasic binding reaction. The sequential order of the binding events involving sites 1 and 2, as well as the molecular interactions corresponding to the fast and slow binding events, is still unknown. In this study we examined the series of events that occur during the binding process with the help of three insulin analogues: insulin, an analogue mutated in site 2 (B17A insulin), and an analogue in which part of site 1 was deleted (Des A1-4 insulin), both with and without a fluorescent probe attached. The binding properties of these analogues were tested using two soluble Midi IR constructs representing the two naturally occurring isoforms of the IR, Midi IR-A and Midi IR-B. Our results showed that in the initial events leading to Midi IR-insulin complex formation, insulin site 2 binds to the IR in a very fast binding event. Subsequent to this initial fast phase, a slower rate-limiting phase occurs, consistent with a conformational change in the insulin-IR complex, which forms the final high-affinity complex. The terminal residues A1-A4 of the insulin A-chain are shown to be important for the slow binding phase, as insulin lacking these amino acids is unable to induce a conformational change of IR and has a severely impaired binding affinity. Moreover, differences in the second phase of the binding process involving insulin site 1 between the IR-A and IR-B isoforms suggest that the additional amino acids encoded by exon 11 in the IR-B isoform influence the binding process.

All‐atom structural models of insulin binding to the insulin receptor in the presence of a tandem hormone‐binding element

“…Also, the orientation of R‐insulin in our model is consistent with ligand crosslinking of receptor subunits76 because the ligand can maintain simultaneous contact at site‐1 with L1 and at site‐2 with the (F1–F2)′ loops. These observations are also in accord with a proposed sequential model of association of insulin‐binding sites 1 and 2 to IR 79. As a next step, this new structural model also helps explain recognition of IR by homologous (to insulin) noncognate ligands such as a single‐chain polypeptide insulin‐like growth factor‐1 (IGF1), which cannot undergo significant separation of its C‐domain due to single‐chain structural constraints.…”

Novel Covalently Linked Insulin Dimer Engineered to Investigate the Function of Insulin Dimerization

“…This is not possible in the dimer form [53]–[56]. Obstruction of the classical binding site by removal of the first four amino acids in the A-chain (desA1-4) was shown to decrease binding affinities to 0.014% relative to HI [57] and fixation of the C-terminal end of the B-chain in SCI resulted in a loss of in vivo activity [55]. In contrast, the second binding site is still fully exposed and available for receptor binding.…”

Role of Insulin and Adenosine in the Human Placenta Microvascular and Macrovascular Endothelial Cell Dysfunction in Gestational Diabetes Mellitus

“…Up to now, two isoforms of the insulin receptor have been described, that is, IR‐A and B (IR‐B) . Both isoforms are expressed in insulin‐sensitive tissues (liver, muscle, and adipose tissue) , but IR‐A is predominantly expressed in the fetus and placenta, where it plays a role in embryonic development (Figure ).…”

“…Thus, an imbalance between the p42/ 44 mapk and Akt signaling pathways could lead to preferential mitogenic or metabolic phenotypes, respectively ( Figure 3). Up to now, two isoforms of the insulin receptor have been described, that is, IR-A and B (IR-B) [7,27,33,35,[75][76][77][78]87,89]. Both isoforms are expressed in insulin-sensitive tissues (liver, muscle, and adipose tissue) [57,59], but IR-A is predominantly expressed in the fetus and placenta, where it plays a role in embryonic development [33] (Figure 3).…”