Use of high affinity insulin analogues to assess the functional relationships between insulin receptor trafficking, mitogenic signaling and mRNA expression in rat liver.

We have investigated the functional relationships between insulin receptor (IR) trafficking, mitogenic signaling and mRNA expression in rat liver and primary hepatocytes. The low-K(d) insulin analogues [His(A8),His(B4), Glu(B10),His(B27)]-human insulin (-HI) (the H2-analogue), [Asp(B10)]HI and [Glu(A13),Glu(B10)]HI, were studied in liver parenchymal cells and compared with wild-type HI and epidermal growth factor (EGF), a mitogenic inducer. The extent and duration of IR endocytosis were markedly increased in response to the H2-analogue and [Asp(B10)]HI compared to wild-type HI, but similar to HI after [Glu(A13),Glu(B10)]HI administration.

Assembly of high-affinity insulin receptor agonists and antagonists from peptide building blocks.

Insulin is thought to elicit its effects by crosslinking the two extracellular alpha-subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked.

Peptides identify the critical hotspots involved in the biological activation of the insulin receptor.

We used phage display to generate surrogate peptides that define the hotspots involved in protein-protein interaction between insulin and the insulin receptor. All of the peptides competed for insulin binding and had affinity constants in the high nanomolar to low micromolar range. Based on competition studies, peptides were grouped into non-overlapping Sites 1, 2, or 3.