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. Importantly, the insulin analogues induced a higher and more prolonged tyrosine phosphorylation of the IR-beta subunit in endosomes compared to authentic HI. A low cell-free endosome-lysosome transfer of the internalized IR was only observed in response to HI and H2-analogue injection. Concomitant with the low endosome-lysosome transfer of the intact IR-beta subunit, 47 and 50 kDa fragments of the IR-beta subunit accumulated in lysosomal fractions. Neither HI nor the insulin analogues promoted the endosomal recruitment and tyrosine phosphorylation of Shc, whereas EGF accessed the Shc signaling pathway. Moreover, EGF induced a fast and prolonged activation of Raf-1 and MAP-kinase pathways whereas HI and insulin analogues displayed a moderate and transient effect. Finally, treatment of primary rat hepatocytes with HI and the protease-resistant H2-analogue did not affect the total level and relative expression of isotype A and B of IR mRNA regardless of time of exposure. These results suggest a lack of relationship between IR trafficking, endosomal tyrosine phosphorylation and mitogenic signaling in rat liver in vivo.

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. An optimized agonist has been shown, both in vitro and in vivo, to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases.

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. Some Site 1 peptides were able to activate the tyrosine kinase activity of the insulin receptor and act as agonists in the insulin-dependent fat cell assay, suggesting that Site 1 marks the hotspot involved in insulin-induced activation of the insulin receptor. On the other hand, Site 2 and 3 peptides were found to act as antagonists in the phosphorylation and fat cell assays. These data show that a peptide display can be used to define the molecular architecture of a receptor and to identify the critical regions required for biological activity in a site-directed manner.