The insulin receptor is a phylogenetically ancient tyrosine kinase receptor found in organisms as primitive as cnidarians and insects. In higher organisms it is essential for glucose homeostasis, whereas the closely related insulin-like growth factor receptor (IGF-1R) is involved in normal growth and development. The insulin receptor is expressed in two isoforms, IR-A and IR-B; the former also functions as a high-affinity receptor for IGF-II and is implicated, along with IGF-1R, in malignant transformation. Here we present the crystal structure at 3.8 A resolution of the IR-A ectodomain dimer, complexed with four Fabs from the monoclonal antibodies 83-7 and 83-14 (ref. 4), grown in the presence of a fragment of an insulin mimetic peptide. The structure reveals the domain arrangement in the disulphide-linked ectodomain dimer, showing that the insulin receptor adopts a folded-over conformation that places the ligand-binding regions in juxtaposition. This arrangement is very different from previous models. It shows that the two L1 domains are on opposite sides of the dimer, too far apart to allow insulin to bind both L1 domains simultaneously as previously proposed. Instead, the structure implicates the carboxy-terminal surface of the first fibronectin type III domain as the second binding site involved in high-affinity binding.

The 42 amino acid Alzheimer's Abeta peptide is involved in the progression of Alzheimer's disease. Here we describe the effects of intracellular Abeta, produced through its attachment to either end of a green fluorescent protein, in yeast. Cells producing Abeta exhibited a lower growth yield and a heat shock response, showing that Abeta fusions promote stress in cells and supporting the notion that intracellular Abeta is a toxic molecule. These studies have relevance in understanding the role of Abeta in the death of neuronal cells, and indicate that yeast may be a new tractable model system for the screening for inhibitors of the stress caused by Abeta.

Latrepirdine (Dimebon™), an anti-histamine, has shown some benefits in trials of neurodegenerative diseases characterized by accumulation of aggregated or misfolded protein such as Alzheimer’s disease (AD) and has been shown to promote the removal of α-synuclein protein aggregates in vivo. An important pathway for removal of aggregated or misfolded proteins is the autophagy-lysosomal pathway, which has been implicated in AD pathogenesis, and enhancing this pathway has been shown to have therapeutic potential in AD and other proteinopathies. Here we use a yeast model Saccharomyces cerevisiae, to investigate whether latrepirdine can enhance autophagy and reduce levels of Aβ42 aggregates. Latrepirdine was shown to up-regulate yeast vacuolar (lysosomal) activity and promote transport of the autophagic marker (Atg8) to the vacuole. Using an in vitro GFP tagged Aβ yeast expression system, we investigated whether latrepirdine-enhanced autophagy was associated with a reduction in levels of intracellular GFP-Aβ42. GFP-Aβ42 was localized into punctate patterns compared to the diffuse cytosolic pattern of GFP and the GFP-Aβ42 (19:34), which does not aggregate. In the autophagy deficient mutant (Atg8Δ), GFP-Aβ42 showed a more diffuse cytosolic localization, reflecting the inability of this mutant to sequester GFP-Aβ42. Similar to rapamycin, we observed that latrepirdine significantly reduced GFP-Aβ42 in wild-type compared to the Atg8Δ mutant. Further, latrepirdine treatment attenuated Aβ42-induced toxicity in wild-type cells but not in the Atg8Δ mutant. Together, our findings provide evidence for a novel mechanism of action for latrepirdine in inducing autophagy and reducing intracellular levels of GFP-Aβ42.

Site-directed mutagenesis has been used to remove 15 of the 18 potential N-linked glycosylation sites, in 16 combinations, from the human exon 11-minus receptor isoform. The three glycosylation sites not mutated were asparagine residues 25, 397 and 894, which are known to be important in receptor biosynthesis or function. The effects of these mutations on proreceptor processing into α and β subunits, cell-surface expression, insulin binding and receptor autophosphorylation were assessed in Chinese hamster ovary cells. The double mutants 16j78, 16j111, 16j215, 16j255, 337j418, the triple mutants 295j337j418, 295j418j514, 337j418j514 and 730j 743j881 and the quadruple mutants 606j730j743j881 and 671j730j743j881 seemed normal by all criteria examined. The triple mutant 16j215j255 showed only low levels of correctly processed receptor on the cell surface, this processed receptor being autophosphorylated in response to insulin. The quadruple mutant 624j730j743j881 showed normal pro-