Revealing Molecular Bases of Signal Transduction through the Drosophila Insulin Receptor: cryoEM and Functional Studies.

We have the opportunity to resolve our understanding of the physiological binding of insulin to the insulin receptor and the resultant molecular signalling signatures of this complex.

Insulin and Insulin-like Growth Factors 1 and 2 (IGF1/2) are the key human protein hormones with a common origin and similar 3-D organisation. Despite similar structures they regulate a wide spectrum of physiological events, with insulin being responsible for broad metabolic control and aging, while IGF1/2 are growth factors involved also in regulation of life span and growth. Insulin and IGFs exert their activities by binding to their highly homologous ca. ~450 kDa homo-dimeric tyrosine-kinase receptors (RTKs): Insulin Receptor (hIR) and IGF-1R, respectively. The close molecular and physiological links between these hormones lie behind the insulin-IGF signalling axis (IIS) responsible for the homeostatic regulations and pathologies in growth, life span, diabetes, cancer and neurodegeneration. IIS is one of the most conserved signalling axis in the animal kingdom showing commonality of similar, orthologues hormones, receptors and signalling nodes from polyps to humans.

Recently, several cryoEM structures of the full length hIR were described. However, they showed a variety of possible, and puzzling, different insulin:hIR stoichiometries (one to four insulins/hIR), and the structural changes in the TK part of the receptor were also not revealed due to its mobility and occlusion by the IR-stabilising lipids. Hence we initiated work on highly homologous to hIR/IGF-1R insect Drosophila melanogaster (Dm) only IR (dmIR) which takes their roles in insects. The key benefits of the proposed research results also from dmIR 60kDa/subunit TK-domain extension (CTD) that is in part homologous to IRS1 - the hIR first downstream phosphorylation protein substrate. Therefore it presents a unique opportunity to expand IR work into IR:IRS1 interaction, and its subsequent association with p85/SH2 domain of PI3K: the next key mediator of IR-based signal transduction. The dmIR complex with CHICO protein (Dm orthologue of human IRS1-4) can be studied as well, as it seems that dmIR binds the downstream PI3K effector in two different ways: one by the CTD - dmIR- internal IRS1-like module, and the other one through the CHICO docking sites in the dmIR juxtamembrane-segment.

The main Aim of this proposal is to resolve our understanding of the physiological binding of insulin to the insulin receptor and define the molecular signalling signatures of this complex. We will use here Drosophila insulin receptor (dmIR), exploit its high homology to the human IR (hIR), and exploit its large intracellular extension which corresponds strongly to the first intracellular substrate of the hIR. Therefore dmIR presents an unique opportunity for the cryoEM and functional studies to study the whole of the hormone:receptor:effector molecular signalling machinery referred to here as the IR signalosome.

Objectives:

1. To provide an independent insight into physiological insulin:IR stoichiometry and the activated conformation of the IR ectodomain (ecd-dmIR)(already advanced cryoEM work).
2. To elucidate the structural transitions of the full-length IR from its hormone-free to activated state (cryoEM studies)
3. To provide an insight into the nature of the IR interaction with, and activation of the first intra-cellular components of its-downstream signalling partners (cryoEM of dmIR with fragments of its PI3K and CHICO signalling partners).
4. To undertake the functional validation of the structural findings through making transgenic and gene replacement flies with mutations on the observed in 1-3 key protein:protein interfaces, and tandem-affinity purification of the downstream complexes.
5. To outline the molecular assembly and signalling steps in the IR-based signalosome by translating findings of 1-4 to the human hIR-based IIS system.

Insulin Receptor (dmIR) with the following objectives. (1) To provide an insight into insulin:IR stoichiometry and the activated conformation of the IR ectodomain (ecto-IR). We have cloned and produced ecto-dmIR (baculovirus system/insects sf9 cells), characterising its binding affinity of Dm insulin (DILP5). The work on the cryoEM structure of the ecto-dmIR:DILP5 (holo-dmIR) complex is already advanced. In (2) we aim to reveal the inactive to active structural transitions of the full-length IR. We will produce the full-length dmIR for its complexes with DILP5. The search for the optimum dmIR:DILP5 lipid environment for the cryoEM work will be crucial for the visualisation of the Tyrosine Kinase (TK). This should be facilitated by its 120kDa IRS-1-like extension modules in comparison with hIR, which aims to provide desired stability to the TK. Next, we will focus on ligand-free cryoEM structure of dmIR; this order of study is dictated by known high flexibility of the IR ectodomain. Objectives (1-2) enable systemic, comparative analysis of the different states of hormone:IR complexes and to outline the physiologically relevant apo-to-holo-IR transitions, followed by (3) elucidation of interactions and activation of the first intra-cellular components of its downstream signalling partners. Objective (2) will be expanded by complex formation between holo-dmIR with p85/SH2 of the PI3K that is the first kinase switch activated by IR:IRS-1 complex, and fragments of CHICO: an insect-homolog of IRS-1. Findings of objectives (2-3) will be underpinned by (4) their functional validation through generation of transgenic and gene replacement flies with mutations on the observed, key protein:protein interfaces. Ultimately, theoretical & molecular graphics comparative analysis of hIR and dmIR systems will allow us to (5) outline the key molecular steps in the IR-based signalosome.