We report the crystal structure of two variants of Drosophila melanogaster insulin-like peptide 5 (DILP5) at a resolution of 1.85 Å . DILP5 shares the basic fold of the insulin peptide family (T conformation) but with a disordered B-chain C terminus. DILP5 dimerizes in the crystal and in solution. The dimer interface is not similar to that observed in vertebrates, i.e. through an anti-parallel ␤-sheet involving the B-chain C termini but, in contrast, is formed through an anti-parallel ␤-sheet involving the B-chain N termini. DILP5 binds to and activates the human insulin receptor and lowers blood glucose in rats. It also lowers trehalose levels in Drosophila. Reciprocally, human insulin binds to the Drosophila insulin receptor and induces negative cooperativity as in the human receptor. DILP5 also binds to insect insulin-binding proteins. These results show high evolutionary conservation of the insulin receptor binding properties despite divergent insulin dimerization mechanisms.The ligands and receptors of the insulin peptide family constitute an ancient metazoan signaling system that plays a crucial pleiotropic role in cell growth, metabolism, reproduction, and longevity (1-7).The mammalian insulin receptor belongs to the family of receptor-tyrosine kinases and is composed of two ␣ subunits and two ␤ subunits linked together by disulfide bonds (for review, see Refs. 4 and 8 -10). The existence of a homologue of the mammalian insulin receptor in Drosophila melanogaster (DIR) 2 was suggested in 1985 by Petruzzelli et al. (11), who identified a glycoprotein of 350 -400 kDa that binds bovine insulin specifically with moderate affinity (15 nM). The cDNA sequence of the DIR is remarkably similar to that of the mammalian insulin and IGF-I receptors (with 33% sequence identity) except for substantial N-and C-terminal extensions (12, 13).In evolution, there is a single receptor from Cnidarians up to and including Amphioxus (Branchiostoma californiense), the phylum closest to vertebrates (for review see Refs. 1, 4 -6). In vertebrates, gene duplications resulted in three related receptors; that is, the insulin receptor, the type I IGF receptor, and the orphan insulin receptor-related receptor (1, 5).In humans, members of the insulin peptide family include insulin, the insulin-like growth factors I and II, and seven relaxin-related peptides (for review, see Ref. 14). The same basic fold is shared for all molecules in the superfamily whose structure is known; the B domain contains a single ␣-helix that lies across the two ␣-helices of the A domain (15) and two canonical disulfide bridges that connect the A-and Bchains, whereas an intrachain disulfide bridge is present in the A-chain.The D. melanogaster genome contains seven insulin-like genes that are expressed in a highly tissue-and stage-specific patterns, dilp1-7 (16). dilp2 is the most related to human insulin with 35% sequence identity, whereas dilp5 has 27.8% identity (16).So far, the structures of only two invertebrate insulin-like peptides have been determined by N...

Insulin and IGF signaling (IIS) is a complex system that controls diverse processes including growth, development, metabolism, stress responses, and aging. Drosophila melanogaster IIS is propagated by eight Drosophila insulin-like peptides (DILPs), homologs of both mammalian insulin and IGFs, with various spatiotemporal expression patterns and functions. DILPs 1–7 are thought to act through a single Drosophila insulin/IGF receptor, InR, but it is unclear how the DILPs thereby mediate a range of physiological phenotypes. We determined the distinct cell signaling effects of DILP2 and DILP5 stimulation upon Drosophila S2 cells. DILP2 and DILP5 induced similar transcriptional patterns but differed in signal transduction kinetics. DILP5 induced sustained phosphorylation of Akt, while DILP2 produced acute, transient Akt phosphorylation. Accordingly, we used phosphoproteomic analysis to identify distinct patterns of non-genomic signaling induced by DILP2 and DILP5. Across all treatments and replicates, 5,250 unique phosphopeptides were identified, representing 1,575 proteins. Among these peptides, DILP2, but not DILP5, dephosphorylated Ser15 on glycogen phosphorylase (GlyP), and DILP2, but not DILP5, was subsequently shown to repress enzymatic GlyP activity in S2 cells. The functional consequences of this difference were evaluated in adult Drosophila dilp mutants: dilp2 null adults have elevated GlyP enzymatic activity relative to wild type, while dilp5 mutants have reduced GlyP activity. In flies with intact insulin genes, GlyP overexpression extended lifespan in a Ser15 phosphorylation-dependent manner. In dilp2 mutants, that are otherwise long-lived, longevity was repressed by expression of phosphonull GlyP that is enzymatically inactive. Overall, DILP2, unlike DILP5, signals to affect longevity in part through its control of phosphorylation to deactivate glycogen phosphorylase, a central modulator of glycogen storage and gluconeogenesis.

Insulin and the insulin-like growth factors (IGFs) bind with high affinity to their cognate receptor and with lower affinity to the noncognate receptor. The major structural difference between insulin and the IGFs is that the IGFs are single chain polypeptides containing A-, B-, C-, and D-domains, whereas the insulin molecule contains separate A-and B-chains. The C-domain of IGF-I is critical for high affinity binding to the insulin-like growth factor I receptor, and lack of a C-domain largely explains the low affinity of insulin for the insulin-like growth factor I receptor. It is less clear why the IGFs have lower affinity for the insulin receptor. In this study, 24 insulin analogues and four IGF analogues were expressed and analyzed to explore the role of amino acid differences in the Aand B-domains between insulin and the IGFs in binding affinity for the insulin receptor. showed affinities similar to that of insulin for the insulin receptor. The mitogenic potency of these analogues correlated well with the binding properties. Thus, a small number of A-and B-domain substitutions that map to the IGF surface equivalent to the classical binding surface of insulin weaken two hotspots that bind to the insulin receptor site 1.

Insulin-like growth factor I (IGF-I) has important anabolic and homeostatic functions in tissues like skeletal muscle, and a decline in circulating levels is linked with catabolic conditions. Whereas IGF-I therapies for musculoskeletal disorders have been postulated, dosing issues and disruptions of the homeostasis have so far precluded clinical application. We have developed a novel IGF-I variant by site-specific addition of polyethylene glycol (PEG) to lysine 68 (PEG-IGF-I). In vitro, this modification decreased the affinity for the IGF-I and insulin receptors, presumably through decreased association rates, and slowed down the association to IGF-I-binding proteins, selectively limiting fast but maintaining sustained anabolic activity. Desirable in vivo effects of PEG-IGF-I included increased half-life and recruitment of IGF-binding proteins, thereby reducing risk of hypoglycemia. PEG-IGF-I was equipotent to IGF-I in ameliorating contraction-induced muscle injury in vivo without affecting muscle metabolism as IGF-I did. The data provide an important step in understanding the differences of IGF-I and insulin receptor contribution to the in vivo activity of IGF-I. In addition, PEG-IGF-I presents an innovative concept for IGF-I therapy in diseases with indicated muscle dysfunction.

The insulin/insulin-like growth factor signalling axis is an evolutionary ancient and highly conserved hormonal system involved in the regulation of metabolism, growth and lifespan in animals. Human insulin is stored in the pancreas, while insulin-like growth factor-1 (IGF-1) is maintained in blood in complexes with IGF-binding proteins (IGFBP1–6). Insect insulin-like polypeptide binding proteins (IBPs) have been considered as IGFBP-like structural and functional homologues. Here, we report structures of the Drosophila IBP Imp-L2 in its free form and bound to Drosophila insulin-like peptide 5 and human IGF-1. Imp-L2 contains two immunoglobulin-like fold domains and its architecture is unrelated to human IGFBPs, suggesting a distinct strategy for bioavailability regulation of insulin-like hormones. Similar hormone binding modes may exist in other insect vectors, as the IBP sequences are highly conserved. Therefore, these findings may open research routes towards a rational interference of transmission of diseases such as malaria, dengue and yellow fevers.