The neural cell adhesion molecule (NCAM) promotes axonal outgrowth, presumably through an interaction with the fibroblast growth factor receptor (FGFR). NCAM also has a little-understood ATPase activity. We here demonstrate for the first time a direct interaction between NCAM (fibronectin type III [F3] modules 1 and 2) and FGFR1 (Ig modules 2 and 3) by surface plasmon resonance (SPR) analysis. The structure of the NCAM F3 module 2 was determined by NMR and the module was shown by NMR to interact with the FGFR1 Ig module 3 and ATP. The NCAM sites binding to FGFR and ATP were found to overlap and ATP was shown by SPR to inhibit the NCAM-FGFR binding, indicating that ATP probably regulates the NCAM-FGFR interaction. Furthermore, we demonstrate that the NCAM module was able to induce activation (phosphorylation) of FGFR and to stimulate neurite outgrowth. In contrast, ATP inhibited neurite outgrowth induced by the module.

The glucagon-like peptide-1 incretin receptor (GLP-1R) of family B G protein-coupled receptors (GPCRs) is a major drug target in type-2-diabetes due to its regulatory effect on post-prandial blood-glucose levels. The mechanism(s) controlling GLP-1R mediated signaling are far from fully understood. A fundamental mechanism controlling the signaling capacity of GPCRs is the post-endocytic trafficking of receptors between recycling and degradative fates. Here, we combined microscopy with novel real-time assays to monitor both receptor trafficking and signaling in living cells. We find that the human GLP-1R internalizes rapidly and with similar kinetics in response to equipotent concentrations of GLP-1 and the stable GLP-1 analogues exendin-4 and liraglutide. Receptor internalization was confirmed in mouse pancreatic islets. GLP-1R is shown to be a recycling receptor with faster recycling rates mediated by GLP-1 as compared to exendin-4 and liraglutide. Furthermore, a prolonged cycling of ligand-activated GLP-1Rs was observed and is suggested to be correlated with a prolonged cAMP signal.

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...

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