Jakob Brandt

Hybrid Receptors Formed by Insulin Receptor (IR) and Insulin-like Growth Factor I Receptor (IGF-IR) Have Low Insulin and High IGF-1 Affinity Irrespective of the IR Splice Variant

Insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) are both from the same subgroup of receptor tyrosine kinases that exist as covalently bound receptor dimers at the cell surface. For both IR and IGF-IR, the most described forms are homodimer receptors. However, hybrid receptors consisting of one-half IR and one-half IGF-IR are also present at the cell surface. Two splice variants of IR are expressed that enable formation of two isoforms of the IGF-IR/IR hybrid receptor. In this study, these two splice variants of hybrid receptors were studied with respect to binding affinities of insulin, insulin-like growth factor I (IGF-I), and insulin-like growth factor II (IGF-II). Unlike previously published data, in which semipurified receptors have been studied, we found that the two hybrid receptor splice variants had similar binding characteristics with respect to insulin, IGF-I, and IGF-II binding. We studied both semipurified and purified hybrid receptors. In all cases we found that IGF-I had at least 50-fold higher affinity than insulin, irrespective of the splice variant. The binding characteristics of insulin and IGF-I to both splice variants of the hybrid receptors were similar to classical homodimer IGF-IR.

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 α-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.

A pathogen-induced gene of barley encodes a HSP90 homologue showing striking similarity to vertebrate forms resident in the endoplasmic reticulum

The full-length nucleotide sequence of a barley (Hordeum vulgare L.) leaf mRNA, found to increase rapidly in amount during infection attempts by the powdery mildew fungus (Erysiphe graminis DC. ex Mérat), is reported. The mRNA encodes a polypeptide of 809 amino acid residues which, by sequence comparison, was identified as a member of the 90 kDa heat shock protein (HSP90) family. The encoded protein most resembles the endoplasmic reticulum (ER) resident HSP90 protein, the 94 kDa glucose-regulated protein (GRP94) of vertebrates, as it possesses both the characteristic N-terminal domain including a signal peptide sequence and the C-terminal ER retention signal (Lys-Asp-Glu-Leu). A transcript cross-hybridizing at high stringency accumulated rapidly in leaves upon heat shock treatment. Genomic DNA blot analysis indicated the presence of a family of related genes in the barley genome.

A removable spacer peptide in an α-factor-leader/insulin precursor fusion protein improves processing and concomitant yield of the insulin precursor in Saccharomyces cerevisiae

An alpha-factor leader/insulin precursor fusion protein was produced in Saccharomyces cerevisiae and metabolically labeled in order to analyse the efficiency of maturation and secretion. A substantial fraction of the secreted material was found in a hyperglycosylated unprocessed form, indicating incomplete Kex2p endopeptidase maturation. Introduction of a spacer peptide (EAEAEAK) after the dibasic Kex2p site, creating a N-terminal extension of the insulin precursor, greatly increased the Kex2p catalytic efficiency and the fermentation yield of insulin precursor. The N-terminal extension features a Lys to allow subsequent proteolytic removal by trypsin or the Achromobacter lyticus Lys-specific protease. Dipeptidyl aminopeptidase A (DPAPA) activity removing Glu-Ala dipeptides from the extension was inhibited by adding a Glu N-terminally to the extension. Unexpectedly, this modified N-terminal extension (EEAEAEAK) was partially cleaved after the Lys during fermentation. This monobasic proteolytic activity was demonstrated to be associated with Yap3p. Yap3p cleavage could be prevented by insertion of a Pro before the Lys (EEAEAEAPK).

Role of Insulin Receptor Dimerization Domains in Ligand Binding, Cooperativity, and Modulation by Anti-receptor Antibodies

To define the structures within the insulin receptor (IR) that are required for high affinity ligand binding, we have used IR fragments consisting of four amino-terminal domains (L1, cysteine-rich, L2, first fibronectin type III domain) fused to sequences encoded by exon 10 (including the carboxyl terminus of the α-subunit). The fragments contained one or both cysteine residues (amino acids 524 and 682) that form disulfides between α-subunits in native IR. A dimeric fragment designated IR593.CT (amino acids 1–593 and 704–719) bound 125I-insulin with high affinity comparable to detergent-solubilized wild type IR and mIR.Fn0/Ex10 (amino acids 1–601 and 650–719) and greater than that of dimeric mIR.Fn0 (amino acids 1–601 and 704–719) and monomeric IR473.CT (amino acids 1–473 and 704–719). However, neither IR593.CT nor mIR.Fn0 exhibited negative cooperativity (a feature characteristic of the native insulin receptor and mIR.Fn0/Ex10), as shown by failure of unlabeled insulin to accelerate dissociation of bound125I-insulin. Anti-receptor monoclonal antibodies that recognize epitopes in the first fibronectin type III domain (amino acids 471–593) and inhibit insulin binding to wild type IR inhibited insulin binding to mIR.Fn0/Ex10 but not IR593.CT or mIR.Fn0. We conclude the following: 1) precise positioning of the carboxyl-terminal sequence can be a critical determinant of binding affinity; 2) dimerization via the first fibronectin domain alone can contribute to high affinity ligand binding; and 3) the second dimerization domain encoded by exon 10 is required for ligand cooperativity and modulation by antibodies.

Dimeric fragment of the insulin receptor alpha-subunit binds insulin with full holoreceptor affinity.

The insulin receptor (IR) is a dimeric receptor, and its activation is thought to involve cross-linking between monomers initiated by binding of a single insulin molecule to separate epitopes on each monomer. We have previously shown that a minimized insulin receptor consisting of the first three domains of the human IR fused to 16 amino acids from the C-terminal of the α-subunit was monomeric and bound insulin with nanomolar affinity (Kristensen, C., Wiberg, F. C., Schäffer, L., and Andersen, A. S. (1998) J. Biol. Chem. 273, 17780–17786). To investigate the insulin binding properties of dimerized α-subunits, we have reintroduced the domains containing α-α disulfide bonds into this minireceptor. When inserting either the first fibronectin type III domain or the full-length sequence of exon 10, the receptor fragments were predominantly secreted as disulfide-linked dimers that both had nanomolar affinity for insulin, similar to the affinity found for the minireceptor. However, when both these domains were included we obtained a soluble dimeric receptor that bound insulin with 1000-fold higher affinity (4–8 pm) similar to what was obtained for the solubilized holoreceptor (14–24 pm). Moreover, dissociation of labeled insulin from this receptor was accelerated in the presence of unlabeled insulin, demonstrating another characteristic feature of the holoreceptor. This is the first direct demonstration showing that the α-subunit of IR contains all the epitopes required for binding insulin with full holoreceptor affinity.

Structural and Biological Properties of the Drosophila Insulin-Like Peptide 5 Show Evolutionary Conservation.

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 β-sheet involving the B-chain C termini but, in contrast, is formed through an anti-parallel β-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.

Recombinant Adiponectin Does Not Lower Plasma Glucose in Animal Models of Type 2 Diabetes

Aims/Hypothesis Several studies have shown that adiponectin can lower blood glucose in diabetic mice. The aim of this study was to establish an effective adiponectin production process and to evaluate the anti-diabetic potential of the different adiponectin forms in diabetic mice and sand rats. Methods Human high molecular weight, mouse low molecular weight and mouse plus human globular adiponectin forms were expressed and purified from mammalian cells or yeast. The purified protein was administered at 10–30 mg/kg i.p. b.i.d. to diabetic db/db mice for 2 weeks. Furthermore, high molecular weight human and globular mouse adiponectin batches were administered at 5–15 mg/kg i.p. b.i.d. to diabetic sand rats for 12 days. Results Surprisingly, none of our batches had any effect on blood glucose, HbA1c, plasma lipids or body weight in diabetic db/db mice or sand rats. In vitro biological, biochemical and biophysical data suggest that the protein was correctly folded and biologically active. Conclusions/Interpretation Recombinant adiponectin is ineffective at lowering blood glucose in diabetic db/db mice or sand rats.

cDNA Cloning and Characterization of mRNAs Induced in Barley by the Fungal Pathogen, Erysiphe Graminis

Several cDNA clones corresponding to barley genes induced following infection attempts by the powdery mildew fungus have been isolated and sequenced. Based on homology, seven of these cDNA clones represent genes encoding peroxidase, sucrose synthase, 14-3-3 protein (protein kinase regulator), GRP94 (a member of the heat shock protein 90 family), and three different PR-proteins: PR1, PR4, and PR5. One cDNA clone (pBH72-Fl) shows weak similarity to plant O-methyl transferases and another (pBH6-12) represents a novel sequence with no homology to available sequences. The transcripts corresponding to all cloned sequences accumulate following inoculation with both avirulent and virulent races of Erysiphe graminis f.sp. hordei. However, the induction pattern during infection time-courses differ between the different clones and between different infection types of the host-pathogen interaction.

A barley cDNA clone encoding a type III chlorophyll a/b-binding polypeptide of the light-harvesting complex II

The nucleotide sequence of a leaf cDNA clone encoding a Type III chlorophyll a/b-binding (CAB) protein of light-harvesting complex II (LHCII) in barley is reported. Sequence comparisons and results from in vitro import into chloroplasts demonstrate that the cDNA clone encodes a functional transit peptide of 45 amino acid residues and a mature polypeptide of 223 residues with a predicted molecular mass of 24.3 kDa. After insertion into thylakoids, the mature protein is resistant to protease attack. Hybridization analysis using a gene-specific probe shows that the gene is expressed in dark-grown seedlings and that the amount of mRNA increases during illumination.