Structure-function analysis of a series of glucagon-like peptide-1 analogs.

We have used NMR in conjunction with measurements of functional bioactivity to define the receptor-binding structure of glucagon-like peptide-1 (GLP-1.) Identification of the important residues for binding was accomplished by the substitution of amino acids at sites that seemed likely, from an examination of the amino acid sequence and from previously published observations, to be important in the three-dimensional (3D) structure of the molecule. Identification of the receptor-bound conformation of GLP-1, because it is a flexible peptide, required constraint of the peptide backbone into a predetermined 3D structure. Constraint was achieved by the introduction of disulfide bonds and specific side chain-side chain cross-links. The biological relevance of the synthetic structure of each rigidified peptide was assessed by measurement of its ability to bind to the receptor present on RINm5F cells and to elicit a functional response, cyclic AMP production. NMR solution structures were obtained for the most biologically relevant of these analogs. The results of this study indicated that the residues necessary for the biological activity of GLP-1 occupy approximately three equally-spaced regions of the peptide 3D structure, at the corners of an equilateral triangle whose sides are, at a minimum estimate, 12-15A.

Molecular characterization of an alpha interferon receptor 1 subunit (IFNaR1) domain required for TYK2 binding and signal transduction.

Binding of alpha interferon (IFNalpha) to its receptors induces rapid tyrosine phosphorylation of the receptor subunits IFNaR1 and IFNaR2, the TYK2 and JAK1 tyrosine kinases, and the Stat1 and Stat2 transcription factors. Previous studies have demonstrated that TYK2 directly and specifically binds to and tyrosine phosphorylates IFNaR1 in vitro. We now report a detailed analysis of the TYK2 binding domain on the IFNaR1 subunit. First, we used an in vitro binding assay to identify the TYK2 binding motif in IFNaR1 as well as the critical residues within this region. The most striking feature is the importance of a number of hydrophobic and acidic residues. A minor role is also ascribed to a region resembling the proline-rich "box 1" sequence. In addition, mutations which disrupt in vitro binding also disrupt the coimmunoprecipitation of the receptor and TYK2. We also provide direct evidence that the binding region is both necessary and sufficient to activate TYK2 in vivo. Specifically, mutations in the binding domain act in a dominant-negative fashion to inhibit the IFNalpha-induced tyrosine phosphorylation of TYK2 and Stat2. Further, introduction of dimerized glutathione S-transferase-IFNaR1 fusion proteins into permeabilized cells is sufficient to induce phosphorylation of TYK2 and the receptor, confirming the role of the binding domain in IFNalpha signal transduction. These studies provide clues to the sequences determining the specificity of the association between JAK family tyrosine kinases and cytokine receptors as well as the functional role of these kinases in cytokine signal transduction.

Effect of the activation of phosphatidylinositol 3-kinase by a thiophosphotyrosine peptide on glucose transport in 3T3-L1 adipocytes.

Insulin causes the activation of phosphatidylinositol 3-kinase (PI 3-kinase) through complexation of tyrosine-phosphorylated YMXM motifs on insulin receptor substrate 1 with the Src homology 2 domains of PI 3-kinase. Previous studies with inhibitors have indicated that activation of PI 3-kinase is necessary for the stimulation of glucose transport in adipocytes. Here, we investigate whether this activation is sufficient for this effect. Short peptides containing two tyrosine-phosphorylated or thiophosphorylated YMXM motifs potently activated PI 3-kinase in the cytosol from 3T3-L1 adipocytes. Introduction of the phosphatase-resistant thiophosphorylated peptide into 3T3-L1 adipocytes through permeabilization with Staphylococcus aureus alpha-toxin stimulated PI 3-kinase as strongly as insulin. However, under the same conditions the peptide increased glucose transport into the permeabilized cells only 20% as well as insulin. Determination of the distribution of the glucose transporter isotype GLUT4 by confocal immunofluorescence showed that GLUT4 translocation to the plasma membrane can account for the effect of the peptide. These results suggest that one or more other insulin-triggered signaling pathways, besides the PI 3-kinase one, participate in the stimulation of glucose transport.

Amylin and CGRP induce insulin resistance via a receptor distinct from cAMP-coupled CGRP receptor.

Amylin and calcitonin gene-related peptide (CGRP) inhibited insulin-stimulated 2-deoxyglucose uptake in L6 myocytes and isolated soleus muscle. Both peptides were maximally active at 10 pM in L6 cells and inhibited insulin action by 40-50%. In soleus muscle amylin and CGRP inhibited insulin-stimulated uptake by 65-85%. Amylin competed with 125I-CGRP for binding to L6 cells but with 100-fold lower potency than CGRP. Occupancy of the CGRP receptor in L6 cells is coupled to adenylyl cyclase. Amylin increased the cellular content of adenosine 3',5'-cyclic monophosphate (cAMP), but consistent with binding, amylin was 100-fold less potent than CGRP. In soleus muscle, 100 nM amylin, which maximally inhibited 2-deoxyglucose uptake, had no effect cAMP content, whereas CGRP at the same concentration increased cAMP by 50%. The effect of CGRP on cAMP levels was completely suppressed by the competitive antagonist, CGRP-(8-37). In contrast, the suppression of insulin-stimulated glycogen synthesis or 2-deoxyglucose uptake by amylin was unaffected by 1 microM CGRP-(8-37). Our results demonstrate that the inhibition of insulin-stimulated glucose transport by amylin is independent of cAMP and may be mediated by a unique receptor that is distinct from the adenylyl cyclase-coupled CGRP receptor.

Fluorescence-based continuous assay for the aspartyl protease of human immunodeficiency virus-1.

5-Dimethylaminoaphthalene-1-sulfonyl-Ser-Gln-Asn-Tyr-Pro-Ile-Val-T rp (Dns-SQNYPIVW) is a fluorescent substrate for the aspartyl protease of human immunodeficiency virus-1. In intact substrate, fluorescence of Trp (lambda ex 290 nm, lambda em 360 nm) was 60% quenched by energy transfer to the dansyl group. Protease-catalyzed cleavage at the Tyr-Pro bond abolished the energy transfer, and the consequent increase in Trp fluorescence was used to follow the enzymatic reaction. At substrate concentrations less than 60 microM, initial reaction velocity increased as a linear function of substrate concentration, with kcat/KM = 9700 M-1 s-1. Limited solubility and internal fluorescence quenching precluded a determination of KM for Dns-SQNYPIVW, but this was clearly greater than 100 microM.