How insulin engages its primary binding site on the insulin receptor (original) (raw)
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Nucleic Acids Research, 1991
The display of proteins on the surface of phage offers a powerful means of selecting for rare genes encoding proteins with binding activities. Recently we found that antibody heavy and light chain variable (V) domains fused as a single polypeptide chain to a minor coat protein of filamentous phage fd, could be enriched by successive rounds of phage growth and panning with antigen. This allows the selection of antigen-binding domains directly from diverse libraries of V-genes. Now we show that heterodimeric Fab fragments can be assembled on the surface of the phage by linking one chain to the phage coat protein, and secreting the other Into the bacterial periplasm. Furthermore by introducing an amber mutation between the antibody chain and the coat protein, we can either display the antibody on phage using supE strains of bacteria, or produce soluble Fab fragment using non-suppressor strains. The use of Fab fragments may offer advantages over single chain Fv fragments for construction of combinatorial libraries.
Characterization of a second ligand binding site of the insulin receptor
Biochemical and Biophysical Research Communications, 2006
Insulin binding to its receptor is characterized by high affinity, curvilinear Scatchard plots, and negative cooperativity. These properties may be the consequence of binding of insulin to two receptor binding sites. The N-terminal L1 domain and the C-terminus of the a subunit contain one binding site. To locate a second site, we examined the binding properties of chimeric receptors in which the L1 and L2 domains and the first Fibronectin Type III repeat of the insulin-like growth factor-I receptor were replaced by corresponding regions of the insulin receptor. Substitutions of the L2 domain and the first Fibronectin Type III repeat together with the L1 domain produced 80-and 300-fold increases in affinity for insulin. Fusion of these domains to human immunoglobulin Fc fragment produced a protein which bound insulin with a K d of 2.9 nM. These data strongly suggest that these domains contain an insulin binding site.
The A-chain of insulin contacts the insert domain of the insulin receptor
Journal of Biological …, 2007
The contribution of the insulin A-chain to receptor binding is investigated by photo-cross-linking and nonstandard mutagenesis. Studies focus on the role of Val A3 , which projects within a crevice between the A-and B-chains. Engineered receptor α-subunits containing specific ...
Proceedings of the National Academy of Sciences, 2010
The C-terminal segment of the human insulin receptor α-chain (designated αCT) is critical to insulin binding as has been previously demonstrated by alanine scanning mutagenesis and photo-crosslinking. To date no information regarding the structure of this segment within the receptor has been available. We employ here the technique of thermal-factor sharpening to enhance the interpretability of the electron-density maps associated with the earlier crystal structure of the human insulin receptor ectodomain. The αCT segment is now resolved as being engaged with the central β-sheet of the first leucine-rich repeat (L1) domain of the receptor. The segment is α-helical in conformation and extends 11 residues N-terminal of the classical αCTsegment boundary originally defined by peptide mapping. This tandem structural element (αCT-L1) thus defines the intact primary insulin-binding surface of the apo-receptor. The structure, together with isothermal titration calorimetry data of mutant αCT peptides binding to an insulin minireceptor, leads to the conclusion that putative "insulin-mimetic" peptides in the literature act at least in part as mimics of the αCT segment as well as of insulin. Photo-cross-linking by novel bifunctional insulin derivatives demonstrates that the interaction of insulin with the αCT segment and the L1 domain occurs in trans, i.e., these components of the primary binding site are contributed by alternate α-chains within the insulin receptor homodimer. The tandem structural element defines a new target for the design of insulin agonists for the treatment of diabetes mellitus.
Selection and characterisation of binders based on homodimerisation of immunoglobulin VH domains
FEBS Letters, 2003
The antigen-binding surface of antibodies is formed by the heterodimerisation of the two variable domains of the light (V L) and heavy (V H) chains. We have previously described the spontaneous formation of V H dimers (VHD) in both bacteria and mammalian cells. The self-association of a single domain produces a homo-VHD, in which the two identical V H domains generate a unique symmetric surface for antigen binding that is never found in the normal V L /V H antibody binding site. We developed a phagemid vector for the construction of phage display libraries in which a cysteine residue, introduced at the C-terminus of the only V H cloned, allowed display of homo-VHDs. Panning of the library on di¡erent proteins yielded antigen speci¢c binders against lysozyme, glutathione S-transferase and streptavidin. A lysozyme speci¢c homo-VHD was further characterised with an apparent a⁄nity determined to be 216 þ 6.6 nM. Importantly, the results showed that its binding activity was fully dependent on the dimerisation of both identical V H domains.
Rapid Identification of Small Binding Motifs with High-Throughput Phage Display
Chemistry & Biology, 2002
and Development for the optimization of loops within proteins [4] and for the identification of novel ligands [2]. M13 lends itself Genentech 1 DNA Way to this process because engineering the viral DNA can lead to the fusion of peptides or proteins of interest South San Francisco, California 94080
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1986
Processing of the insulin receptor by hepatocytes was studied using a t251-1abeiled photoreactive insulin derivative which could be covalently attached to the receptor and facilitate the analysis of receptor structure in isolated subcellular fractions by SDS-polyacrylamide gel electrophoresis. Following binding at the cell surface, the label was rapidly internalised and located in a low-density subcellular fraction ('endosomes'). The intact receptor (350000 molecular weight) and binding (a) subunit (135000), produced by in vitro disulphide reduction of the samples, were found in the plasma membrane fraction but not in endosomes. In endosomes, the label was concentrated in a band at 140000 (non-reduced) which on reduction generated species of 100000 and 68000 predominantly. The insulin receptor therefore undergoes an early structural change during endocytosis. This modification does not involve complete disulphide reduction and may be due to a proteolytic event.
Insulin Receptor: Covalent Labeling and Identification of Subunits
Proceedings of The National Academy of Sciences, 1979
Two methods were used to label insulin receptors covalently with 125I. In the first, an aryl azide derivative of insulin, 125I-labeled 4-azido-2-nitrophenyl-insulin, was synthesized and used to photolabel the binding region of the insulin receptor in rat liver membranes and human placenta membranes. In the second, insulin receptors were purified from rat liver membranes and labeled with 125I by use of chloramine-T; this method presumably has no specificity for the binding region of the receptor. The proteins labeled by both methods were anal zed by sodium dodecyl sulfate/poyacrylamide gel elec-