Structural insights into ligand-induced activation of the insulin receptor.

The current model for insulin binding to the insulin receptor proposes that there are two binding sites, referred to as sites 1 and 2, on each monomer in the receptor homodimer and two binding surfaces on insulin, one involving residues predominantly from the dimerization face of insulin (the classical binding surface) and the other residues from the hexamerization face. High-affinity binding involves one insulin molecule using its two surfaces to make bridging contacts with site 1 from one receptor monomer and site 2 from the other. Whilst the receptor dimer has two identical site 1-site 2 pairs, insulin molecules cannot bridge both pairs simultaneously. Our structures of the insulin receptor (IR) ectodomain dimer and the L1-CR-L2 fragments of IR and insulin-like growth factor receptor (IGF-1R) explain many of the features of ligand-receptor binding and allow the two binding sites on the receptor to be described. The IR dimer has an unexpected folded-over conformation which places the C-terminal surface of the first fibronectin-III domain in close juxtaposition to the known L1 domain ligand-binding surface suggesting that the C-terminal surface of FnIII-1 is the second binding site involved in high-affinity binding. This is very different from previous models based on three-dimensional reconstruction from scanning transmission electron micrographs. Our single-molecule images indicate that IGF-1R has a morphology similar to that of IR. In addition, the structures of the first three domains (L1-CR-L2) of the IR and IGF-1R show that there are major differences in the two regions governing ligand specificity. The implications of these findings for ligand-induced receptor activation will be discussed. This review summarizes the key findings regarding the discovery and characterization of the insulin receptor, the identification and arrangement of its structural domains in the sequence and the key features associated with ligand binding. The remainder of the review deals with a description of the receptor structure and how it explains much of the large body of biochemical data in the literature on insulin binding and receptor activation.

Identification of a determinant of epidermal growth factor receptor ligand-binding specificity using a truncated, high-affinity form of the ectodomain.

Murine and human epidermal growth factor receptors (EGFRs) bind human EGF (hEGF), mouse EGF (mEGF), and human transforming growth factor alpha (hTGF-alpha) with high affinity despite the significant differences in the amino acid sequences of the ligands and the receptors. In contrast, the chicken EGFR can discriminate between mEGF (and hEGF) and hTGF-alpha and binds the EGFs with approximately 100-fold lower affinity. The regions responsible for this poor binding are known to be Arg(45) in hEGF and the L2 domain in the chicken EGFR. In this study we have produced a truncated form of the hEGFR ectodomain comprising residues 1-501 (sEGFR501), which, unlike the full-length hEGFR ectodomain (residues 1-621, sEGFR621), binds hEGF and hTGF-alpha with high affinity (K(D) = 13-21 and 35-40 nM, respectively). sEGFR501 was a competitive inhibitor of EGF-stimulated mitogenesis, being almost 10-fold more effective than the full-length EGFR ectodomain and three times more potent than the neutralizing anti-EGFR monoclonal antibody Mab528. Analytical ultracentrifugation showed that the primary EGF binding sites on sEGFR501 were saturated at an equimolar ratio of ligand and receptor, leading to the formation of a 2:2 EGF:sEGFR501 dimer complex. We have used sEGFR501 to generate three mutants with single position substitutions at Glu(367), Gly(441), or Glu(472) to Lys, the residue found in the corresponding positions in the chicken EGFR. All three mutants bound hTGF-alpha and were recognized by Mab528. However, mutant Gly(441)Lys showed markedly reduced binding to hEGF, implicating Gly(441), in the L2 domain, as part of the binding site that recognizes Arg(45) of hEGF.

The three dimensional structure of the type I insulin-like growth factor receptor.

Ever since the discovery of insulin and its role in the regulation of glucose metabolism, there has been great interest in the molecule itself, the insulin-like growth factors (IGFs), and their receptors (IR and IGF-R). These receptors form a subfamily of tyrosine kinase receptors which are large, transmembrane proteins consisting of several structural domains. Their ectodomains have a similar arrangement of two homologous domains (L1 and L2) separated by a Cys rich region. The C-terminal half of their ectodomains consists of three fibronectin type 3 repeats, and an insert domain that contains the alpha-beta cleavage site. This review summarises the key developments in the understanding of the structure of this family of receptors and their relation to other multidomain proteins. Data presented will include multiple sequence analyses, single molecule electron microscope images of the IGF-1R, insulin receptor (IR), and IR-Fab complexes, and the three dimensional structure of the first three domains of the IGF-1R determined to 2.6 A resolution by x ray crystallography. The L domains each adopt a compact shape consisting of a single stranded, right handed beta-helix. The Cys rich region is composed of eight disulphide bonded modules, seven of which form a rod shaped domain with modules associated in an unusual manner.

Single-molecule imaging of human insulin receptor ectodomain and its Fab complexes.

The insulin receptor (IR) is a four-chain, transmembrane dimer held together by disulfide bonds. To gain information about the molecular envelope and the organization of its domains, single-molecule images of the IR ectodomain and its complexes with three Fabs have been analyzed by electron microscopy. The data indicate that the IR ectodomain resembles a U-shaped prism of approximate dimensions 90 x 80 x 120 A. The width of the cleft (assumed membrane-distal) between the two side arms is sufficient to accommodate ligand. Fab 83-7, which recognizes the cys-rich region of IR, bound halfway up one end of each side arm in a diametrically opposite manner, indicating a twofold axis of symmetry normal to the membrane surface. Fabs 83-14 and 18-44, which have been mapped respectively to the first fibronectin type III domain (residues 469-592) and residues 765-770 in the insert domain, bound near the base of the prism at opposite corners. These images, together with the data from the recently determined 3D structure of the first three domains of the insulin-like growth factor type I receptor, suggest that the IR dimer is organized into two layers with the L1/cys-rich/L2 domains occupying the upper (membrane distal) region of the U-shaped prism and the fibronectin type III domains and the insert domains located predominantly in the membrane-proximal region.

Crystal structure of the first three domains of the type-1 insulin-like growth factor receptor.

The type-1 insulin-like growth-factor receptor (IGF-1R) and insulin receptor (IR) are closely related members of the tyrosine-kinase receptor superfamily. IR is essential for glucose homeostasis, whereas IGF-1R is involved in both normal growth and development and malignant transformation. Homologues of these receptors are found in animals as simple as cnidarians. The epidermal growth-factor receptor (EGFR) family is closely related to the IR family and has significant sequence identity to the extracellular portion we describe here. We now present the structure of the first three domains of IGF-IR (L1-Cys-rich-L2) determined to 2.6 A resolution. The L domains each consist of a single-stranded right-handed beta-helix. The Cys-rich region is composed of eight disulphide-bonded modules, seven of which form a rod-shaped domain with modules associated in an unusual manner. The three domains surround a central space of sufficient size to accommodate a ligand molecule. Although the fragment (residues 1-462) does not bind ligand, many of the determinants responsible for hormone binding and ligand specificity map to this central site. This structure therefore shows how the IR subfamily might interact with their ligands.

Crystallization of the first three domains of the human insulin-like growth factor-1 receptor.

The insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor of central importance in cell proliferation. A fragment (residues 1-462) comprising the L1-cysteine rich-L2 domains of the human IGF-1R ectodomain has been overexpressed in glycosylation-deficient Lec8 cells and has been affinity-purified via a c-myc tag followed by gel filtration. The fragment was recognized by two anti-IGF-1R monoclonal antibodies, 24-31 and 24-60, but showed no detectable binding of IGF-1 or IGF-2. Isocratic elution of IGF-1R/462 on anion-exchange chromatography reduced sample heterogeneity, permitting the production of crystals that diffracted to 2.6 A resolution with cell dimensions a = 77.0 A, b = 99.5 A, c = 120.1 A, and space group P2(1)2(1)2(1).

Purification and properties of insulin receptor ectodomain from large-scale mammalian cell culture.

Ectodomain of the exon 11+ form of the human insulin receptor (hIR) was expressed in the mammalian cell secretion vector pEE6.HCMV-GS, containing the glutamine synthetase gene. Following transfection of the hIR ectodomain gene into Chinese hamster ovary (CHO-K1) cells, clones were isolated by selecting for glutamine synthetase expression with methionine sulphoximine. The expression levels of ectodomain were subsequently increased by gene amplification. Production was scaled up using a 40-liter airlift fermenter in which the transfected CHO-K1 cells were cultured on microcarrier beads, initially in medium containing 10% fetal calf serum (FCS). By continuous perfusion of serum-free medium into the bioreactor, cell viability was maintained during reduction of FCS, which enabled soluble hIR ectodomain to be harvested for at least 22 days. Harvests were concentrated 20-fold by anion-exchange chromatography. Optimal recovery of ectodomain from early harvests containing large quantities of serum proteins was achieved by insulin-affinity chromatography, whereas in later harvests purification was achieved by multistep chromatography. Analysis of the purified hIR ectodomain showed that it had a molecular weight by sedimentation equilibrium analysis of 269,500. Amino-terminal amino acid sequence analysis showed that the ectodomain was correctly processed to alpha and beta chains and that glycosylation characteristics were similar to those of native hIR. The integrity of the ectodomain was demonstrated by the recognition of conformation-dependent anti-hIR antibodies and by its binding of insulin (Kd approximately 2 x 10(-9) M). These results demonstrate the successful production and purification of hIR ectodomain by processes amenable to scale-up and in a form appropriate for structure/function studies of the ligand-binding domain of the receptor.

Use of on-line gas analysis to monitor recombinant mammalian cell cultures.

The development of a system capable of accurately measuring the oxygen uptake and carbon dioxide production rates during mammalian cell cultures is described. A detailed study of the specifications of the various components used in the system for the measurement of gas flow rates and composition, coupled with the validation of the system independent of the bioreactor was carried out. The aim of this study was to identify and eliminate where possible the errors controlling the accuracy of determination of gaseous metabolic rates. This study showed the importance of controlling the temperature of gaseous oxygen entering the system. With such temperature control, it was possible to obtain data with an accuracy of +/- 5% at the 95% confidence level. Another source of error, the use of bi-carbonate buffer, was studied. A mathematical model was used to compensate for the affect of such buffers on the determination of carbon dioxide production rates. The use of the system for the continuous determination of gaseous metabolism during the growth and production phase for recombinant CHO cell cultures is described.