The relationship between the L1 and L2 domains of the insulin and epidermal growth factor receptors and leucine-rich repeat modules.

BACKGROUND: Leucine-rich repeats are one of the more common modules found in proteins. The leucine-rich repeat consensus motif is LxxLxLxxNxLxxLxxLxxLxx- where the first 11-12 residues are highly conserved and the remainder of the repeat can vary in size Leucine-rich repeat proteins have been subdivided into seven subfamilies, none of which include members of the epidermal growth factor receptor or insulin receptor families despite the similarity between the 3D structure of the L domains of the type I insulin-like growth factor receptor and some leucine-rich repeat proteins. RESULTS: Here we have used profile searches and multiple sequence alignments to identify the repeat motif Ixx-LxIxx-Nx-Lxx-Lxx-Lxx-Lxx- in the L1 and L2 domains of the insulin receptor and epidermal growth factor receptors. These analyses were aided by reference to the known three dimensional structures of the insulin-like growth factor type I receptor L domains and two members of the leucine rich repeat family, porcine ribonuclease inhibitor and internalin 1B. Pectate lyase, another beta helix protein, can also be seen to contain the sequence motif and much of the structural features characteristic of leucine-rich repeat proteins, despite the existence of major insertions in some of its repeats. CONCLUSION: Multiple sequence alignments and comparisons of the 3D structures has shown that right-handed beta helix proteins such as pectate lyase and the L domains of members of the insulin receptor and epidermal growth factor receptor families, are members of the leucine-rich repeat superfamily.

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.

Structure and function of the type 1 insulin-like growth factor receptor.

The type 1 insulin-like growth factor receptor (IGF-1R), a transmembrane tyrosine kinase, is widely expressed across many cell types in foetal and postnatal tissues. Activation of the receptor following binding of the secreted growth factor ligands IGF-1 and IGF-2 elicits a repertoire of cellular responses including proliferation, and the protection of cells from programmed cell death or apoptosis. As a result, signalling through the IGF-1R is the principal pathway responsible for somatic growth in foetal mammals, whereas somatic growth in postnatal animals is achieved through the synergistic interaction of growth hormone and the IGFs. Forced overexpression of the IGF-1R results in the malignant transformation of cultured cells: conversely, downregulation of IGF-1R levels can reverse the transformed phenotype of tumour cells, and may render them sensitive to apoptosis in vivo. Elevated levels of IGF-IR are observed in a variety of human tumour types, whereas epidemiological studies implicate the IGF-1 axis as a predisposing factor in the pathogenesis of human breast and prostate cancer. The IGF-1R has thus emerged as a therapeutic target for the development of antitumour agents. Recent progress towards the elucidation of the three-dimensional structure of the extracellular domain of the IGF-1R represents an opportunity for the rational assembly of small molecule antagonists of receptor function for clinical use.

Properties of an insulin receptor with an IGF-1 receptor loop exchange in the cysteine-rich region.

The insulin receptor (IR) and the insulin-like growth factor-I receptor (IGF-1R) show differential binding of insulin and IGFs. The specificity determinants for IGF-1 binding are known to be located in the cysteine-rich (Cys-rich) region between residues 223 and 274 of human IGF-1R, which includes a loop that protrudes into the putative ligand binding site. In this report we have replaced residues 260-277 of human IR with residues 253-266 of the human IGF-1R to produce an IR-based, cysteine loop exchange chimaera, termed hIR-Cys loop exchange (CLX), in which all 14 amino acid residues in the exchanged loop differ from wild-type insulin receptor. This loop exchange had a detrimental effect on the efficiency of pro-receptor processing and on the binding of the mouse monoclonal antibody 83-7. However, this antibody, which binds hIR but not hIGF-1R, was still capable of immunoprecipitating the mature chimaeric receptor, indicating that the conformational epitope recognised by this antibody is not primarily determined by the loop region exchanged. The loop exchange did not significantly affect the ability of insulin to displace bound radiolabelled insulin, but increased the capacity of IGF-1 to competitively displace labelled insulin by at least 10 fold.

Characterization of a comparative model of the extracellular domain of the epidermal growth factor receptor.

The Epidermal Growth Factor (EGF) receptor is a tyrosine kinase that mediates the biological effects of ligands such as EGF and transforming growth factor alpha. An understanding of the molecular basis of its action has been hindered by a lack of structural and mutational data on the receptor. We have constructed comparative models of the four extracellular domains of the EGF receptor that are based on the structure of the first three domains of the insulin-like growth factor-1 (IGF-1) receptor. The first and third domains of the EGF receptor, L1 and L2, are right-handed beta helices. The second and fourth domains of the EGF receptor, S1 and S2, consist of the modules held together by disulfide bonds, which, except for the first module of the S1 domain, form rod-like structures. The arrangement of the L1 and S1 domains of the model are similar to that of the first two domains of the IGF-1 receptor, whereas that of the L2 and S2 domains appear to be significantly different. Using the EGF receptor model and limited information from the literature, we have proposed a number of regions that may be involved in the functioning of the receptor. In particular, the faces containing the large beta sheets in the L1 and L2 domains have been suggested to be involved with ligand binding of EGF to its receptor.

Congenital insulin resistance associated with a conformational alteration in a conserved beta-sheet in the insulin receptor L1 domain.

The hormone binding site of members of the insulin receptor family is contained within a highly conserved extracellular region of the receptor. Recent crystallization of the N-terminal region of the binding site revealed two large domains (L1, L2), each organized as a single-stranded right-handed beta-helix, connected by a rod-shaped cysteine-rich domain. Here, we analyze two new naturally occurring mutations in a single beta-sheet within L1, D59G and L62P, that we previously identified in a young woman with classic congenital insulin resistance (type A). Substitution of D59G, a beta-sheet connecting loop residue, caused decreased hormone binding but did not disrupt overall folding, assembly, or movement to the cell surface. In contrast, replacement of the adjacent residue L62P, which is located within the beta-sheet, and positioned in a hormone binding surface, completely disrupted intracellular folding, oligomerization, and trafficking and resulted in aberrant proteolytic degradation. Immunohistochemistry in combination with biosynthetic studies showed that misfolded receptors were retained in an incorrect cellular location and that they colocalized with the resident endoplasmic reticulum chaperone calnexin. This study, together with other mutagenesis data, shows that formation of beta-sheet elements within the L1 beta-helix are critical for the folding of the entire extracellular domain of the receptor and that the hormone contact site is composed in part by residues in this domain.

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.

Functional aspects of skeletal muscle contractile apparatus and sarcoplasmic reticulum after fatigue.

This study examined the effects of fatigue on the functional aspects of the contractile apparatus and sarcoplasmic reticulum (SR). Frog semitendinosus muscles were stimulated to fatigue, and skinned fibers or a homogenate fraction was prepared from both fatigued and rested contralateral muscles. In fatigued fibers, maximal Ca2+-activated force of the contractile apparatus was unaltered, whereas maximal actomyosin-ATPase activity was depressed by 20%. The Ca2+ sensitivity of force was increased, whereas that of actomyosin-ATPase was not altered. Also, the rate constant for tension redevelopment was decreased at submaximal Ca2+ concentration. These latter findings suggest that fatigue slows the dissociation of force-generating myosin cross bridges. Ca2+ uptake and Ca2+-ATPase activity of the SR were depressed by 46 and 21%, respectively, in the fatigued muscles. Fatigue also reduced the rates of SR Ca2+ release evoked by AgNO3 and 4-chloro-m-cresol by 38 and 45%, respectively. During fatigue, the contractile apparatus and SR undergo intrinsic functional alterations. These changes likely result in altered force production and energy consumption by the intact muscle.

Effects of varied fatigue protocols on sarcoplasmic reticulum calcium uptake and release rates.

The purpose of this investigation was to examine changes in sarcoplasmic reticulum (SR) function in muscles subjected to different patterns of muscle activity. Frog sartorius muscles were stimulated with tetanic trains (100 ms, 100 Hz) delivered at rates of 2.0, 0.5, and 0.2 trains/s. In one set of experiments, stimulation was continued until force had declined to approximately 17% of initial (constant fatigue), whereas in the other set, stimulation was continued for 1 min (constant duration). In the constant-fatigue experiments, Ca2+ uptake (1 mM MgATP) and release rates (25 microM AgNO3, 5 mM 4-chloro-m-cresol) were depressed by similar extents following each protocol. This occurred despite 1, 4, and 17 min of stimulation, respectively, used to induce fatigue. In the constant-duration experiments, larger reductions in SR function occurred following the highest frequency stimulation protocol. These data suggest that when muscles are fatigued to similar extents, depressions in SR function are independent of the activity protocol. On the other hand, when a constant duration of activity is imposed, changes in SR function are closely linked to the extent of force reduction.

Glucose 6-phosphate alters rat skeletal muscle contractile apparatus and sarcoplasmic reticulum function.

We investigated the effects of glucose 6-phosphate (G6P) on skeletal muscle contractile apparatus and sarcoplasmic reticulum (SR) function. Using rat extensor digitorum longus fibres, the presence of 5 mM G6P decreased the Ca2+ sensitivity of both force production and actomyosin ATPase (AM-ATPase) activity. Conversely, maximal Ca(2+)-activated force was unaffected while maximal AM-ATPase activity was increased by 37%. In SR vesicles isolated from rat gastrocnemius, G6P markedly altered Ca2+ handling. It increased Ca(2+)-stimulated Ca(2+)-ATPase activity but depressed the net rate of Ca2+ uptake. This latter effect appears to be due to G6P-stimulated Ca2+ release. When G6P was added to Ca(2+)-loaded vesicles, a small, transient release of Ca2+ was elicited. In addition, G6P lowered the threshold for Ca(2+)-induced Ca2+ release but depressed the net rates of both AgNO3- and caffeine-induced releases. It is possible that the accumulation of G6P during muscular activity may adversely affect muscle force production and contribute to the fatigue process via its action on the contractile apparatus and SR.

Changes in skeletal muscle sarcoplasmic reticulum function and force production following myocardial infarction in rats.

In patients following myocardial infarction (MI), exercise tolerance and muscular strength are often markedly reduced. The purpose of this investigation was to determine if calcium (Ca2+) uptake and release by skeletal muscle sarcoplasmic reticulum (SR) are altered following MI and if such changes are associated with diminished muscular performance. SR vesicles were isolated from rat gastrocnemius muscle at 6 and 12 weeks following MI (via left coronary artery ligation) or sham surgery. At both post-surgery intervals, the rates of Ca2+ uptake (measured using fura-2) were 35% greater in the MI group. In addition, the rates of Ca2+ release were increased in the MI group by 10 and 30% at 6 and 12 weeks, respectively. At 12 weeks post-surgery, animals of the MI group showed significant reduction in in situ twitch and tetanic forces and significant elevations in the rates of tension increase and decrease. These data indicate that SR Ca2+ exchange is altered following MI. In addition, changes in SR function are associated with changes in force production by the whole muscle.

The disulfide bonds in the C-terminal domains of the human insulin receptor ectodomain.

The human insulin receptor is a homodimer consisting of two monomers linked by disulfide bonds. Each monomer comprises an alpha-chain that is entirely extracellular and a beta-chain that spans the cell membrane. The alpha-chain has a total of 37 cysteine residues, most of which form intrachain disulfide bonds, whereas the beta-chain contains 10 cysteine residues, four of which are in the extracellular region. There are two classes of disulfide bonds in the insulin receptor, those that can be reduced under mild reducing conditions to give alpha-beta monomers (class I) and those that require stronger reducing conditions (class II). The number of class I disulfides is small and includes the alpha-alpha dimer bond Cys524. In this report we describe the use of cyanogen bromide and protease digestion of the exon 11 plus form of the receptor ectodomain to identify disulfide linkages between the beta-chain residues Cys798 and Cys807 and between the alpha-chain Cys647 and the beta-chain Cys872. The latter bond is the sole alpha-beta link in the molecule and implies a side-by-side alignment of the two fibronectin III domains of the receptor. Also presented is evidence for additional alpha-alpha dimer bond(s) involving at least one of the cysteine residues of the triplet at positions 682, 683, and 685. Evidence is also presented to show that Cys884 exists as a buried thiol in the soluble ectodomain.

Novel isoform of syntaxin 1 is expressed in mammalian cells.

Syntaxin 1A has been identified previously as a neural-cell-specific, membrane-anchored receptor protein required for docking and fusion of synaptic vesicles with the presynaptic plasma membrane. Syntaxin 1A consists of 288 amino acid residues including a 265-residue N-terminal region exposed to the cytoplasm and a C-terminal hydrophobic stretch of 23 residues believed to anchor syntaxin to the plasma membrane. Using a human fat-cell library we have isolated a novel cDNA clone of syntaxin 1A containing an insert of 91 bp in codon 226. This insert and subsequent frame shift generated a cDNA that codes for a truncated protein of 260 residues without the C-terminal transmembrane domain characteristic of the syntaxin family. Analysis of the deduced amino acid sequence of the new cDNA clone, termed syntaxin 1C, showed that it was identical for the first 226 residues with the previously described neural syntaxin 1A, and diverged thereafter. The truncated protein lacked the botulinum neurotoxin C cleavage site (Lys253-Ala254), a feature of the syntaxin 1A protein, because of the novel C-terminal domain of 34 residues. The new C-terminal region contained a single cysteine residue and was moderately rich in proline, with three repeats of a PXP motif. The insert occurred within the region encoding the coiled-coil motifs required for interactions with synaptobrevin, alpha-SNAP (SNAP being soluble N-ethylmaleimide-sensitive factor attachment protein) and n-Sec1/Munc-18 (n-Sec1 being the rat brain homologue of yeast Sec1p and Munc-18 the mammalian homologue of Caenorhabditis elegans unc-18, but five residues outside the domain previously mapped as being required for binding SNAP-25. Interaction studies in vitro suggested that unlike syntaxin 1A, which binds to both Munc-18a and- 18b, syntaxin 1C binds only to Munc-18b. The new isoform syntaxin 1C, which might be generated by alternative splicing of the syntaxin 1 gene, was expressed in several human tissues, including brain. Immuno-precipitation and immunoblotting with the monoclonal antibody HPC-1 and a polyclonal antibody raised against a peptide corresponding to the unique C-terminal 35 residues of syntaxin 1C failed to detect syntaxin 1C at the protein level in extracts of muscle, fat or brain.

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).

Decreased contraction economy in mouse EDL muscle injured by eccentric contractions.

The objective of this study was to find out whether basal and/or active energy metabolism are altered in isolated mouse extensor digitorum longus muscle injured by eccentric (Ecc) contractions. Measurements of basal O2 consumption and isometric tetanus O2 recovery cost were made at 25 degrees C on muscles that had done either 10 Ecc, 10 isometric (Iso), or no contractions (No). In parallel experiments, rates of lactate and pyruvate production were measured to estimate the anaerobic contribution. Basal O2 consumption was unaffected by the type of protocol performed (P = 0.07). However, the tetanus O2 cost per force-time integral was elevated by 30-36% for the Ecc protocol muscles over that for the Iso and No protocol muscles. When including the increased lactate production by the Ecc protocol muscles, the total energetic cost per force-time integral was 53% higher than that for the Iso protocol muscles [2.35 +/- 0.17 vs. 1.54 +/- 0.18 mumol O2/(N.m.s)]. The decreased economy was attributed to two factors. First, in skinned fibers isolated from the injured muscles, the ratio of maximal actomyosin adenosinetriphosphatase activity to force production was up by 37.5%, suggesting uncoupling of ATP hydrolysis from force production. Second, increased reliance on anaerobic metabolism along with the fluorescent microscopic study of mitochondrial membrane potential and histochemical study of ATP synthase suggested an uncoupling of oxidative phosphorylation in the injured muscles.

Systematic mapping of potential binding sites for Shc and Grb2 SH2 domains on insulin receptor substrate-1 and the receptors for insulin, epidermal growth factor, platelet-derived growth factor, and fibroblast growth factor.

Multipin peptide synthesis has been employed to produce biotinylated 11-mer phosphopeptides that account for every tyrosine residue in insulin receptor substrate-1 (IRS-1) and the cytoplasmic domains of the insulin-, epidermal growth factor-, platelet-derived growth factor- and basic fibroblast growth factor receptors. These phosphopeptides have been screened for their capacity to bind to the SH2 domains of Shc and Grb in a solution phase enzyme-linked immunosorbent assay. The data revealed new potential Grb2 binding sites at Tyr-1114 (epidermal growth factor receptor (EGFR) C-tail); Tyr-743 (platelet-derived growth factor receptor (PDGFR) insert region), Tyr-1110 from the E-helix of the catalytic domain of insulin receptor (IR), and Tyr-47, Tyr-939, and Tyr-727 in IRS-1. None of the phosphopeptides from the juxtamembrane or C-tail regions of IR bound Grb2 significantly, and only one phosphopeptide from the basic fibroblast growth factor receptor (Tyr-556) bound Grb2 but with medium strength. Tyr-1068 and -1086 from the C-tail of EGFR, Tyr-684 from the kinase insert region of PDGFR, and Tyr-895 from IRS-1 were confirmed as major binding sites for the Grb2 SH2 domain. With regard to Shc binding, the data revealed new potential binding sites at Tyr-703 and Tyr-789 from the catalytic domain of EGFR and at Tyr-557 in the juxtamembrane region of PDGFR. It also identified new potential Shc binding sites at Tyr-764, in the C-tail of basic fibroblast growth factor receptor, and Tyr-960, in the juxtamembrane of IR, a residue previously known to be required for Shc phosphorylation in response to insulin. The study confirmed the previous identification of Tyr-992 and Tyr-1173 in the C-tail of EGFR and several phosphopeptides from the PDGFR as medium strength binding sites for the SH2 domain of Shc. None of the 34 phosphopeptides from IRS-1 bound Shc strongly, although Tyr-690 showed medium strength binding. The specificity characteristics of the SH2 domains of Grb2 and Shc are discussed. This systematic peptide mapping strategy provides a way of rapidly scanning candidate proteins for potential SH2 binding sites as a first step to establishing their involvement in kinase-mediated signaling pathways.

Insulin and epidermal growth factor receptors contain the cysteine repeat motif found in the tumor necrosis factor receptor.

The insulin receptor (INSR) and epidermal growth factor receptor (EGFR) are representatives of two structurally related subfamilies of tyrosine kinase receptors. Using the Wisconsin GCG sequence analysis programs, we have demonstrated that the cysteine-rich regions of INSR and EGFR conform to the structural motif found in the tumor necrosis factor receptor (TNFR) family. The study also revealed that these regions were not composed of simple repeats of eight cysteine residues as previously proposed and that the second Cys-rich region of EGFR contained one fewer TNFR repeat than the first. The sequence alignments identified two cysteine residues in INSR that could be responsible for the additional disulfide bonds known to be involved in dimer formation. The published data on the alignments for the fibronectin type III repeat region of the INSR together with previous cysteine mutagenesis studies indicated that there were two disulfide bonds linking the alpha and beta chains of the INSR, but only one alpha-beta linkage in the insulin-like growth factor 1 receptor (IG1R). Database searches and sequence alignments showed that the TNFR motif is also found in the cysteine-rich repeats of laminins and the noncatalytic domains of furin-like proteases. If the starting position of the repeat is altered the characteristic laminin repeat of eight cysteine residues can be shown to consist of a TNFR-like motif fused to the last half of an EGF-like repeat. The overlapping regions of these two motifs are known to have identical disulfide bonding patterns and similar protein folds.

Mutagenic structure/function analysis of the cytoplasmic cysteines of the insulin receptor.

Native human insulin receptor (hIR) has been reported to contain only one free thiol group proposed to lie near the ATP-binding. domain of its beta-subunit [Finn, Ridge and Hofmann (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 419-423]. The present study investigated the role of the six cytoplasmic cysteines of the beta-subunit of the hIR using a mutagenic approach in which insulin receptors, mutated at each cytoplasmic cysteine (to alanine) in turn, were transfected into Chinese hamster ovary (CHO) cells. Cell lines expressing hIR mutation at high level were obtained which, by both flow-cytometric analysis towards an hIR-specific monoclonal antibody (83-7) and insulin-binding analysis, were similar to the well-characterized CHOT cell line which overexpresses native hIR. The ED50 and Kd values of the mutant receptors were the same as those of the wild-type hIR. Each of the mutant receptors signalled insulin action to stimulate receptor autophosphorylation and kinase activity as well as glucose utilization to levels appropriate for the receptor level expressed. In contrast, insulin-stimulated thymidine uptake and glucose-transport responses of two of the six mutant cell lines, those expressing Cys981Ala and Cys1245Ala, were impaired compared with that of the native hIR-expressing cell line, CHOT. The beta-subunits of each of the hIR cytoplasmic cysteine mutant cell lines could be alkylated specifically with N-[3H]ethylmaleimide. The kinase activity of each receptor was inhibited by N-ethylmaleimide and stimulated by iodoacetamide, indicating that none of the cytoplasmic cysteines alone contributes the single free thiol group to the hIR structure. We conclude that the cytoplasmic cysteines of the hIR have a predominantly passive role in hIR activity although Cys-981 and Cys-1245 do affect mitogenic and glucose-transport responses of the receptor. Our findings indicate that the stoicheiometry of a single free thiol group/mol of insulin-binding activity noted in previous studies is either spread fractionally over a number of the cytoplasmic cysteines or is one of the four cysteines in the ectodomain of the hIR beta-subunit. Alternatively, the mutagenesis performed in the present study may enable differential exposure of a second titratable cysteine in wild-type and mutant receptors.

Evaluation of complete genome sequences and sequences of individual gene products for the classification of hepatitis C viruses.

Comparisons of genome and polyprotein sequences of hepatitis C virus (HCV) isolates world-wide has led to the identification of nine major genotypes and many subtypes. This classification is based on either complete genome/polyprotein sequences or sequence data from the 5' noncoding region, core, E1, NS3 or NS5B genes. The relative merit of different gene segments as taxonomic markers and the validity of the resulting assignments is not clear at this stage. To resolve the taxonomy of HCV genotypes and subtypes, we have compared the complete genome and polyprotein sequences of 19 HCV isolates available in the databases as well as sequences of individual genes and gene products of these isolates. Based on the correlation between sequence relationships and taxonomic assignments of other RNA viruses, we show that the nine major genotypes of HCV represent nine distinct virus species and their subtypes subspecies. Our sequence comparison of the 5' noncoding regions and the individual gene products suggests that E2, NS2, NS5B, E1, NS4A, NS4B and NS5A (in that order) are the most appropriate regions for the discrimination between species, subspecies and strains of HCV. The 5' noncoding, core and NS3 regions are less effective in distinguishing between species, subspecies and strains. Based on a comparison of the polymerase sequence identities of HCVs, pestiviruses and flaviviruses as well as the recent information on the size and morphology of HCV virions, we propose that HCVs, pestiviruses and flaviviruses should be classified into three separate families, named Hepciviridae, Pestiviridae and Flaviviridae, respectively rather than three genera of the Flaviviridae as currently classified. We also propose "Hepcivirus" as the genus name for HCVs.

Cysteine-524 is not the only residue involved in the formation of disulphide-bonded dimers of the insulin receptor.

The human insulin receptor (hIR) is a member of the transmembrane tyrosine kinase receptor family. It is a disulphide-linked homodimer which can be reduced to two insulin-binding monomers by mild reduction of class-I disulphide bonds. The number of disulphide bonds between the alpha- and beta-chains within the monomer or between the monomers in the dimer is not known, although one dimer bond involving hIR Cys-524 has recently been identified [Schaffer and Ljungqvist (1992) Biochem. Biophys. Res. Commun. 189, 650-653]. In the present report hIR Cys-524 was converted into alanine by site-directed mutagenesis and expressed at high levels in Chinese hamster ovary (CHO) cells. The mutant receptor was processed normally and shown to bind insulin normally, with ED50 and KD values not different from those of the wild-type hIR. It was still a disulphide-linked dimer as judged by SDS/PAGE, indicating that there are alpha-alpha-chain disulphide bonds additional to the Cys-524 linkage in the insulin receptor dimer. Insulin-stimulated receptor autophosphorylation and kinase activity of the mutated receptor were both impaired compared with that of the wild-type receptor by 49% and 53% respectively. CHO cells overexpressing the mutant receptor, however, did not show a reduced capacity to stimulate glucose utilization, indicative that the level of receptor expression was sufficient to saturate downstream insulin action. These findings indicate that alpha-alpha disulphides additional to that provided by Cys-524 hold the receptor dimer together and that mutagenesis of Cys-524 reduces the ability of the receptor to signal insulin action subsequent to hormone binding.