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).

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.

Sequence diversity in the surface-exposed amino-terminal region of the coat proteins of seven strains of sugarcane mosaic virus correlates with their host range.

The N-terminal region of the coat proteins of five strains (Isis, Brisbane, Sabi, Bundaberg, and BC) of sugarcane mosaic virus (SCMV) isolated from four different plant species (sugarcane, sabi grass, wild sorghum, and blue couch grass) have been compared with the previously published data for SCMV-SC and SCMV-MDB, isolated from sugarcane and maize, respectively. The region, beginning at residue 11 and ending 16 residues beyond the second trypsin cleavage site of the coat protein, varied in size from 68 amino acid residues (Bundaberg) to 115 residues (BC) and contained repeat sequence motifs. Comparisons of the sequence identity and the nature of the repeats in the seven sequences showed that there were five different sequence patterns. These could be grouped further into three subsets which appeared to correlate with the host range of the strains. SCMV-Brisbane, SC, and Isis, isolated from sugarcane, showed almost identical sequence patterns and formed one subset. The other four strains had different sequence patterns and could be grouped further into a Sabi and Bundaberg subset (isolated from sabi grass), and a BC and MDB subset.

The isolation, characterization and cloning of a globin-like, host-protective antigen from the excretory-secretory products of Trichostrongylus colubriformis.

An 18-kDa component from the excretory-secretory (ES) products of adults of Trichostrongylus colubriformis was isolated and characterized, and was shown to induce 60-84% protection of guinea pigs from challenge infection following a single intraperitoneal injection. Amino-terminal sequence analysis of gel-purified protein enabled oligonucleotides to be synthesized and used to screen a lambda gt10 cDNA library made from young adult worm mRNA, and to synthesize full-length clones from cDNA using the polymerase chain reaction (PCR). The full-length clones coded for a 20-kDa precursor protein of 173 amino acids which had a strongly hydrophobic leader sequence of 15 residues. The mature protein sequence of 158 amino acid residues was rich in charged amino acids (32%), including 8 oppositely charged pairs of amino acids. The protein sequence contained no half-cystine residues and no potential N-glycosylation sites. Unlike 2 other fully characterized ES components which are expressed only in the parasitic stages, mRNA coding for the 20-kDa component was present in both the parasitic and free-living stages of T. colubriformis. The parasite protein had approximately 20% identity with globins from human and from the larvae of the insect Chironomus thummi thummi. The homology included the invariant distal histidine and phenylalanine, and a number of other residues highly conserved in globins.

Characterization, cloning and host-protective activity of a 30-kilodalton glycoprotein secreted by the parasitic stages of Trichostrongylus colubriformis.

The helminth Trichostrongylus colubriformis is a parasitic nematode infecting the small intestine of sheep. We report the isolation and characterization of a 30-kDa glycoprotein capable of partially protecting guinea-pigs against the parasite. This glycoprotein is secreted by the L4 and adult parasitic stages of the worm. The sequence of three separate cDNA clones predicts the polypeptide to be about 15 kDa, with four N-linked carbohydrate chains and an internal disulphide bond. The clones also indicate the existence of sequence variability in this antigen. Limited sequence homology to a porcine intestinal peptide suggests an influence on host gut physiology.

The use of 3' non-coding nucleotide sequences in the taxonomy of potyviruses: application to watermelon mosaic virus 2 and soybean mosaic virus-N.

The sequence of the 3' 1106 nucleotides of the watermelon mosaic virus 2 (WMV 2) genome has been determined. The sequence contains the complete coding region of the viral coat protein followed by a 3' untranslated sequence of 251 nucleotides. When these sequences were compared with the equivalent regions of the N strain of soybean mosaic virus (SMV-N), the coat protein coding regions were 82% homologous, whereas the 3' untranslated sequences were 78% homologous. Optimal alignment of the 3' untranslated regions of RNA from 13 strains of seven other distinct potyviruses revealed that the degree of homology between strains was in the range 83 to 99%. In contrast, the sequences from distinct viruses had identities in the range 39 to 53%, comparable to the level of identity found between the 3' non-coding regions of viruses from unrelated plant virus groups. On the basis of these results, WMV 2 and SMV-N could be regarded as strains of one virus. These results also suggest that the sequence of the 3' untranslated region of the potyvirus genome may be an accurate marker of genetic relatedness and could serve as an aid to identification and classification of potyviruses.

Coat protein of potyviruses. 6. Amino acid sequences suggest watermelon mosaic virus 2 and soybean mosaic virus-N are strains of the same potyvirus.

The amino acid sequence of the coat protein of watermelon mosaic virus 2 (WMV 2) was determined by a combination of peptide and nucleic acid sequencing. The coat protein of WMV 2 contained 281 amino acid residues including a single cysteine at position 132 and a blocked amino terminus. Comparison with the coat protein sequences of 20 strains of ten distinct potyviruses showed sequence homologies ranging from 43% to 69% except for the N strain of soybean mosaic virus (SMV-N), where the sequence homology with WMV 2 was 83%. This degree of homology and the location of sequence differences between WMV 2 and SMV-N is much closer to that observed between strains of the same virus than that found between distinct potyviruses. These data suggest that WMV 2 and SMV-N may be strains of the same virus.

The proteins of the keratin component of bird's beaks.

Birds' beaks have an outer shell of hard keratin which consists almost entirely of proteins which are very rich in glycine [about 30 residues per 100 residues (residues %)], contain moderate levels of tyrosine and serine (each about 8 residues %), and which have relatively low contents of cystine (about 2-5 residues %), lysine, histidine, isoleucine and methionine. Major protein fractions in the S-carboxymethyl form isolated from the beaks of six different orders of birds have similar amino acid compositions, isoelectric points (pH 4-2-4-9) and molecular weights (13,000-14,500). Detailed chromatographic electrophoretic and compositional studies of the proteins of kookaburra beak reveal them to be a family of closely related proteins with only limited heterogeneity, in contrast to mammalian keratin systems. The major kookaburra beak fraction is similar in overall composition and molecular weight to fowl epidermal scale, kookaburra claw and turtle scute proteins and shows some resemblance to reptile claw protein. Beaks also contain small amounts of protein which are distinctly different from the major fraction but which resemble feather keratin proteins in composition and size.