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.

Mutational analysis of the N-linked glycosylation sites of the human insulin receptor.

Site-directed mutagenesis has been used to remove 15 of the 18 potential N-linked glycosylation sites, in 16 combinations, from the human exon 11-minus receptor isoform. The three glycosylation sites not mutated were asparagine residues 25, 397 and 894, which are known to be important in receptor biosynthesis or function. The effects of these mutations on proreceptor processing into alpha and beta subunits, cell-surface expression, insulin binding and receptor autophosphorylation were assessed in Chinese hamster ovary cells. The double mutants 16+78, 16+111, 16+215, 16+255, 337+418, the triple mutants 295+337+418, 295+418+514, 337+418+514 and 730+743+881 and the quadruple mutants 606+730+743+881 and 671+730+743+881 seemed normal by all criteria examined. The triple mutant 16+215+255 showed only low levels of correctly processed receptor on the cell surface, this processed receptor being autophosphorylated in response to insulin. The quadruple mutant 624+730+743+881 showed normal processing and ligand binding but exhibited a constitutively active tyrosine kinase as judged by autophosphorylation. Three higher-order mutants were constructed, two of which, 16+337+418+730+743+881 (Delta6) and 16+295+337+418+730+743+881 (Delta7a), seemed normal. The third construct, 16+337+418+514+730+743+881 (Delta7b), was expressed at high levels on the cell surface, essentially as uncleaved proreceptor with only the small proportion of Delta7b that was correctly processed showing insulin-stimulated autophosphorylation. The mutations of Delta6 and Delta7a were incorporated into soluble ectodomains, which had affinities for insulin that were 4-fold that of wild-type ectodomain. The Delta6 ectodomain expressed in Lec8 cells was produced in quantity in a bioreactor for subsequent structural analysis.

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).

Insulin-responsive tissues contain the core complex protein SNAP-25 (synaptosomal-associated protein 25) A and B isoforms in addition to syntaxin 4 and synaptobrevins 1 and 2.

SNAP-25 (synaptosomal-associated protein 25), syntaxin and synaptobrevin are the three SNARE [soluble NSF attachment protein receptor (where NSF = N-ethylmaleimide-sensitive fusion protein)] proteins that form the core complex involved in synaptic vesicle docking and subsequent fusion with the target membrane. The present study is aimed at understanding the mechanisms of fusion of vesicles carrying glucose transporter proteins with the plasma membrane in human insulin-responsive tissues. It describes the isolation and characterization of cDNA molecules encoding SNAP-25 A and B isoforms, syntaxin 4 and synaptobrevins (also known as vehicle-associated membrane proteins) from two major human insulin-responsive tissues, skeletal muscle and fat. The DNA and deduced amino acid sequences of SNAP-25 revealed perfect identity with the previously reported human neural SNAP-25 A and B isoforms. Our results indicate the presence of both isoforms both in insulin-responsive tissues and in in vitro cultured 3T3-L1 cells, but suggest a differential pattern of gene expression: isoform A is the major species in adipose tissue, and isoform B is the major species in skeletal muscle. The presence of SNAP-25 protein in 3T3-L1 cells was demonstrated by immunofluorescence microscopy using an anti-SNAP-25 monoclonal antibody. Immunoprecipitation experiments using the same monoclonal antibody also revealed the presence of SNAP-25 protein in plasma membrane fractions from rat epididymal fat pads. The syntaxin 4-encoding region from skeletal muscle contains five nucleotide differences from the previously reported placental cDNA sequence, two of which result in amino acid changes: Asp-174 to Glu and Val-269 to Ala. The synaptobrevin 1 cDNA from skeletal muscle contains two nucleotide differences when compared with the corresponding clone from neural tissues, one of which is silent and the other resulting in the amino acid change Thr-102 to Ala. The cDNA sequence of the protein from fat is identical with that of human synaptobrevin 1 from neural tissues. Furthermore, we have confirmed the presence of syntaxin 4 in fat and of synaptobrevin 2 in skeletal muscle by PCR amplification and Southern hybridization analysis. Using the yeast two-hybrid system, an interaction was observed between the full-length cytoplasmic domains of syntaxin 4 and synaptobrevin 2, a vesicle membrane SNARE previously shown by others to be associated with vesicles carrying the GLUT4 glucose transporter protein, but no interaction was seen with synaptobrevin 1. Flow cytometry of low-density microsomes isolated from fat cells was used to demonstrate the binding of syntaxin 4 to a subset of vesicles carrying GLUT4 protein; whereas SNAP-25 on its own bound poorly to these vesicles, the syntaxin 4-SNAP-25 complex gave a strong interaction.

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.

Nucleotide sequence of the 3'-terminal region of the genome confirms that pea mosaic virus is a strain of bean yellow mosaic potyvirus.

The 1,035 nucleotides at the 3'end of the I strain of pea mosaic potyvirus (PMV-I) genomic RNA, encoding the coat protein, have been cloned and sequenced. A comparison of the derived coat protein sequence with those of the bean yellow mosaic virus (BYMV) strains, CS, S, D and GDD, indicates that PMV-I is a strain of BYMV. Sequence comparisons and hybridisation studies using the 3'-noncoding region support this classification. The nucleotide and protein sequence data also suggest that PMV-I and BYMV-CS form one subset of BYMV strains while the other three strains form another.

Characterization of the mRNA encoding a proline-rich 37-kilodalton glycoprotein from the excretory-secretory products of Trichostrongylus colubriformis.

A glycoprotein, with apparent molecular weight in SDS-polyacrylamide gels of 37 kDa, has been isolated from the excretory-secretory (ES) products of the adult stage of Trichostrongylus colubriformis, a parasitic nematode. This protein is the major ES product recognized in immunoblots by lymph from a naturally infected sheep. A synthetic oligonucleotide, based on peptide sequence data from a digest of the purified protein was used to successfully screen a cDNA library. A cDNA clone was isolated which encoded a presumptive protein precursor of 220 amino acids that contained a 63 amino acid region of which more than 35% of the residues were proline, three peptide sequences determined from the natural component, and three potential N-glycosylation sites, consistent with the protein being isolated from the lectin-bound fraction of the adult ES products. The presumptive, processed, amino terminus encoded by the cDNA clone was preceded by a signal-like, hydrophobic-rich region of 16 amino acids.

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.

A polymerase chain reaction method adapted for selective amplification and cloning of 3' sequences of potyviral genomes: application to dasheen mosaic virus.

'Universal' degenerate oligonucleotide primers were used to amplify cDNA sequences containing the 3' untranslated region (3' UTR) and a portion of the coat protein gene sequence of dasheen mosaic potyvirus (DMV). These primers were based on the conserved WCIEN and QMKAAA 'boxes' of the potyviral coat protein and the poly-A tail found at the 3' end of the genome. The forward genome-sense primers were designed taking into consideration the codon degeneracy of the WCIEN and QMKAAA residues for several potyviruses. The anti-sense reverse primer has 21 T residues followed by either A, C or G at the 3' end to ensure specific priming at the end of the 3' UTR and beginning of the poly-A tail. The specificity of amplification was verified using the known potyviruses (watermelon mosaic 2 and soybean mosaic viruses). To demonstrate the applicability of this method, the 3' UTR of the unsequenced DMV was amplified, cloned and sequenced. Sequence comparisons with other potyviral 3' UTRs revealed DMV to be quite distinct: nucleotide sequence similarities of only 34% to 44% were found with sequenced viruses indicating no close affinities with any other potyvirus. The potyvirus 3' sequence amplification procedure is simple and rapid, is potentially useful in developing virus specific probes and may be used to differentiate strains and species of potyviruses on the basis of the 3' UTR sequences.

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.

Differentiation of potyviruses and their strains by hybridization with the 3' non-coding region of the viral genome.

Nucleic acid hybridization with the 3' non-coding region of the potyvirus genome as the probe was shown to be a relatively simple means of distinguishing between distinct potyviruses and their strains. Comparisons of the nucleotide sequences of potyvirus genomes (ignoring gaps) showed that the degree of identity between equivalent genes of strains was greater than 96%, while between distinct potyviruses the identity ranged from 42% to 65%, suggesting that any extended sequence could be considered representative of the whole genome and be suitable as a diagnostic probe. The comparisons however, also revealed that some parts of the genome, but not the 3' non-coding region, had local regions of high sequence identity that could lead to cross-hybridization between distinct potyviruses. For this reason, and because its location immediately upstream of the poly(A) tail makes it the most accessible region for the purpose of cloning and sequencing, the 3' non-coding sequence should be most suitable for use as a diagnostic probe. Successful hybridizations (using radiolabeled, polymerase chain reaction-amplified 3' non-coding sequences) have been achieved by probing recombinant clones, purified potyviral RNA, partially purified total RNA from infected plants, and a crude extract of infected plant tissue. The method has been used to support the proposals that watermelon mosaic virus 2 and soybean mosaic virus-N are both strains of the same virus, and to discriminate between several isolates previously believed to be strains of sugarcane mosaic virus. The method should have wide application as a means of differentiating distinct potyviruses from strains.

Transient expression of the coat protein of sugarcane mosaic virus in sugarcane protoplasts and expression in Escherichia coli.

The coat protein (CP) of strain SC of sugarcane mosaic virus (SCMV-SC) was expressed transiently in sugarcane protoplasts after electroporation with one of two plasmids encoding the CP gene. The CP gene was fused with either the cauliflower mosaic virus 35S promoter or the synthetic monocotyledon promoter "Emu". The coat protein gene was also inducibly expressed in Escherichia coli when fused to the trc promoter. The protein expressed in both systems had the same electrophoretic mobility and antigenic specificity as purified SCMV-SC coat protein. Transient expression of the 35S-CP gene in protoplasts could only be demonstrated in Western blots developed with the chemiluminescence enzyme substrate luminol.

Sequence data as the major criterion for potyvirus classification.

Recent knowledge of the structure of the potyvirus particle and its components appears to have resolved what was thought to be an intractable problem of plant virology. This review describes how coat-protein and gene sequence data can be used to provide an hierarchical classification of potyviruses. This classification puts the aphid and non-aphid-transmitted potyviruses into a single family, divides this family into four genera that correspond to the four modes of vector transmission, discriminates distinct potyvirus species from strains, and provides a basis for the formation of subgroups composed of closely related species within a genus.

Present status of the sugarcane mosaic subgroup of potyviruses.

Until recently, sugarcane mosaic virus (SCMV) was believed to be a single potyvirus consisting of a large number of strains, differing from each other in certain biological and antigenic properties. The use of affinity-purified polyclonal antibodies directed towards the surface-located, virus-specific amino termini of the coat proteins showed that 17 strains from Australia and the United States represented four distinct potyviruses, namely johnsongrass mosaic virus (JGMV), maize dwarf mosaic virus (MDMV), sorghum mosaic virus (SrMV) and SCMV. Comparisons of strains from each of these four viruses on the basis of reactions on differential sorghum and oat cultivars, cell-free translation of RNAs, morphology and serology of cytoplasmic cylindrical inclusions, amino acid sequence and peptide profiling of coat proteins, 3' non-coding nucleotide sequences, and molecular hybridization with probes corresponding to the 3' non-coding regions, resulted in exactly the same taxonomic assignments as obtained using amino-terminal serology. These results further confirm that the former sugarcane mosaic virus actually consists of four distinct viruses and show that MDMV, SrMV, and SCMV are more closely related to each other than they are to JGMV. Because these four viruses are closely related but distinct, formation of a sugarcane mosaic subgroup in the genus Potyvirus would be appropriate.

Bean yellow mosaic, clover yellow vein, and pea mosaic are distinct potyviruses: evidence from coat protein gene sequences and molecular hybridization involving the 3' non-coding regions.

The sequences of the 3' 1019 nucleotides of the genome of an atypical strain of bean yellow mosaic virus (BYMV-S) and of the 3' 1018 nucleotides of the clover yellow vein virus (CYVV-B) genome have been determined. These sequences contain the complete coding region of the viral coat protein followed by a 3' non-coding region of 173 and 178 nucleotides for BYMV-S and CYVV-B, respectively. When the deduced amino acid sequences of the coat protein coding regions were compared, a sequence identity of 77% was found between the two viruses, and optimal alignment of the 3' untranslated regions of BYMV-S and CYVV-B gave a 65% identity. However, the degree of homology of the amino acid sequences of coat proteins of BYMV-S with the published sequences for three other strains of BYMV ranged from 88% to 94%, while the sequence homology of the 3' untranslated regions between the four strains of BYMV ranged between 86% and 95%. Amplified DNA probes corresponding to the 3' non-coding regions of BYMV-S and CYVV-B showed strong hybridization only with the strains of their respective viruses and not with strains of other potyviruses, including pea mosaic virus (PMV). The relatively low sequence identities between the BYMV-S and CYVV-B coat proteins and their 3' non-coding regions, together with the hybridization results, indicate that BYMV, CYVV, and PMV are distinct potyviruses.

Molecular characterisation of a protective, 11-kDa excretory-secretory protein from the parasitic stages of Trichostrongylus colubriformis.

An 11-kDa protein occurring as a major component of the non-glycosylated fraction of 4th larval stage (L4) and adult Trichostrongylus colubriformis excretory-secretory (ES) fluid has been found to be highly protective in guinea pigs, an alternate host for T. colubriformis. The protein has been purified, characterised and partly sequenced. With a reverse-complement oligonucleotide based on the carboxy-terminal sequence of the protein, recombinant lambda gt11 clones were detected in an L4 cDNA library. The DNA sequence from one clone has a single extended open reading frame coding for a highly charged 11-kDa protein which lacks a leader sequence and contains a potential N-glycosylation site. Expression of the cloned DNA in Escherichia coli was detected with an antibody, raised in rabbits against gel-purified 11-kDa protein.

Unexpected sequence diversity in the amino-terminal ends of the coat proteins of strains of sugarcane mosaic virus.

The sequence of the 3'-terminal 1343 nucleotides of the SC strain of the sugarcane mosaic virus (SCMV-SC) genome was compared with the 1376 nucleotides at the 3' terminus of maize dwarf mosaic virus B (MDMV-B). The SCMV-SC sequence includes an open reading frame which codes for the viral coat protein of 313 amino acids (nucleotides 157 to 1116), followed by a 3' non-coding region of 235 nucleotides and a poly(A) tail. The MDMV-B sequence codes for the capsid protein (nucleotides 157 to 1139) of 328 amino acids and has a 3' non-coding region of 236 nucleotides. The coat protein of SCMV-SC has 92% identity with that of MDMV-B except for the region between amino acid residues 27 and 70 of SCMV-SC. This region of SCMV-SC is smaller (44 residues) than the equivalent region in MDMV-B (59 residues) and has only 22% identity with the MDMV-B sequence. Possible mechanisms for the generation of this sequence diversity are discussed. Despite this diversity, the sequence identities of both the major part of the coat proteins and the 3' non-coding regions confirm the proposal, based on previously described serological data, that SCMV-SC and MDMV-B are strains of SCMV.

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.