Activation of Neu (ErbB-2) Mediated by Disulfide Bond-Induced Dimerization Reveals a Receptor Tyrosine Kinase Dimer Interface

Receptor dimerization is a crucial intermediate step in activation of signaling by receptor tyrosine kinases (RTKs). However, dimerization of the RTK Neu (also designated ErbB-2, HER-2, and p185^neu), while necessary, is not sufficient for signaling. Earlier work in our laboratory had shown that introduction of an ectopic cysteine into the Neu juxtamembrane domain induces Neu dimerization but not signaling. Since Neu signaling does require dimerization, we hypothesized that there are additional constraints that govern signaling ability. With the importance of the interreceptor cross-phosphorylation reaction, a likely constraint was the relative geometry of receptors within the dimer. We have tested this possibility by constructing a consecutive series of cysteine substitutions in the Neu juxtamembrane domain in order to force dimerization along a series of interreceptor faces. Within the group that dimerized constitutively, a subset had transforming activity. The substitutions in this subset all mapped to the same face of a predicted alpha helix, the most likely conformation for the intramembrane domain. Furthermore, this face of interaction aligns with the projected Neu* V664E substitution and with a predicted amphipathic interface in the Neu juxtamembrane domain. We propose that these results identify an RTK dimer interface and that dimerization of this RTK induces an extended contact between juxtamembrane and intramembrane alpha helices.     

Use of azo dye ligand chromatography for the partial purification of a novel extracellular peroxidase from Streptomyces viridosporus T7A

Crude peroxidase preparations from the lignocellulose-degrading actinomycete, Streptomyces viridosporus T7A, were shown to decolorize several azo dye isomers and showed a correlation of dye structure to degradability similar to that shown by fungal Mn-peroxidase, an enzyme not previously described in actinomycetes. Addition of the heme-peroxidase inhibitor KCN did not significantly change the ability of the T7A enzyme(s) to decompose the dyes. These results suggest that T7A may produce a Mn- or other peroxidase with similar substrate specificity to Mn-peroxidase. Affinity chromatography using immobilized azo dye isomers was used for purifying peroxidases from T7A. A significantly purified peroxidase preparation was obtained irrespective of the azo dye used. In comparison, concanavalin A lectin affinity chromatography showed very poor binding and resolution for T7A peroxidases. Azo dye affinity purification gave preparations sufficiently purified to allow amino acid microsequencing for two of the bound proteins. N-terminal amino acid sequences were found to share significant homology with a fungal Mn-peroxidase and actinomycete cellulases. Received: 20 May 1997 / Received revision: 17 December 1997 / Accepted: 2 January 1998     

Selection of the 3′-splice site in group I introns

A model for selection of 3′-splice sites in splicing of RNA precursors containing group I introns is presented. The key feature of this model is a newly identified tertiary interaction between the catalytic core of the intron and the 3′-splice site. This tertiary pairing would bring the 3′-splice site into the core of the intron, which is known to contain RNA sequences and structures essential for catalyzing the splicing reactions. The proposed tertiary interaction can coexist with P10, a pairing between 3′-exon sequences and the ‘internal guide sequence’ near the 5′-end of the intron. The model predicts that three RNA-RNA interactions are important in selection of 3′-splice sites: (i) binding of intron sequences with the core; (ii) pairing of exon sequences with the internal guide sequence; and (iii) binding of the terminal guanosine to an unknown site within the core.     

Tubular lysosomes accompany stimulated pinocytosis in macrophages

A network of tubular lysosomes extends through the cytoplasm of J774.2 macrophages and phorbol ester-treated mouse peritoneal macrophages. The presence of this network is dependent upon the integrity of cytoplasmic microtubules and correlates with high cellular rates of accumulation of Lucifer Yellow (LY), a marker of fluid phase pinocytosis. We tested the hypothesis that the efficiency of LY transfer between the pinosomal and lysosomal compartments is increased in the presence of tubular lysosomes by asking how conditions that deplete the tubular lysosome network affect pinocytic accumulation of LY. Tubular lysosomes were disassembled in cells treated with microtubule-depolymerizing drugs or in cells that had phagocytosed latex beads. In unstimulated peritoneal macrophages, which normally contain few tubular lysosomes and which exhibit relatively inefficient transfer of pinocytosed LY to lysosomes, such treatments had little effect on pinocytosis. However, in J774 macrophages and phorbol ester-stimulated peritoneal macrophages, these treatments markedly reduced the efficiency of pinocytic accumulation of LY. We conclude that a basal level of solute accumulation via pinocytosis proceeds independently of the tubular lysosomes, and that an extended tubular lysosomal network contributes to the elevated rates of solute accumulation that accompany macrophage stimulation. Moreover, we suggest that the transformed mouse macrophage cell line J774 exhibits this stimulated pinocytosis constitutively.     

Preservation of thrombomodulin antigen on vascular and extravascular surfaces

The protein C anticoagulant system is mediated by thrombin and is highly accelerated by thrombomodulin. We studied the distribution of thrombomodulin antigen (TM Ag) in the rabbit using an affinity-purified antibody raised in a goat against rabbit thrombomodulin. The preservation of TM Ag was highly dependent on immediate fixation of the surface on which it is located. TM Ag was found on the endothelium of the entire vasculature, whereas it was absent from all connective tissue, smooth and striated muscle, secretory epithelia, cartilage, bone, neural tissue, and all parenchyma examined. A new finding was the presence of TM Ag on nonvascular surfaces of body cavities (the mesothelia of pleura, pericardium, and peritoneum, the synovial membrane, and the arachnoid enveloping the central nervous system). By use of a functional assay, TM activity was recovered in buffered saline/detergent solution which was either injected into the intraperitoneal cavity of rabbits in vivo or incubated with the surface of the arachnoid in vitro. These findings extend the importance of anticoagulant mechanisms to the systems of slowly circulating fluids, in which they might be required for maintenance of the flow, and to mesothelial cavities, in which they could be necessary for preventing adherence between the surfaces, in conditions associated with pathological exudation.     

Analysis of mutations occurring during replication of a SV40 shuttle vector in mammalian cells

To analyse mutations that arise in mammalian cells we have used a SV40::plasmid shuttle vector containing a portion of the E. coli lacZ gene. We have found that following transfection into monkey Cos-7 cell mutations are not detected in the recovered plasmids at 24 h post transfection, but are found at 48 h post transfection, after the onset of DNA replication. Analysis of the mutant plasmids shows that in almost all cases the mutant phenotype is caused by a deletion or rearrangement of the lacZ gene in the shuttle vector.     

Unusually high-level expression of a foreign gene (hepatitis B virus core antigen) in Saccharomyces cerevisiae

As a model system for the study of factors affecting gene expression, hepatitis B virus core antigen (HBcAg) has been expressed in the yeast Saccharomyces cerevisiae. The singularly high levels of expression achieved are approx. 40% of the soluble yeast protein. The HBcAg polypeptides are present as 28-nm particles which are morphologically indistinguishable from HBcAg particles in human plasma and are highly immunogenic in mice. The plasmid construction employed to achieve these very high levels of expression utilizes the constitutively active yeast promoter from the GAP491 gene which is fused in a way that all non-translated sequences flanking the HBcAg coding region are yeast-derived. Hybrid constructions containing 3'-nontranslated viral DNA (yeast 5') or 5'-nontranslated viral DNA (yeast 3') as well as a construction with both 5'- and 3'-nontranslated viral DNA also have been made. A comparison of these constructions for levels of HBcAg expression indicates that the strongest contributor to the high levels of protein is the presence of 5'-flanking sequences which are yeast-derived; secondarily, a significant improvement can be achieved if the 3'-flanking sequences also are yeast-derived. The high abundance of HBcAg in the highest producer is explicable in part on the basis of the very high stability in yeast cells of HBcAg polypeptides. Analysis of the HBcAg coding sequence reveals a very low index of codon bias for S. cerevisiae, largely discounting codon usage as a contributor to the high level of protein obtained.     

Substructure of skeletal myosin subfragment 1. Preferential destabilization of a domain by methanol and its effect on catalytic activity

Evidence is presented that in increasing concentrations of methanol the structure of the subfragment 1 is perturbed in such a way that the Mr = 50,000 central portion of the associated heavy chain is preferentially unfolded. This unfolding is accompanied by the loss in ATPase function where the rate of inactivation can be correlated with the loss in the amount of the Mr = 50,000 fragment generated under standard tryptic digestion conditions. The residual protein appears to be a soluble aggregate of a complex of the Mr = 27,000, 21,000, and light chain with no intact Mr = 50,000 fragment. Tryptic digestions in the presence of MgATP are restricted to the usual linker regions and the Mr = 50,000 fragment is completely protected from attack. Binding of actin to subfragment 1 also results in the protection of the Mr = 50,000 segment and of the Mr = 50,000/21,000 junction from tryptic attack. The data suggest that, in terms of methanolic perturbation, the subfragment 1 appears to be comprised of two domains with differential stability. One domain appears to be the central Mr = 50,000 segment of the heavy chain which is preferentially unfolded by methanol and requires the presence of MgATP or of actin for stabilization. The other domain is more stable and appears to consist of the interacting Mr = 27,000, 21,000, and light chain. The results also suggest that the integrity of the Mr = 50,000 segment is essential for the ATPase function of the protein.     

Structural changes in myosin subfragment 1 by mild denaturation and proteolysis probed by antibodies

The perturbations in the structure of myosin subfragment 1 (S1) by mild denaturation or proteolysis were investigated by measuring the inhibition of the binding of antibodies to immobilized S1 by treated S1 in a solution-phase competitive immunochemical assay. The structural changes in S1 were probed by using anti-50-kDa segment, anti-N-terminus, anti-27-kDa segment, and anti-A1 light chain monoclonal antibodies (MAbs). Methanol and heat denaturation increased MAb binding to the 50-kDa segment. MAb binding to regions in the 27-kDa segment was also promoted, slightly by methanol and more drastically by heat. Proteolysis also induced structural alterations in 50- and 27-kDa segments as shown by increased MAb binding to these regions in cleaved S1. These results indicate that mild denaturation and proteolysis induce structural perturbations which alter the epitope accessibility in 50- and 27-kDa segments of S1 and that antibody binding studies afford a sensitive probe to such perturbations.     

Procaine isothiocyanate: An irreversible inhibitor of the specific binding of [3H]batrachotoxinin-A benzoate to sodium channels

[³H]Batrachotoxinin-A benzoate ([³H]BTX-B) binds with high affinity to sites on voltage sensitive sodium channels in synaptoneurosomes from guinea pig cerebral cortex. Local anesthetics competitively antagonize the binding of [³H]BTX-B. An irreversible local anesthetic, procaine isothiocyanate (PRIT) and a tritiated derivative ([³H]PRIT) have been prepared. PRIT inhibits the binding of [³H]BTX-B in a noncompetitive, irreversible manner (apparent K_i=13 M) whereas the parent compound, procaine, inhibits in a competitive, reversible manner (K_i=40 M). The dissociation rate of [³H]BTX-B from sites on the sodium channel is greatly accelerated in a concentration dependent manner in the presence of PRIT. A 50% increase in the dissociation rate of [³H]BTX-B is achieved in the presence of 0.98 M PRIT. [³H]PRIT binds irreversibly to three proteins in synaptoneurosomes with apparent molecular weights of 20, 42, and 68 kDa. Protection studies with procaine and other local anesthetics suggest that only the 68 kDa species was related to local anesthetic binding.