Activating mutations in the extracellular domain of the fibroblast growth factor receptor 2 function by disruption of the disulfide bond in the third immunoglobulin-like domain

Multiple human skeletal and craniosynostosis disorders, including Crouzon, Pfeiffer, Jackson–Weiss, and Apert syndromes, result from numerous point mutations in the extracellular region of fibroblast growth factor receptor 2 (FGFR2). Many of these mutations create a free cysteine residue that potentially leads to abnormal disulfide bond formation and receptor activation; however, for noncysteine mutations, the mechanism of receptor activation remains unclear. We examined the effect of two of these mutations, W290G and T341P, on receptor dimerization and activation. These mutations resulted in cellular transformation when expressed as FGFR2/Neu chimeric receptors. Additionally, in full-length FGFR2, the mutations induced receptor dimerization and elevated levels of tyrosine kinase activity. Interestingly, transformation by the chimeric receptors, dimerization, and enhanced kinase activity were all abolished if either the W290G or the T341P mutation was expressed in conjunction with mutations that eliminate the disulfide bond in the third immunoglobulin-like domain (Ig-3). These results demonstrate a requirement for the Ig-3 cysteine residues in the activation of FGFR2 by noncysteine mutations. Molecular modeling also reveals that noncysteine mutations may activate FGFR2 by altering the conformation of the Ig-3 domain near the disulfide bond, preventing the formation of an intramolecular bond. This allows the unbonded cysteine residues to participate in intermolecular disulfide bonding, resulting in constitutive activation of the receptor.

Nedd4 mediates control of an epithelial Na+ channel in salivary duct cells by cytosolic Na+

Epithelial Na+ channels are expressed widely in absorptive epithelia such as the renal collecting duct and the colon and play a critical role in fluid and electrolyte homeostasis. Recent studies have shown that these channels interact via PY motifs in the C terminals of their α, β, and γ subunits with the WW domains of the ubiquitin-protein ligase Nedd4. Mutation or deletion of these PY motifs (as occurs, for example, in the heritable form of hypertension known as Liddle’s syndrome) leads to increased Na+ channel activity. Thus, binding of Nedd4 by the PY motifs would appear to be part of a physiological control system for down-regulation of Na+ channel activity. The nature of this control system is, however, unknown. In the present paper, we show that Nedd4 mediates the ubiquitin-dependent down-regulation of Na+ channel activity in response to increased intracellular Na+. We further show that Nedd4 operates downstream of Go in this feedback pathway. We find, however, that Nedd4 is not involved in the feedback control of Na+ channels by intracellular anions. Finally, we show that Nedd4 has no influence on Na+ channel activity when the Na+ and anion feedback systems are inactive. We conclude that Nedd4 normally mediates feedback control of epithelial Na+ channels by intracellular Na+, and we suggest that the increased Na+ channel activity observed in Liddle’s syndrome is attributable to the loss of this regulatory feedback system.

Tsetse thrombin inhibitor: Bloodmeal-induced expression of an anticoagulant in salivary glands and gut tissue of Glossina morsitans morsitans

The tsetse thrombin inhibitor, a potent and specific low molecular mass (3,530 Da) anticoagulant peptide, was purified previously from salivary gland extracts of Glossina morsitans morsitans (Diptera: Glossinidae). A 303-bp coding sequence corresponding to the inhibitor has now been isolated from a tsetse salivary gland cDNA library by using degenerate oligonucleotide probes. The full-length cDNA contains a 26-bp untranslated segment at its 5′ end, followed by a 63-bp sequence corresponding to a putative secretory signal peptide. A 96-bp segment codes for the mature tsetse thrombin inhibitor, whose predicted molecular weight matches that of the purified native protein. Based on its lack of homology to any previously described family of molecules, the tsetse thrombin inhibitor appears to represent a unique class of naturally occurring protease inhibitors. Recombinant tsetse thrombin inhibitor expressed in Escherichia coli and the chemically synthesized peptide are both substantially less active than the purified native protein, suggesting that posttranslational modification(s) may be necessary for optimal inhibitory activity. The tsetse thrombin inhibitor gene, which is present as a single copy in the tsetse genome, is expressed at high levels in salivary glands and midguts of adult tsetse flies, suggesting a possible role for the anticoagulant in both feeding and processing of the bloodmeal.

A novel pair of immunoglobulin-like receptors expressed by B cells and myeloid cells

An Fcα receptor probe of human origin was used to identify novel members of the Ig gene superfamily in mice. Paired Ig-like receptors, named PIR-A and PIR-B, are predicted from sequence analysis of the cDNAs isolated from a mouse splenic library. Both type I transmembrane proteins possess similar ectodomains with six Ig-like loops, but have different transmembrane and cytoplasmic regions. The predicted PIR-A protein has a short cytoplasmic tail and a charged Arg residue in the transmembrane region that, by analogy with the FcαR relative, suggests the potential for association with an additional transmembrane protein to form a signal transducing unit. In contrast, the PIR-B protein has an uncharged transmembrane region and a long cytoplasmic tail containing four potential immunoreceptor tyrosine-based inhibitory motifs. These features are shared by the related killer inhibitory receptors. PIR-A proteins appear to be highly variable, in that predicted peptide sequences differ for seven randomly selected PIR-A clones, whereas PIR-B cDNA clones are invariant. Southern blot analysis with PIR-B and PIR-A-specific probes suggests only one PIR-B gene and multiple PIR-A genes. The PIR-A and PIR-B genes are expressed in B lymphocytes and myeloid lineage cells, wherein both are expressed simultaneously. The characteristics of the highly-conserved PIR-A and PIR-B genes and their coordinate cellular expression suggest a potential regulatory role in humoral, inflammatory, and allergic responses.

Sequence dependent hypermutation of the immunoglobulin heavy chain in cultured B cells

The variable (V) regions of immunoglobulin heavy and light chains undergo high rates of somatic mutation during the immune response. Although point mutations accumulate throughout the V regions and their immediate flanking sequences, analysis of large numbers of mutations that have arisen in vivo reveal that the triplet AGC appears to be most susceptible to mutation. We have stably transfected B cell lines with γ2a heavy chain constructs containing TAG nonsense codons in their V regions that are part of either a putative (T)AGC hot spot or a (T)AGA non-hot spot motif. Using an ELISA spot assay to detect revertants and fluctuation analysis to determine rates of mutation, the rate of reversion of the TAG nonsense codon has been determined for different motifs in different parts of the V region. In the NSO plasma cell line, the (T)AGC hot spot motif mutates at rates of ≈6 × 10−4/bp per generation and ≈3 × 10−5/bp per generation at residues 38 and 94 in the V region. At each of these locations, the (T)AGC hot spot motif is 20–30 times more likely to undergo mutation than the (T)AGA non-hot spot motif. Moreover, the AGA non-hot spot motif mutates at as high a rate as the hot spot motif when it is located adjacent to hot spot motifs, suggesting that more extended sequences influence susceptibility to mutation.

Transduction of Basolateral-to-Apical Signals across Epithelial Cells: Ligand-stimulated Transcytosis of the Polymeric Immunoglobulin Receptor Requires Two Signals

Transcytosis of the polymeric immunoglobulin receptor (pIgR) is stimulated by binding of its ligand, dimeric IgA (dIgA). During this process, dIgA binding at the basolateral surface of the epithelial cell transmits a signal to the apical region of the cell, which in turn stimulates the transport of dIgA–pIgR complex from a postmicrotubule compartment to the apical surface. We have previously reported that the signal of stimulation was controlled by a protein-tyrosine kinase (PTK) activated upon dIgA binding. We now show that this signal of stimulation moves across the cell independently of pIgR movement or microtubules and acts through the tyrosine kinase activity by releasing Ca++ from inositol trisphosphate–sensitive intracellular stores. Surprisingly we have found that a second independent signal is required to achieve dIgA-stimulated transcytosis of pIgR. This second signal depends on dIgA binding to the pIgR solely at the basolateral surface and the ability of pIgR to dimerize. This enables pIgR molecules that have bound dIgA at the basolateral surface to respond to the signal of stimulation once they reach the postmicrotubule compartment. We propose that the use of two signals may be a general mechanism by which signaling receptors maintain specificity along their signaling and trafficking pathways.

Dimerization of the Polymeric Immunoglobulin Receptor Controls Its Transcytotic Trafficking

Binding of dimeric immunoglobulin (Ig)A to the polymeric Ig receptor (pIgR) stimulates transcytosis of pIgR across epithelial cells. Through the generation of a series of pIgR chimeric constructs, we have tested the ability of ligand to promote receptor dimerization and the subsequent role of receptor dimerization on its intracellular trafficking. Using the cytoplasmic domain of the T cell receptor-ζ chain as a sensitive indicator of receptor oligomerization, we show that a pIgR:ζ chimeric receptor expressed in Jurkat cells initiates a ζ-specific signal transduction cascade when exposed to dimeric or tetrameric IgA, but not when exposed to monomeric IgA. In addition, we replaced the pIgR’s transmembrane domain with that of glycophorin A to force dimerization or with a mutant glycophorin transmembrane domain to prevent dimerization. Forcing dimerization stimulated transcytosis of the chimera, whereas preventing dimerization abolished ligand-stimulated transcytosis. We conclude that binding of dimeric IgA to the pIgR induces its dimerization and that this dimerization is necessary and sufficient to stimulate pIgR transcytosis.

WRS-85D: A Tryptophanyl-tRNA Synthetase Expressed to High Levels in the Developing Drosophila Salivary Gland

In a screen for genes expressed in the Drosophila embryonic salivary gland, we identified a tryptophanyl-tRNA synthetase gene that maps to cytological position 85D (WRS-85D). WRS-85D expression is dependent on the homeotic gene Sex combs reduced (Scr). In the absence of Scr function, WRS-85D expression is lost in the salivary gland primordia; conversely, ectopic expression of Scr results in expression of WRS-85D in new locations. Despite the fact that WRS-85D is a housekeeping gene essential for protein synthesis, we detected both WRS-85D mRNA and protein at elevated levels in the developing salivary gland. WRS-85D is required for embryonic survival; embryos lacking the maternal contribution were unrecoverable, whereas larvae lacking the zygotic component died during the third instar larval stage. We showed that recombinant WRS-85D protein specifically charges tRNATrp, and WRS-85D is likely to be the only tryptophanyl-tRNA synthetase gene in Drosophila. We characterized the expression patterns of all 20 aminoacyl-tRNA synthetases and found that of the four aminoacyl-tRNA synthetase genes expressed at elevated levels in the salivary gland primordia, WRS-85D is expressed at the highest level throughout embryogenesis. We also discuss the potential noncanonical activities of tryptophanyl-tRNA synthetase in immune response and regulation of cell growth.

BiP and Immunoglobulin Light Chain Cooperate to Control the Folding of Heavy Chain and Ensure the Fidelity of Immunoglobulin Assembly

The immunoglobulin (Ig) molecule is composed of two identical heavy chains and two identical light chains (H2L2). Transport of this heteromeric complex is dependent on the correct assembly of the component parts, which is controlled, in part, by the association of incompletely assembled Ig heavy chains with the endoplasmic reticulum (ER) chaperone, BiP. Although other heavy chain-constant domains interact transiently with BiP, in the absence of light chain synthesis, BiP binds stably to the first constant domain (CH1) of the heavy chain, causing it to be retained in the ER. Using a simplified two-domain Ig heavy chain (VH-CH1), we have determined why BiP remains bound to free heavy chains and how light chains facilitate their transport. We found that in the absence of light chain expression, the CH1 domain neither folds nor forms its intradomain disulfide bond and therefore remains a substrate for BiP. In vivo, light chains are required to facilitate both the folding of the CH1 domain and the release of BiP. In contrast, the addition of ATP to isolated BiP–heavy chain complexes in vitro causes the release of BiP and allows the CH1 domain to fold in the absence of light chains. Therefore, light chains are not intrinsically essential for CH1 domain folding, but play a critical role in removing BiP from the CH1 domain, thereby allowing it to fold and Ig assembly to proceed. These data suggest that the assembly of multimeric protein complexes in the ER is not strictly dependent on the proper folding of individual subunits; rather, assembly can drive the complete folding of protein subunits.

Unexpected homology between inducible cell wall protein QID74 of filamentous fungi and BR3 salivary protein of the insect Chironomus

A gene, qid74, of mycoparasitic filamentous fungus Trichoderma harzianum and its allies encodes a cell wall protein that is induced by replacing glucose in the culture medium with chitin (simulated mycoparasitism conditions). Because no trace of this gene can be detected in related species such as Gibberella fujikuroi and Saccharomyces cerevisiae, the qid74 gene appears to have arisen de novo within the genus Trichoderma. Qid74 protein, 687 residues long, is now seen as highly conserved tandem repeats of the 59-residue-long unit. This unit itself, however, may have arisen as tandem repeats of the shorter 13-residue-long basic unit. Within the genus Trichoderma, the amino acid sequence of Qid74 proteins has been conserved in toto. The most striking is the fact that Qid74 shares 25.3% sequence identity with the carboxyl-terminal half of the 1,572-residue-long BR3 protein of the dipteran insect Chironomus tentans. BR3 protein is secreted by the salivary gland of each aquatic larva of Chironomus to form a tube to house itself. Furthermore, the consensus sequence derived from these 59-residue-long repeating units resembles those of epidermal growth factor-like domains found in divergent invertebrate and vertebrate proteins as to the positions of critical cysteine residues and homology of residues surrounding these cysteines.

Ongoing immunoglobulin somatic mutation in germinal center B cell-like but not in activated B cell-like diffuse large cell lymphomas

B cell diffuse large cell lymphoma (B-DLCL) is a heterogeneous group of tumors, based on significant variations in morphology, clinical presentation, and response to treatment. Gene expression profiling has revealed two distinct tumor subtypes of B-DLCL: germinal center B cell-like DLCL and activated B cell-like DLCL. In a separate study, we determined that B-DLCL can also be subdivided into two groups based on the presence or absence of ongoing Ig gene hypermutation. Here, we evaluated the correlation between these B-DLCL subtypes established by the two different methods. Fourteen primary B-DLCL cases were studied by gene expression profiling using DNA microarrays and for the presence of ongoing mutations in their Ig heavy chain gene. All seven cases classified as germinal center B cell-like DLCL by gene expression showed the presence of ongoing mutations in the Ig genes. Five of the seven cases classified by gene expression as activated B cell-like DLCL had no ongoing somatic mutations, whereas, in the remaining two cases, a single point mutation was observed in only 2 of 15 and 21 examined molecular clones of variable heavy (VH) chain gene, respectively. These two cases were distantly related to the rest of the activated B cell-like DLCL tumors by gene expression. Our findings validate the concept that lymphoid malignancies are derived from cells at discrete stages of normal lymphocyte maturation and that the malignant cells retain the genetic program of those normal cells.

Vaccination with syngeneic, lymphoma-derived immunoglobulin idiotype combined with granulocyte/macrophage colony-stimulating factor primes mice for a protective T-cell response.

The idiotype of the Ig expressed by a B-cell malignancy (Id) can serve as a unique tumor-specific antigen and as a model for cancer vaccine development. In murine models of Id vaccination, formulation of syngeneic Id with carrier proteins or adjuvants induces an anti-idiotypic antibody response. However, inducing a potent cell-mediated response to this weak antigen instead would be highly desirable. In the 38C13 lymphoma model, we observed that low doses of free granulocyte/macrophage colony-stimulating factor (GM-CSF) 10,000 units i.p. or locally s.c. daily for 4 days significantly enhanced protective antitumor immunity induced by s.c. Id-keyhole limpet hemocyanin (KLH) immunization. This effect was critically dependent upon effector CD4+ and CD8+ T cells and was not associated with any increased anti-idiotypic antibody production. Lymphocytes from spleens and draining lymph nodes of mice primed with Id-KLH plus GM-CSF, but not with Id-KLH alone, demonstrated significant proliferation to Id in vitro without any biased production of interferon gamma or interleukin 4 protein or mRNA. As a further demonstration of potency, 50% of mice immunized with Id-KLH plus GM-CSF on the same day as challenge with a large s.c. tumor inoculum remained tumor-free at day 80, compared with 17% for Id-KLH alone, when immunization was combined with cyclophosphamide. Taken together, these results demonstrate that GM-CSF can significantly enhance the immunogenicity of a defined self-antigen and that this effect is mediated exclusively by activating the T-cell arm of the immune response.

Inhibition of immunoglobulin folding and secretion by dominant negative BiP ATPase mutants.

A group of resident ER proteins have been identified that are proposed to function as molecular chaperones. The best characterized of these is BiP/GRP78, an hsp70 homologue that binds peptides containing hydrophobic residues in vitro and unfolded or unassembled proteins in vivo. However, evidence that mammalian BiP plays a direct role in protein folding remains circumstantial. In this study, we examine how BiP interacts with a particular substrate, immunoglobulin light chain (lambda LC), during its folding. Wild-type hamster BiP and several well-characterized BiP ATPase mutants were used in transient expression experiments. We demonstrate that wild-type lambda LCs showed prolonged association with mutant BiP which inhibited their secretion. Both wild-type and mutant BiP bound only to unfolded and partially folded LCs. The wild-type BiP was released from the incompletely folded LCs, allowing them to fold and be secreted, whereas the mutant BiP was not released. As a result, the LCs that were bound to BiP mutants were unable to undergo complete disulfide bond formation and were retained in the ER. Our experiments suggest that LCs undergo both BiP-dependent and BiP-independent folding steps, demonstrating that both ATP binding and hydrolysis activities of BiP are essential for the completion of LC folding in vivo and reveal that BiP must release before disulfide bond formation can occur in that domain.