Domain-specific gene disruption reveals critical regulation of neuregulin signaling by its cytoplasmic tail

Neuregulins are a multi-isoform family of growth factors that activate members of the erbB family of receptor tyrosine kinases. The membrane-anchored isoforms contain the receptor-activating ligand in their extracellular domain, a single membrane-spanning region, and a long cytoplasmic tail. To evaluate the potential biological role of the intracellular domain of the membrane-anchored neuregulin isoforms, we used a domain-specific gene disruption approach to produce a mouse line in which only the region of the neuregulin gene encoding almost the entire intracellular domain was disrupted. Consistent with previous reports in which all neuregulin isoforms were disrupted, the resulting homozygous neuregulin mutants died at E10.5 of circulatory failure and displayed defects in neural and cardiac development. To further understand these in vivo observations, we evaluated a similarly truncated neuregulin construct after transient expression in COS-7 cells. This cytoplasmic tail-deleted mutant, unlike wild-type neuregulin isoforms, was resistant to proteolytic release of its extracellular-domain ligand, a process required for erbB receptor activation. Thus, proteolytic processing of the membrane-bound neuregulin isoforms involved in cranial ganglia and heart embryogenesis is likely developmentally regulated and is critically controlled by their intracellular domain. This observation indicates that erbB receptor activation by membrane-bound neuregulins most likely involves a unique temporally and spatially regulated “inside-out” signaling process that is critical for processing and release of the extracellular-domain ligand.

The parathyroid hormone/parathyroid hormone-related peptide receptor coordinates endochondral bone development by directly controlling chondrocyte differentiation

During vertebrate limb development, growth plate chondrocytes undergo temporally and spatially coordinated differentiation that is necessary for proper morphogenesis. Parathyroid hormone-related peptide (PTHrP), its receptor, the PTH/PTHrP receptor, and Indian hedgehog are implicated in the regulation of chondrocyte differentiation, but the specific cellular targets of these molecules and specific cellular interactions involved have not been defined. Here we generated chimeric mice containing both wild-type and PTH/PTHrP receptor (−/−) cells, and analyzed cell–cell interactions in the growth plate in vivo. Abnormal differentiation of mutant cells shows that PTHrP directly signals to the PTH/PTHrP receptor on proliferating chondrocytes to slow their differentiation. The presence of ectopically differentiated mutant chondrocytes activates the Indian hedgehog/PTHrP axis and slows differentiation of wild-type chondrocytes. Moreover, abnormal chondrocyte differentiation affects mineralization of cartilaginous matrix in a non-cell autonomous fashion; matrix mineralization requires a critical mass of adjacent ectopic hypertrophic chondrocytes. Further, ectopic hypertrophic chondrocytes are associated with ectopic bone collars in adjacent perichondrium. Thus, the PTH/PTHrP receptor directly controls the pace and synchrony of chondrocyte differentiation and thereby coordinates development of the growth plate and adjacent bone.

Cystic fibrosis transmembrane conductance regulator is an epithelial cell receptor for clearance of Pseudomonas aeruginosa from the lung

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel, but its relationship to the primary clinical manifestation of CF, chronic Pseudomonas aeruginosa pulmonary infection, is unclear. We report that CFTR is a cellular receptor for binding, endocytosing, and clearing P. aeruginosa from the normal lung. Murine cells expressing recombinant human wild-type CFTR ingested 30–100 times as many P. aeruginosa as cells lacking CFTR or expressing mutant ΔF508 CFTR protein. Purified CFTR inhibited ingestion of P. aeruginosa by human airway epithelial cells. The first extracellular domain of CFTR specifically bound to P. aeruginosa and a synthetic peptide of this region inhibited P. aeruginosa internalization in vivo, leading to increased bacterial lung burdens. CFTR clears P. aeruginosa from the lung, indicating a direct connection between mutations in CFTR and the clinical consequences of CF.

EphA4 (Sek1) receptor tyrosine kinase is required for the development of the corticospinal tract

Members of the Eph family of tyrosine kinase receptors have been implicated in the regulation of developmental processes and, in particular, axon guidance in the developing nervous system. The function of the EphA4 (Sek1) receptor was explored through creation of a null mutant mouse. Mice with a null mutation in the EphA4 gene are viable and fertile but have a gross motor dysfunction, which is evidenced by a loss of coordination of limb movement and a resultant hopping, kangaroo-like gait. Consistent with the observed phenotype, anatomical studies and anterograde tracing experiments reveal major disruptions of the corticospinal tract within the medulla and spinal cord in the null mutant animals. These results demonstrate a critical role for EphA4 in establishing the corticospinal projection.

Identification and analysis of the plant peroxisomal targeting signal 1 receptor NtPEX5

Protein translocation into peroxisomes takes place via recognition of a peroxisomal targeting signal present at either the extreme C termini (PTS1) or N termini (PTS2) of matrix proteins. In mammals and yeast, the peroxisomal targeting signal receptor, Pex5p, recognizes the PTS1 consisting of -SKL or variants thereof. Although many plant peroxisomal matrix proteins are transported through the PTS1 pathway, little is known about the PTS1 receptor or any other peroxisome assembly protein from plants. We cloned tobacco (Nicotiana tabacum) cDNAs encoding Pex5p (NtPEX5) based on the protein’s interaction with a PTS1-containing protein in the yeast two-hybrid system. Nucleotide sequence analysis revealed that the tobacco Pex5p contains seven tetratricopeptide repeats and that NtPEX5 shares greater sequence similarity with its homolog from humans than from yeast. Expression of NtPEX5 fusion proteins, consisting of the N-terminal part of yeast Pex5p and the C-terminal region of NtPEX5, in a Saccharomyces cerevisiae pex5 mutant restored protein translocation into peroxisomes. These experiments confirmed the identity of the tobacco protein as a PTS1 receptor and indicated that components of the peroxisomal translocation apparatus are conserved functionally. Two-hybrid assays showed that NtPEX5 interacts with a wide range of PTS1 variants that also interact with the human Pex5p. Interestingly, the C-terminal residues of some of these peptides deviated from the established plant PTS1 consensus sequence. We conclude that there are significant sequence and functional similarities between the plant and human Pex5ps.

Role of the Y5 neuropeptide Y receptor in limbic seizures

Neuropeptide Y (NPY) is an inhibitory neuromodulator expressed abundantly in the central nervous system that is suspected of being an endogenous antiepileptic agent that can control propagation of limbic seizures. Electrophysiological and pharmacological data suggest that these actions of NPY are mediated by G protein-coupled NPY Y2 and NPY Y5 receptors. To determine whether the NPY Y5 receptor (Y5R) is required for normal control of limbic seizures, we examined hippocampal function and responsiveness to kainic acid-induced seizures in Y5R-deficient (Y5R−/−) mice. We report that Y5R−/− mice do not exhibit spontaneous seizure-like activity; however, they are more sensitive to kainic acid-induced seizures. Electrophysiological examination of hippocampal slices from mutant mice revealed normal function, but the antiepileptic effects of exogenously applied NPY were absent. These data demonstrate that Y5R has an important role in mediating NPY’s inhibitory actions in the mouse hippocampus and suggest a role for Y5R in the control of limbic seizures.

Acetylcholine receptor ɛ-subunit deletion causes muscle weakness and atrophy in juvenile and adult mice

In mammalian muscle a postnatal switch in functional properties of neuromuscular transmission occurs when miniature end plate currents become shorter and the conductance and Ca2+ permeability of end plate channels increases. These changes are due to replacement during early neonatal development of the γ-subunit of the fetal acetylcholine receptor (AChR) by the ɛ-subunit. The long-term functional consequences of this switch for neuromuscular transmission and motor behavior of the animal remained elusive. We report that deletion of the ɛ-subunit gene caused in homozygous mutant mice the persistence of γ-subunit gene expression in juvenile and adult animals. Neuromuscular transmission in these animals is based on fetal type AChRs present in the end plate at reduced density. Impaired neuromuscular transmission, progressive muscle weakness, and atrophy caused premature death 2 to 3 months after birth. The results demonstrate that postnatal incorporation into the end plate of ɛ-subunit containing AChRs is essential for normal development of skeletal muscle.

Truncated BRCA2 is cytoplasmic: Implications for cancer-linked mutations

BRCA2 mutations predispose carriers mainly to breast cancer. The vast majority of BRCA2 mutations are predicted to result in a truncated protein product. The smallest known cancer-associated deletion removes from the C terminus only 224 of the 3,418 residues constituting BRCA2, suggesting that these terminal amino acids are crucial for BRCA2 function. A series of green fluorescent protein (GFP)-tagged BRCA2 deletion mutants revealed that nuclear localization depends on two nuclear localization signals that reside within the final 156 residues of BRCA2. Consistent with this observation, an endogenous truncated BRCA2 mutant (6174delT) was found to be cytoplasmic. Together, these studies provide a simple explanation for why the vast majority of BRCA2 mutants are nonfunctional: they do not translocate into the nucleus.

Recruitment of IRAK to the interleukin 1 receptor complex requires interleukin 1 receptor accessory protein

The proinflammatory cytokine interleukin 1 (IL-1) activates the transcription of many genes encoding acute phase and proinflammatory proteins, a function mediated primarily by the transcription factor NF-κB. An early IL-1 signaling event is the recruitment of the Ser/Thr kinase IRAK to the type I IL-1 receptor (IL-1RI). Here we describe the function of a previously identified IL-1 receptor subunit designated IL-1 receptor accessory protein (IL-1RAcP). IL-1 treatment of cells induces the formation of a complex containing both IL-1RI and IL-1RAcP. IRAK is recruited to this complex through its association with IL-1RAcP. Overexpression of an IL-1RAcP mutant lacking its intracellular domain, the IRAK-binding domain, prevented the recruitment of IRAK to the receptor complex and blocked IL-1-induced NF-κB activation.

cAMP receptor protein–cAMP plays a crucial role in glucose–lactose diauxie by activating the major glucose transporter gene in Escherichia coli

The inhibition of β-galactosidase expression in a medium containing both glucose and lactose is a typical example of the glucose effect in Escherichia coli. We studied the glucose effect in the lacL8UV5 promoter mutant, which is independent of cAMP and cAMP receptor protein (CRP). A strong inhibition of β-galactosidase expression by glucose and a diauxic growth were observed when the lacL8UV5 cells were grown on a glucose–lactose medium. The addition of isopropyl β-d-thiogalactoside to the culture medium eliminated the glucose effect. Disruption of the crr gene or overproduction of LacY also eliminated the glucose effect. These results are fully consistent with our previous finding that the glucose effect in wild-type cells growing in a glucose–lactose medium is not due to the reduction of CRP–cAMP levels but is due to the inducer exclusion. We found that the glucose effect in the lacL8UV5 cells was no longer observed when either the crp or the cya gene was disrupted. Evidence suggested that CRP–cAMP may not enhance directly the lac repressor action in vivo. Northern blot analysis revealed that the mRNA for ptsG, a major glucose transporter gene, was markedly reduced in a Δcrp or Δcya background. The constitutive expression of the ptsG gene by the introduction of a multicopy plasmid restored the glucose effect in Δcya or Δcrp cells. We conclude that CRP–cAMP plays a crucial role in inducer exclusion, which is responsible for the glucose–lactose diauxie, by activating the expression of the ptsG gene.

Smad8 mediates the signaling of the receptor serine kinase

Smad proteins are critical intracellular mediators of signaling by growth and differentiation factors of the transforming growth factor β superfamily. We have isolated a member of the Smad family, Smad8, from a rat brain cDNA library and biochemically and functionally characterized its ability to transduce signals from serine kinase receptors. In Xenopus embryo, Smad8 is able to transcriptionally activate a subset of mesoderm target genes similar to those induced by the receptor serine kinase, activin receptor-like kinase (ALK)-2. Smad8 can be specifically phosphorylated by a constitutively active ALK-2 but not the related receptor serine kinase, ALK-4. In response to signaling from ALK-2, Smad8 associates with a common regulatory molecule, Smad4, and this association leads to a synergistic effect on gene transcription. Furthermore, Smad8 is able to rescue the expression of mesoderm genes blocked by truncated ALK-2 in the embryo. These results indicate that Smad8 can function as a downstream signaling mediator of ALK-2.

Disruption of the m1 receptor gene ablates muscarinic receptor-dependent M current regulation and seizure activity in mice

Muscarinic acetylcholine receptors are members of the G protein-coupled receptor superfamily expressed in neurons, cardiomyocytes, smooth muscle, and a variety of epithelia. Five subtypes of muscarinic acetylcholine receptors have been discovered by molecular cloning, but their pharmacological similarities and frequent colocalization make it difficult to assign functional roles for individual subtypes in specific neuronal responses. We have used gene targeting by homologous recombination in embryonic stem cells to produce mice lacking the m1 receptor. These mice show no obvious behavioral or histological defects, and the m2, m3, and m4 receptors continue to be expressed in brain with no evidence of compensatory induction. However, the robust suppression of the M-current potassium channel activity evoked by muscarinic agonists in sympathetic ganglion neurons is completely lost in m1 mutant mice. In addition, both homozygous and heterozygous mutant mice are highly resistant to the seizures produced by systemic administration of the muscarinic agonist pilocarpine. Thus, the m1 receptor subtype mediates M current modulation in sympathetic neurons and induction of seizure activity in the pilocarpine model of epilepsy.

The mutationally activated Met receptor mediates motility and metastasis

Mutations in Met have been identified in human papillary renal carcinomas. We have shown previously that these mutations deregulate the enzymatic activity of Met and that NIH 3T3 cells expressing mutationally activated Met are transformed in vitro and are tumorigenic in vivo. In the present investigation, we find that mutant Met induces the motility of Madin-Darby canine kidney cells in vitro and experimental metastasis of NIH 3T3 cells in vivo, and that the Ras-Raf-MEK-ERK signaling pathway, which has been implicated previously in cellular motility and metastasis, is constitutively activated by the Met mutants. We also report that transgenic mice harboring mutationally activated Met develop metastatic mammary carcinoma. These data confirm the tumorigenic activity of mutant Met molecules and demonstrate their ability to induce the metastatic phenotype.

An enhancer-blocking element between α and δ gene segments within the human T cell receptor α/δ locus

T cell receptor (TCR) α and δ gene segments are organized within a single genetic locus but are differentially regulated during T cell development. An enhancer-blocking element (BEAD-1, for blocking element alpha/delta 1) was localized to a 2.0-kb region 3′ of TCR δ gene segments and 5′ of TCR α joining gene segments within this locus. BEAD-1 blocked the ability of the TCR δ enhancer (Eδ) to activate a promoter when located between the two in a chromatin-integrated construct. We propose that BEAD-1 functions as a boundary that separates the TCR α/δ locus into distinct regulatory domains controlled by Eδ and the TCR α enhancer, and that it prevents Eδ from opening the chromatin of the TCR α joining gene segments for VDJ recombination at an early stage of T cell development.

Peptidyl prolyl cis-trans isomerase activity of cyclophilin A in functional homo-oligomeric receptor expression

The functional expression of homo-oligomeric α7 neuronal nicotinic and type 3 serotonin receptors is dependent on the activity of a cyclophilin. In this paper we demonstrate that the mechanism of cyclophilin action during functional homo-oligomeric receptor expression in Xenopus oocytes is distinct from the calcineurin-dependent immunosuppressive mechanism by showing that a nonimmunosuppressive analog of cyclosporin A (CsA), SDZ 211–811, reduces functional receptor expression to the same extent as CsA. The cytoplasmic subtype of cyclophilin, cyclophilin A (CyPA), appears to be required for functional receptor expression. This is because overexpression of CyPA and a CyPA mutant that is deficient in CsA binding activity reverses CsA-induced reduction in functional receptor expression. The mechanism of action of CyPA is likely to involve its prolyl isomerase activity because a mutant CyPA with a single amino acid substitution (arginine 55 to alanine) that is predicted to produce a 1000-fold attenuation in isomerase activity fails to reverse the cyclosporin A effect. Our data also suggest that CyPA does not form a stable complex with receptor subunits.