An intact zinc ring finger is required for tumor necrosis factor receptor-associated factor-mediated nuclear factor-kappaB activation but is dispensable for c-Jun N-terminal kinase signaling

The diverse biological effects of the tumor necrosis factor (TNF) receptor superfamily are believed to be mediated in part through TNF receptor-associated factors (TRAFs), a family of cytoplasmic adaptor proteins which can activate intracellular signaling pathways, including the nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK) pathways. TRAFs 2, 5, and 6 strongly activate both pathways when overexpressed; however, TRAF 3 (a close homologue of TRAF 5) does not significantly activate either pathway. The current study addresses the structural basis for this difference by substituting corresponding domains of TRAF 5 into TRAF 3 and testing activation of both pathways. A small region of TRAF 5 (the first zinc finger and 10 residues of the second zinc finger) is sufficient to convert TRAF 3 into an activator of both pathways. Also, an intact zinc ring finger is required for NF-kappaB activation but not JNK activation. In agreement with this finding, TRAF 2A, a TRAF 2 splice variant with an altered ring finger, is a specific activator of JNK. These findings suggest that different domains of TRAFs may be involved in NF-kappaB and JNK signaling. Also, alternative splicing of TRAFs may represent a novel mechanism whereby TNF family receptors can mediate distinct downstream effects in different tissues.     

Downstream of Crk adaptor signaling pathway: activation of Jun kinase by v-Crk through the guanine nucleotide exchange protein C3G

Crk, which belongs to the adaptor family of proteins composed of Src homology 2 (SH2) and SH3 domains, has a putative role in signaling. However, the downstream events of Crk signaling remain unclear. In this study, we found that Jun kinase (JNK) is moderately activated by v-Crk in both NIH 3T3 cells and chicken embryo fibroblasts. Transient expression of v-Crk, c-Crk-I, or c-Crk-II activated JNK1 in human embryo kidney cells, 293T. Coexpression of a guanine nucleotide exchange protein C3G, which specifically binds to Crk's SH3 domain, further enhanced the JNK activity as well as growth rate and anchorage-independent growth of v-Crk NIH 3T3 cells. Furthermore, overexpression of a dominant-negative form of C3G lacking the guanine nucleotide exchange domain abolished both the JNK activity and the colony forming potential of v-Crk NIH 3T3 cells. The requirement for JNK activation in v-Crk induced transformation was demonstrated by the suppression of colony forming activity of v-Crk NIH 3T3 cells when a dominant-negative form of JNK kinase, Sek1/MKK4 is expressed in these cells. These data strongly suggest the existence of a novel signaling cascade involving an adaptor protein v-Crk, which transmits signals through C3G toward JNK activation.     

TNF receptor-associated factor-3 signaling mediates activation of p38 and Jun N-terminal kinase, cytokine secretion, and Ig production following ligation of CD40 on human B cells

CD40 engagement induces a variety of functional outcomes following association with adaptor molecules of the TNF receptor-associated factor (TRAF) family. Whereas TRAF2, -5, and -6 initiate NF-kappaB activation, the outcomes of TRAF3-initiated signaling are less characterized. To delineate CD40-induced TRAF3-dependent events, Ramos B cells stably transfected with a dominant negative TRAF3 were stimulated with membranes expressing recombinant CD154/CD40 ligand. In the absence of TRAF3 signaling, activation of p38 and control of Ig production were abrogated, whereas Jun N-terminal kinase activation and secretion of IL-10, lymphotoxin-alpha, and TNF-alpha were partially blocked. By contrast, induction of apoptosis, activation of NF-kappaB, generation of granulocyte-macrophage CSF, and up-regulation of CD54, MHC class II, and CD95 were unaffected by the TRAF3 dominant negative. Together, these results indicate that TRAF3 initiates independent signaling pathways via p38 and JNK that are associated with specific functional outcomes.     

Extracellular signal-regulated kinase-2, but not c-Jun NH2-terminal kinase, activation correlates with surface IgM-mediated apoptosis in the WEHI 231 B cell line

Both extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) have been implicated in mediating the signaling events that precede apoptosis. We studied the activation of these kinases during apoptosis of WEHI 231 B cells. Surface IgM ligation induces apoptosis of WEHI 231 cells. This effect is augmented by simultaneous engagement of CD95 and is inhibited by costimulation with either CD40 or IL-4R. We determined that surface IgM ligation activates ERK2 to a much greater level than JNK, and that IgM-mediated ERK2 activation is enhanced by costimulation with anti-CD95. Costimulation with either IL-4 or anti-CD40 interferes with anti-IgM-stimulated ERK2 activation. Transient expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) inhibits both ERK2 activation and cell death following stimulation with anti-IgM and the combination of anti-IgM plus anti-CD95. CD40 engagement alone activates JNK, but IL-4 stimulation does not. N-acetyl-L-cysteine pretreatment, which blocks CD40-mediated JNK activation, does not affect the ability of CD40 to inhibit anti-IgM-mediated ERK2 activation and apoptosis. Together, these data suggest that JNK activation is not required for CD40 inhibition of surface IgM-induced cell death and that ERK2 plays an active role in mediating anti-IgM-induced apoptosis of WEHI 231 B cells.     

Requirement for activation of the serine-threonine kinase Akt (protein kinase B) in insulin stimulation of protein synthesis but not of glucose transport

A wide variety of biological activities including the major metabolic actions of insulin is regulated by phosphatidylinositol (PI) 3-kinase. However, the downstream effectors of the various signaling pathways that emanate from PI 3-kinase remain unclear. Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, is thought to be one such downstream effector. A mutant Akt (Akt-AA) in which the phosphorylation sites (Thr308 and Ser473) targeted by growth factors are replaced by alanine has now been shown to lack protein kinase activity and, when overexpressed in CHO cells or 3T3-L1 adipocytes with the use of an adenovirus vector, to inhibit insulin-induced activation of endogenous Akt. Akt-AA thus acts in a dominant negative manner in intact cells. Insulin-stimulated protein synthesis, which is sensitive to wortmannin, a pharmacological inhibitor of PI 3-kinase, was abolished by overexpression of Akt-AA without an effect on amino acid transport into the cells, suggesting that Akt is required for insulin-stimulated protein synthesis. Insulin activation of p70 S6 kinase was inhibited by approximately 75% in CHO cells and approximately 30% in 3T3-L1 adipocytes, whereas insulin-induced activation of endogenous Akt was inhibited by 80 to 95%, by expression of Akt-AA. Thus, Akt activity appears to be required, at least in part, for insulin stimulation of p70 S6 kinase. However, insulin-stimulated glucose uptake in both CHO cells and 3T3-L1 adipocytes was not affected by overexpression of Akt-AA, suggesting that Akt is not required for this effect of insulin. These data indicate that Akt acts as a downstream effector in some, but not all, of the signaling pathways downstream of PI 3-kinase.     

IFN-gamma induces cell growth inhibition by Fas-mediated apoptosis: requirement of STAT1 protein for up-regulation of Fas and FasL expression

The mechanism by which IFN-gamma inhibits tumor cell growth has not been fully understood. Here we report that IFN-gamma up-regulated the expression of Fas and Fas ligand (FasL) on HT29 cells, a human colon adenocarcinoma cell line, and subsequently induced apoptosis of these cells. The kinetics of cell death in IFN-gamma-treated HT29 cells paralleled the increase in the levels of Fas and FasL expression. We further show that IFN-gamma up-regulated the expression of Fas and FasL in STAT1-transfected U3A cells but not in STAT1-deficient U3A cells. Correspondingly, IFN-gamma induced cell death in STAT1-transfected U3A cells but not in STAT1-deficient U3A cells. IFN-gamma-induced cell death was inhibited by caspase-1 inhibitors. Our results suggest that cell growth inhibition by IFN-gamma is due to apoptosis mediated by Fas and FasL interaction.     

Requirement of N-terminal cysteines of PSD-95 for PSD-95 multimerization and ternary complex formation, but not for binding to potassium channel Kv1.4

The PSD-95 family of PSD-95/Discs large/ZO-1 (PDZ) domain-containing proteins plays a role in the clustering and localization of specific ion channels and receptors at synapses. Previous studies have shown that PSD-95 forms multimers through an N-terminal region (termed the N-segment) and that the multimerization of PSD-95 is critical for its ability to cluster Shaker-type potassium channel Kv1.4 in heterologous cells. We show here that the PSD-95 N-segment functions as a multimerization domain only when located at the N-terminal end of a heterologous protein. A pair of N-terminal cysteines, Cys3 and Cys5, is essential for the ability of PSD-95 to self-associate and to form cell surface clusters with Kv1.4. However, PSD-95 mutants lacking these cysteine residues retain their ability to associate with membranes and to bind to Kv1.4. Unlike wild type PSD-95, the cysteine mutant of PSD-95 cannot form a ternary complex with Kv1.4 and the cell adhesion molecule Fasciclin II. These results suggest that the N-terminal cysteines are essential for PSD-95 multimerization and that multimerization is required for simultaneous binding of multiple membrane protein ligands by PSD-95.     

Gi-mediated activation of the Ras/MAP kinase pathway involves a 100 kDa tyrosine-phosphorylated Grb2 SH3 binding protein, but not Src nor Shc

Mitogenic G protein-coupled receptors, such as those for lysophosphatidic acid (LPA) and thrombin, activate the Ras/MAP kinase pathway via pertussis toxin (PTX)-sensitive Gi, tyrosine kinase activity and recruitment of Grb2, which targets guanine nucleotide exchange activity to Ras. Little is known about the tyrosine phosphorylations involved, although Src activation and Shc phosphorylation are thought to be critical. We find that agonist-induced Src activation in Rat-1 cells is not mediated by Gi and shows no correlation with Ras/MAP kinase activation. Furthermore, LPA-induced tyrosine phosphorylation of Shc is PTX-insensitive and Ca2+-dependent in COS cells, but undetectable in Rat-1 cells. Expression of dominant-negative Src or Shc does not affect MAP kinase activation by LPA. Thus, Gi-mediated Ras/MAP kinase activation in fibroblasts and COS cells involves neither Src nor Shc. Instead, we detect a 100 kDa tyrosine-phosphorylated protein (p100) that binds to the C-terminal SH3 domain of Grb2 in a strictly Gi- and agonist-dependent manner. Tyrosine kinase inhibitors and wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, prevent p100-Grb2 complex formation and MAP kinase activation by LPA. Our results suggest that the p100-Grb2 complex, together with an upstream non-Src tyrosine kinase and PI 3-kinase, couples Gi to Ras/MAP kinase activation, while Src and Shc act in a different pathway.     

Activation of CPP32-like proteases is not sufficient to trigger apoptosis: inhibition of apoptosis by agents that suppress activation of AP24, but not CPP32-like activity

The 24-kD apoptotic protease (AP24) is a serine protease that is activated during apoptosis and has the capacity to activate internucleosomal DNA fragmentation in isolated nuclei. This study examined the following: (a) the functional relationship between AP24 and the CPP32-like proteases of the caspase family; and (b) whether activation of CPP32-like proteases is sufficient to commit irreversibly a cell to apoptotic death. In three different leukemia cell lines, we showed that agents that directly (carbobenzoxy-Ala-Ala-borophe (DK120) or indirectly inhibit activation of AP24 (protein kinase inhibitors, basic fibroblast growth factor, tosylphenylalaninechloromethylketone, and caspase inhibitors) protected cells from apoptosis induced by TNF or UV light. Only the caspase inhibitors, however, prevented activation of CPP32-like activity as revealed by cleavage of the synthetic substrate, DEVD-pNa, by cell cytosols, and also by in vivo cleavage of poly (ADP-ribosyl) polymerase, a known substrate of CPP32. Activation of DEVD-pNa cleaving activity without apoptosis was also demonstrated in two variants derived from the U937 monocytic leukemia in the absence of exogenous inhibitors. Cell-permeable peptide inhibitors selective for CPP32-like proteases suppressed AP24 activation and apoptotic death. These findings indicate that CPP32-like activity is one of several upstream signals required for AP24 activation. Furthermore, activation of CPP32-like proteases alone is not sufficient to commit irreversibly a cell to apoptotic death under conditions where activation of AP24 is inhibited.     

Double-stranded (ds) RNA binding and not dimerization correlates with the activation of the dsRNA-dependent protein kinase (PKR)

Upon binding to double-stranded (ds) RNA, the dsRNA-dependent protein kinase (PKR) sequentially undergoes autophosphorylation and activation. Activated PKR may exist as a dimer and phosphorylates the eukaryotic translation initiation factor 2 alpha subunit (cIF-2 alpha) to inhibit polypeptide chain initiation. Transfection of COS-1 cells with a plasmid cDNA expression vector encoding a marker gene, activates endogenous PKR, and selectively inhibits translation of the marker mRNA, dihydrofolate reductase (DHFR). This system was used to study the dsRNA binding and dimerization requirements for over-expressed PKR mutants and subdomains to affect DHFR translation. DHFR translation was rescued by expression of either an ATP hydrolysis defective mutant PKR K296P, the amino-terminal 1-243 fragment containing two dsRNA binding motifs, or the isolated first RNA binding motif (amino acids 1-123). Mutation of K64E within the dsRNA binding motif 1 destroyed dsRNA binding and the ability to rescue DHFR translation. Immunoprecipitation of T7 epitope-tagged PKR derivatives from cell lysates detected interaction between intact PKR and the amino-terminal 1-243 fragment as well as a 1-243 fragment harboring the K64E mutation. Expression of adenovirus VAI RNA, a potent inhibitor of PKR activity, did not disrupt this interaction. In contrast, intact PKR did not interact with fragments containing the first dsRNA binding motif (1-123), the second dsRNA binding motif (98-243), or the isolated PKR kinase catalytic domain (228-551). These results demonstrate that the translational stimulation mediated by the dominant negative PKR mutant does not require dimerization, but requires the ability to bind dsRNA and indicate these mutants act by competition for binding to activators.     

Requirement of atypical protein kinase clambda for insulin stimulation of glucose uptake but not for Akt activation in 3T3-L1 adipocytes

Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKCzeta and PKClambda) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKClambda in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKClambda (lambdaKD or lambdaDeltaNKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKClambda, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone- or hyperosmolarity-induced glucose uptake, were inhibited by lambdaKD or lambdaDeltaNKD in a dose-dependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKClambda was approximately 50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKClambda mutant that lacks the pseudosubstrate domain (lambdaDeltaPD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of lambdaDeltaPD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKClambda. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKClambda pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.     

Death activation does not stop tumor growth: quantitative evidence for concomitant activation of apoptosis in tumor growth in vitro

A protein-independent fibrosarcoma, Gc-4 PF, grows exponentially in a protein-free medium. The doubling time (approximately 26 h) was similar to that of the serum-dependent parental clone, Gc-4 SD cultivated in the presence of fetal calf serum (FCS). We demonstrated here that the protein-free cultivation of Gc-4 PF cells concomitantly activates apoptotic phenotypes (one third of total cell population), including typical morphology, high uptake of Hoechst 33342 dye, and cleavage of DNA to large fragments, as observed in protein-deprived Gc-4 SD cell previously. Gc-4 SD cells arrested in the G0/G1-phase in response to the protein-free condition. In contrast, Gc-4 PF cells did not reach G0/G1 arrest in the protein-free condition; instead the durations of both G0/G1 and G2-phases were markedly reduced. The estimation of one cell cycle duration revealed that the cell division cycle was accelerated to 1.7 (27 h/15.4 h)-fold. Then the growth kinetics was able to be verified quantitatively by both the cell division rate and apoptotic cell loss. Protein-free cultivation resulted in slight down-regulation of c-myc protein in both cell types, while the down-regulation of p34cdc2, shown clearly in Gc-4 SD cells, was avoided in Gc-4 PF cells. Interestingly, while the expression of p53 was not affected in Gc-4 SD cells in response to the protein-free condition, the suppressor gene product expression was suppressed markedly in Gc-4 PF cells. These results suggest that Gc-4 PF cells may have acquired an ability to accelerate cell division by shortening the cell cycle duration to maintain a proper growth rate in response to intrinsic apoptosis activation with, at least in part, a suppression of p53 expression as well as an escape of down-regulation of p34cdc2.     

DNA-binding domains of Fos and Jun do not induce DNA curvature: an investigation with solution and gel methods

We demonstrate the use of a DNA minicircle competition binding assay, together with DNA cyclization kinetics and gel-phasing methods, to show that the DNA-binding domains (dbd) of the heterodimeric leucine zipper protein Fos-Jun do not bend the AP-1 target site. Our DNA constructs contain an AP-1 site phased by 1-4 helical turns against an A-tract-directed bend. Competition binding experiments reveal that (dbd)Fos-Jun has a slight preference for binding to linear over circular AP-1 DNAs, independent of whether the site faces in or out on the circle. This result suggests that (dbd)Fos-Jun slightly stiffens rather than bends its DNA target site. A single A-tract bend replacing the AP-1 site is readily detected by its effect on cyclization kinetics, in contrast to the observations for Fos-Jun bound at the AP-1 locus. In contrast, comparative electrophoresis reveals that Fos-Jun-DNA complexes, in which the A-tract bend is positioned close (1-2 helical turns) to the AP-1 site, show phase-dependent variations in gel mobilities that are comparable with those observed when a single A-tract bend replaces the AP-1 site. Whereas gel mobility variations of Fos-Jun-DNA complexes decrease linearly with increasing Mg2+ contained in the gel, the solution binding preference of (dbd)Fos-Jun for linear over circular DNAs is independent of Mg2+ concentration. Hence, gel mobility variations of Fos-Jun-DNA complexes are not indicative of (dbd)Fos-Jun-induced DNA bending (upper limit 5 degrees) in the low salt conditions of gel electrophoresis. Instead, we propose that the gel anomalies depend on the steric relationship of the leucine zipper region with respect to a DNA bend.     

Integrin-mediated activation of MAP kinase is independent of FAK: evidence for dual integrin signaling pathways in fibroblasts

Integrin-mediated cell adhesion causes activation of MAP kinases and increased tyrosine phosphorylation of focal adhesion kinase (FAK). Autophosphorylation of FAK leads to the binding of SH2-domain proteins including Src-family kinases and the Grb2-Sos complex. Since Grb2-Sos is a key regulator of the Ras signal transduction pathway, one plausible hypothesis has been that integrin-mediated tyrosine phosphorylation of FAK leads to activation of the Ras cascade and ultimately to mitogen activated protein (MAP) kinase activation. Thus, in this scenario FAK would serve as an upstream regulator of MAP kinase activity. However, in this report we present several lines of evidence showing that integrin-mediated MAP kinase activity in fibroblasts is independent of FAK. First, a beta1 integrin subunit deletion mutant affecting the putative FAK binding site supports activation of MAP kinase in adhering fibroblasts but not tyrosine phosphorylation of FAK. Second, fibroblast adhesion to bacterially expressed fragments of fibronectin demonstrates that robust activation of MAP kinase can precede tyrosine phosphorylation of FAK. Finally, we have used FRNK, the noncatalytic COOH-terminal domain of FAK, as a dominant negative inhibitor of FAK autophosphorylation and of tyrosine phosphorylation of focal contacts. Using retroviral infection, we demonstrate that levels of FRNK expression sufficient to completely block FAK tyrosine phosphorylation were without effect on integrin-mediated activation of MAP kinase. These results strongly suggest that integrin-mediated activation of MAP kinase is independent of FAK and indicate the probable existence of at least two distinct integrin signaling pathways in fibroblasts.     

Kit signaling through PI 3-kinase and Src kinase pathways: an essential role for Rac1 and JNK activation in mast cell proliferation

The receptor tyrosine kinase Kit plays critical roles in hematopoiesis, gametogenesis and melanogenesis. In mast cells, Kit receptor activation mediates several cellular responses including cell proliferation and suppression of apoptosis induced by growth factor deprivation and gamma-irradiation. Kit receptor functions are mediated by kinase activation, receptor autophosphorylation and association with various signaling molecules. We have investigated the role of phosphatidylinositol 3'-kinase (PI 3-kinase) and Src kinases in Kit-mediated cell proliferation and suppression of apoptosis induced both by factor deprivation and irradiation in bone marrow-derived mast cells (BMMC). Analysis of Kit-/- BMMC expressing mutant Kit receptors and the use of pharmacological inhibitors revealed that both signaling pathways contribute to these Kit-mediated responses and that elimination of both pathways abolishes them. We demonstrate that the PI 3-kinase and Src kinase signaling pathways converge to activate Rac1 and JNK. Analysis of BMMC expressing wild-type and dominant-negative mutant forms of Rac1 and JNK revealed that the Rac1/JNK pathway is critical for Kit ligand (KL)-induced proliferation of mast cells but not for suppression of apoptosis. In addition, KL was shown to inhibit sustained activation of JNK induced by gamma-irradiation and concomitant irradiation-induced apoptosis.     

Glycerol kinase from Escherichia coli and an Ala65-->Thr mutant: the crystal structures reveal conformational changes with implications for allosteric regulation

BACKGROUND: Glycerol kinase (GK) from Escherichia coli is a velocity-modulated (V system) enzyme that has three allosteric effectors with independent mechanisms: fructose-1,6-bisphosphate (FBP); the phosphocarrier protein IIAGlc; and adenosine nucleotides. The enzyme exists in solution as functional dimers that associate reversibly to form tetramers. GK is a member of a superfamily of ATPases that share a common ATPase domain and are thought to undergo a large conformational change as an intrinsic step in their catalytic cycle. Members of this family include actin, hexokinase and the heat shock protein hsc70. RESULTS: We report here the crystal structures of GK and a mutant of GK (Ala65-->Thr) in complex with glycerol and ADP. Crystals of both enzymes contain the same 222 symmetric tetramer. The functional dimer is identical to that described previously for the IIAGlc-GK complex structure. The tetramer interface is significantly different, however, with a relative 22.3 degrees rotation and 6.34 A translation of one functional dimer. The overall monomer structure is unchanged except for two regions: the IIAGlc-binding site undergoes a structural rearrangement and residues 230-236 become ordered and bind orthophosphate at the tetramer interface. We also report the structure of a second mutant of GK (IIe474-->Asp) in complex with IIAGlc; this complex crystallized isomorphously to the wild type IIAGlc-GK complex. Site-directed mutants of GK with substitutions at the IIAGlc-binding site show significantly altered kinetic and regulatory properties, suggesting that the conformation of the binding site is linked to the regulation of activity. CONCLUSIONS: We conclude that the new tetramer structure presented here is an inactive form of the physiologically relevant tetramer. The structure and location of the orthophosphate-binding site is consistent with it being part of the FBP-binding site. Mutational analysis and the structure of the IIAGlc-GK(IIe474-->Asp) complex suggest the conformational transition of the IIAGlc-binding site to be an essential aspect of IIAGlc regulation.     

The N-terminal extracellular segments of the chemokine receptors CCR1 and CCR3 are determinants for MIP-1alpha and eotaxin binding, respectively, but a second domain is essential for efficient receptor activation

CCR1 and CCR3 are seven-transmembrane domain G protein-coupled receptors specific for members of the CC chemokine subgroup of leukocyte chemoattractants. Both have been implicated in the inflammatory response, and CCR3, through its expression on eosinophils, basophils, and Th2 lymphocytes, may be especially important in allergic inflammation. CCR1 and CCR3 are 54% identical in amino acid sequence and share some ligands but not others. In particular, macrophage inflammatory protein 1alpha (MIP-1alpha) is a ligand for CCR1 but not CCR3, and eotaxin is a ligand for CCR3 but not CCR1. To map ligand selectivity determinants and to guide rational antagonist design, we analyzed CCR1:CCR3 chimeric receptors. When expressed in mouse pre-B cells, chimeras in which the N-terminal extracellular segments were switched were both able to bind both MIP-1alpha and eotaxin, but in each case, binding occurred via separate sites. Nevertheless, neither MIP-1alpha nor eotaxin were effective agonists at either chimeric receptor in either calcium flux or chemotaxis assays. These data are consistent with a multi-site model for chemokine-chemokine receptor interaction in which one or more subsites determine chemokine selectivity, but others are needed for receptor activation. Agents that bind to the N-terminal segments of CCR1 and CCR3 may be useful in blocking receptor function.     

Analysis of liver regeneration in mice lacking type 1 or type 2 tumor necrosis factor receptor: requirement for type 1 but not type 2 receptor

We used KO mice lacking either TNF receptor 1 (TNFR-1) or receptor 2 (TNFR-2) to determine whether signaling at the start of liver regeneration after partial hepatectomy (PH) involves only one or both TNF receptors and to analyze in more detail the abnormalities caused by lack of TNFR-1 receptor, which is required for the initiation of liver regeneration. Lack of TNFR-2 had little effect on NF-kappaB and STAT3 binding, and no effect in interleukin-6 production after PH, but caused a delay in AP-1 and C/EBP binding and in the expression of c-jun and c-myc messenger RNA (mRNA). In contrast to mice lacking TNFR-1, which had deficient hepatocyte DNA synthesis and massive lipid accumulation in hepatocytes, TNFR-2 KO mice had normal liver structure and similar levels of hepatocyte DNA replication as those of wild type mice. We conclude that TNFR-1, but not TNFR-2, is necessary for liver regeneration, and that NF-kappaB and STAT3 binding are activated by signals transduced by TNFR-1. Inhibition of AP-1 and C/EBP binding and in the expression of c-jun and c-myc mRNA in the first 4 hours after PH, as well as the apparent lack of Fos in AP-1 complexes, had no effect on the timing or extent of DNA replication.     

Phonological activation of category coordinates during speech planning is observable in children but not in adults: Evidence for cascaded processing.

There is a long-standing debate in the area of speech production on the question of whether only words selected for articulation are phonologically activated (as maintained by serial-discrete models) or whether this is also true for their semantic competitors (as maintained by forward-cascading and interactive models). Past research has addressed this issue by testing whether retrieval of a target word (e.g., cat) affects--or is affected by--the processing of a word that is phonologically related to a semantic category coordinate of the target (e.g., doll, related to dog) and has consistently failed to obtain such mediated effects in adult speakers. The authors present a series of experiments demonstrating that mediated effects are present in children (around age 7) and diminish with increasing age. This observation provides further evidence for cascaded models of lexical retrieval. (PsycINFO Database Record (c) 2010 APA, all rights reserved)