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The cytokine receptor gp130 is the common signaling subunit of receptors used by the interleukin (IL)-6 cytokine family. gp130 is widely expressed in breast cancer cell lines and primary tumors. The role of gp130 in breast cancer in vivo is unknown. To study the effect of gp130 inhibition in breast cancer, endogenous gp130 signaling in breast cancer cell lines was blocked with a dominant-negative gp130 protein (DN gp130). DN gp130 inhibited constitutive Stat3 activation in breast cancer cells. Both gp130 and epidermal growth factor receptor (EGFR) have been implicated in constitutive Stat3 activation in breast cancer. There are known physical and functional interactions between gp130 and EGFR. Consistent with this, we show that DN gp130 inhibits signaling downstream of the EGFR in breast cancer cells. The effect of DN gp130 on breast cancer in vivo was assessed with an orthotopic nude mouse model. DN gp130 MDA-231 cells had markedly decreased engraftment, size, and metastasis compared with control cells. These results are particularly striking considering that DN gp130-expressing breast cancer cells grow faster in vitro. We hypothesized that DN gp130 expression results in inhibition of invasion and metastasis in vivo. Marked angiogenesis was present in tumors from control animals and was absent in tumors from DN gp130 animals. We additionally show that tissue inhibitor of metalloproteinase-3, an inhibitor of tumor invasion and angiogenesis, is up-regulated in both MDA-231 DN gp130 cells and tumors. These results, in light of the availability of several potential pharmacological inhibitors of gp130, suggest novel approaches to breast cancer therapy.

The Janus kinases are considered to be cytoplasmic kinases that constitutively associate with the cytoplasmic region of cytokine receptors, and the Janus kinases (Jaks) are crucial for cytokine signal transduction. We investigated Jak1 localization using subcellular fractionation techniques and fluorescence microscopy (immunofluorescence and yellow fluorescent protein-tagged Jaks). In the different experimental approaches we found Jak1 (as well as Jak2 and Tyk2) predominantly located at membranes. In contrast to previous reports we did not observe Jak proteins in significant amounts within the nucleus or in the cytoplasm. The cytoplasmic localization observed for the Jak1 mutant L80A/Y81A, which is unable to associate with cytokine receptors, indicates that Jak1 does not have a strong intrinsic membrane binding potential and that only receptor binding is crucial for the membrane recruitment. Finally we show that Jak1 remains a membrane-localized protein after cytokine stimulation. These data strongly support the hypothesis that cytokine receptor.Janus kinase complexes can be regarded as receptor tyrosine kinases.

The transcription factor STAT3 is most important for the signal transduction of interleukin-6 and related cytokines. Upon stimulation cytoplasmic STAT3 is phosphorylated at tyrosine 705, translocates into the nucleus, and induces target genes. Notably, STAT proteins are also detectable in the nuclei of unstimulated cells. In this report we introduce a new method for the real time analysis of STAT3 nucleocytoplasmic shuttling in living cells which is based on the recently established fluorescence localization after photobleaching (FLAP) approach. STAT3 was C-terminally fused with the cyan (CFP) and yellow (YFP) variants of the green fluorescent protein. In the resulting STAT3-CFP-YFP (STAT3-CY) fusion protein the YFP can be selectively bleached using the 514-nm laser of a confocal microscope. This setting allows studies on the dynamics of STAT3 nucleocytoplasmic transport by monitoring the subcellular distribution of fluorescently labeled and selectively bleached STAT3-CY. By this means we demonstrate that STAT3-CY shuttles continuously between the cytosol and the nucleus in unstimulated cells. This constitutive shuttling does not depend on the phosphorylation of tyrosine 705 because a STAT3(Y705F)-CY mutant shuttles to the same extent as STAT3-CY. Experiments with deletion mutants reveal that the N-terminal moiety of STAT3 is essential for shuttling. Further studies suggest that a decrease in STAT3 nuclear export contributes to the nuclear accumulation of STAT3 in response to cytokine stimulation. The new approach presented in this study is generally applicable to any protein of interest for analyzing nucleocytoplasmic transport mechanisms in real time.

The human muscle chloride channel ClC-1 has a 398-amino acid carboxyl-terminal domain that resides in the cytoplasm and contains two CBS (cystathionine-beta-synthase) domains. To examine the role of this region, we studied various carboxyl-terminal truncations by heterologous expression in mammalian cells, whole-cell patch clamp recording, and confocal imaging. Channel constructs lacking parts of the distal CBS domain, CBS2, did not produce functional channels, whereas deletion of CBS1 was tolerated. ClC channels are dimeric proteins with two ion conduction pathways (protopores). In heterodimeric channels consisting of one wild type subunit and one subunit in which the carboxyl terminus was completely deleted, only the wild type protopore was functional, indicating that the carboxyl terminus supports the function of the protopore. All carboxyl-terminal-truncated mutant channels fused to yellow fluorescent protein were translated and the majority inserted into the plasma membrane as revealed by confocal microscopy. Fusion proteins of cyan fluorescent protein linked to various fragments of the carboxyl terminus formed soluble proteins that could be redistributed to the surface membrane through binding to certain truncated channel subunits. Stable binding only occurs between carboxyl-terminal fragments of a single subunit, not between carboxyl termini of different subunits and not between carboxyl-terminal and transmembrane domains. However, an interaction with transmembrane domains can modify the binding properties of particular carboxyl-terminal proteins. Our results demonstrate that the carboxyl terminus of ClC-1 is not necessary for intracellular trafficking but is critical for channel function. Carboxyl termini fold independently and modify individual protopores of the double-barreled channel.

The immediate early response of cells treated with IL-6 (interleukin-6) is the activation of the signal transducer and activator of transcription (STAT)3. The Src homology domain 2 (SH2)-containing protein tyrosine phosphatase SHP2 and the feedback inhibitor SOCS3 (suppressor of cytokine signalling) are potent inhibitors of IL-6 signal transduction. Impaired function of SOCS3 or SHP2 leads to enhanced and prolonged IL-6 signalling. The inhibitory function of both proteins depends on their recruitment to the tyrosine motif 759 within glycoprotein gp130. In contrast to inactivation, desensitization of signal transduction is regarded as impaired responsiveness due to prestimulation. Usually, after activation the sensing receptor becomes inactivated by modifications such as phosphorylation, internalization or degradation. We designed an experimental approach which allows discrimination between desensitization and inactivation of IL-6 signal transduction. We observed that pre-stimulation with IL-6 renders cells less sensitive to further stimulation with IL-6. After several hours, the cells become sensitive again. We show that not only signal transduction through previously activated receptors is affected by desensitization but signalling through receptors which were not targeted by the first stimulation was also attenuated ( trans -desensitization). Interestingly, in contrast to inhibition, desensitization does not depend on the presence of functional SHP2. Furthermore, cells lacking SOCS3 show constitutive STAT3 activation which is not affected by pre-stimulation with IL-6. All these observations suggest that desensitization and inhibition of signalling are mechanistically distinct.

The kidney tubulointerstitium contains numerous bone marrow-derived antigen-presenting cells, which are often referred to as resident tissue macrophages, although several previous studies had demonstrated characteristics of dendritic cells (DC). In this study, we describe a subset of tubulointerstitial cells expressing the DC marker CD11c. A protocol was established to isolate these cells for in vitro analysis. Renal CD11c(+) cells resembled splenic DC, but not peritoneal macrophages, in morphology, lysosomal content, phagocytic activity, microbicidal effector functions, expression of T cell costimulatory molecules, and ability to activate T cells. Nevertheless, many CD11c(+) renal cells expressed low or intermediate levels of F4/80 and CD11b, indicating that both markers are not absolutely specific for macrophages in the kidney. Subpopulations of renal DC could be distinguished based on their expression of MHC class II and costimulatory molecules and may represent different maturation stages. In nephrotoxic glomerulonephritis, increased numbers of CD11c(+) cells showing DC functionality were found in the tubulointerstitium. Focal accumulation was seen within tubulointerstitial mononuclear infiltrates and adjacent to, but not within, inflamed glomeruli. These results are the first to identify and characterize renal CD11c(+) cells as DC and to demonstrate marked changes in experimental glomerulonephritis.

A novel method employing filter arrays of a cDNA expression library for the identification of substrates for protein kinases was developed. With this technique, we identified a new member of the cyclin family, cyclin L2, as a substrate of the nuclear protein kinase DYRK1A. Cyclin L2 contains an N-terminal cyclin domain and a C-terminal arginine/serine-rich domain (RS domain), which is a hallmark of many proteins involved in pre-mRNA processing. The gene for cyclin L2 encodes the full-length cyclin L2, which is predominantly expressed in testis, as well as a truncated splicing variant (cyclin L2S) that lacks the RS domain and is ubiquitously expressed in human tissues. Full-length cyclin L2, but not cyclin L2S, was associated with the cyclin-dependent kinase PITSLRE. Cyclin L2 interacted with splicing factor 2 in vitro and was co-localized with the splicing factor SC35 in the nuclear speckle compartment. Photobleaching experiments showed that a fusion protein of green fluorescent protein and cyclin L2 in nuclear speckles rapidly exchanged with unbleached molecules in the nucleus, similar to other RS domain-containing proteins. In striking contrast, the closely related green fluorescent protein-cyclin L1 was immobile in the speckle compartment. DYRK1A interacted with cyclin L2 in pull-down assays, and overexpression of DYRK1A stimulated phosphorylation of cyclin L2 in COS-7 cells. These data characterize cyclin L2 as a highly mobile component of nuclear speckles and suggest that DYRK1A may regulate splicing by phosphorylation of cyclin L2.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is involved in a variety of biological functions. It is essential for the signal transduction of interleukin-6 (IL-6) and related cytokines. In response to IL-6 stimulation STAT3 becomes phosphorylated and translocates into the nucleus where it binds to enhancer sequences of target genes. We found that activated STAT3 is enriched in dot-like structures within the nucleus, which we termed STAT3 nuclear bodies. To examine the dynamics of STAT3 nuclear body formation, a fusion protein of STAT3 and yellow fluorescent protein (YFP) was constructed. Studies in living cells have shown that the appearance of STAT3 nuclear bodies is transient, correlating with the timecourse of tyrosine-phosphorylation of STAT3. Furthermore, we show by fluorescence recovery after photobleaching (FRAP) analysis that STAT3 within nuclear bodies consists of a highly mobile and an immobile fraction. Colocalization studies provided evidence that these bodies are accompanied with CREB binding protein (CBP) and acetylated histone H4, which are markers for transcriptionally active chromatin. Moreover, STAT3 nuclear bodies in HepG2 cells are not colocalized with promyelocytic leukemia oncoprotein (PML)-containing bodies; neither is a sumoylation of activated STAT3 detectable. Taken together, our data suggest that STAT3 nuclear bodies are either directly involved in active gene transcription or they serve as reservoirs of activated STAT3.

Signal transduction through cytokine receptors is mediated mainly by non-covalently associated Jak tyrosine kinases. By confocal microscopy, the cytokine receptor gp130 and Jak1, fused with either yellow (YFP) or cyan (CFP) fluorescent protein, were found to be colocalized predominantly at intracellular vesicular structures and at the plasma membrane. Quantitative fluorescence recovery after photobleaching (FRAP) analysis at the plasma membrane revealed equal mobilities for gp130-YFP and Jak1-YFP. Thus, Jak1-YFP diffuses like a transmembrane protein indicating that membrane-bound Jak1 does not exchange rapidly with cytosolic Jaks. Applying a novel dual-color FRAP approach we found that immobilization of gp130-CFP by a pair of monoclonal antibodies led to a corresponding immobilization of co-transfected Jak1-YFP. We conclude from these findings that Jak1, once bound to a gp130 molecule, does not exchange between different receptors at the plasma membrane neither via the cytoplasmic compartment nor via a membrane-associated state.

Diverse cytokines can signal through receptor complexes containing the gp130 subunit. These cytokines, which include interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), and CLC (cardiotrophin-like cytokine), are implicated in inflammatory and immune responses, heart development, and fertility. The gp130 receptor-ligand complexes are contrasted with the complex formed by erythropoietin (Epo) and its receptor, EpoR. Also discussed are insights provided by the structural analysis of IL-6, its alpha receptor (IL6Ralpha), and the signal transducer gp130 for understanding receptor-ligand assembly, for predicting the structures of ligand-receptor complexes of the other IL-6-type cytokines, and for rational drug design.

The IL (interleukin)-6-type cytokines IL-6, IL-11, LIF (leukaemia inhibitory factor), OSM (oncostatin M), ciliary neurotrophic factor, cardiotrophin-1 and cardiotrophin-like cytokine are an important family of mediators involved in the regulation of the acute-phase response to injury and infection. Besides their functions in inflammation and the immune response, these cytokines play also a crucial role in haematopoiesis, liver and neuronal regeneration, embryonal development and fertility. Dysregulation of IL-6-type cytokine signalling contributes to the onset and maintenance of several diseases, such as rheumatoid arthritis, inflammatory bowel disease, osteoporosis, multiple sclerosis and various types of cancer (e.g. multiple myeloma and prostate cancer). IL-6-type cytokines exert their action via the signal transducers gp (glycoprotein) 130, LIF receptor and OSM receptor leading to the activation of the JAK/STAT (Janus kinase/signal transducer and activator of transcription) and MAPK (mitogen-activated protein kinase) cascades. This review focuses on recent progress in the understanding of the molecular mechanisms of IL-6-type cytokine signal transduction. Emphasis is put on the termination and modulation of the JAK/STAT signalling pathway mediated by tyrosine phosphatases, the SOCS (suppressor of cytokine signalling) feedback inhibitors and PIAS (protein inhibitor of activated STAT) proteins. Also the cross-talk between the JAK/STAT pathway with other signalling cascades is discussed.

Interleukin (IL)-6 is involved in the maintenance and progression of several diseases such as multiple myeloma, rheumatoid arthritis, or osteoporosis. The present work aims at the development of an IL-6 inhibitor for the use in anti-cytokine therapies. The IL-6 receptor is composed of two different subunits, an alpha-subunit (IL-6Ralpha) that binds IL-6 with low affinity and a beta-subunit (gp130) that binds the IL-6.IL-6Ralpha complex with high affinity and as a result triggers intracellular signaling. In its soluble form, gp130 is a natural antagonist that neutralizes IL-6.soluble IL-6Ralpha complexes. It was our strategy to appropriately fuse the two receptor subunit fragments involved in IL-6 receptor complex formation to bind IL-6 with high affinity and to antagonize its effects. The ligand-binding domains of gp130 (D1-D2-D3) and IL-6Ralpha (D2-D3) were connected using three different linkers. The resulting constructs were expressed in stably transfected insect cells and tested for their ability to inhibit IL-6 activity in several in vitro systems. All fusion proteins were strong inhibitors of IL-6 signaling and abrogated IL-6-induced phosphorylation of STAT3, proliferation of transfected Ba/F3 cells, and induction of acute-phase protein synthesis. As intended, the fused receptors were much more effective than the separately expressed soluble receptor proteins. The fusion protein strategy presented here can also be applied to other cytokines that signal via receptors composed of two different subunits to design new potent inhibitors for anti-cytokine therapies.

Idiopathic generalized epilepsy (IGE) is an inherited neurological disorder affecting about 0.4% of the world's population. Mutations in ten genes causing distinct forms of idiopathic epilepsy have been identified so far, but the genetic basis of many IGE subtypes is still unknown. Here we report a gene associated with the four most common IGE subtypes: childhood and juvenile absence epilepsy (CAE and JAE), juvenile myoclonic epilepsy (JME), and epilepsy with grand mal seizures on awakening (EGMA; ref. 8). We identified three different heterozygous mutations in the chloride-channel gene CLCN2 in three unrelated families with IGE. These mutations result in (i) a premature stop codon (M200fsX231), (ii) an atypical splicing (del74-117) and (iii) a single amino-acid substitution (G715E). All mutations produce functional alterations that provide distinct explanations for their pathogenic phenotypes. M200fsX231 and del74-117 cause a loss of function of ClC-2 channels and are expected to lower the transmembrane chloride gradient essential for GABAergic inhibition. G715E alters voltage-dependent gating, which may cause membrane depolarization and hyperexcitability.

On human macrophages IL-10 acts as a more potent anti-inflammatory cytokine than IL-6, although both cytokines signal mainly via activation of the transcription factor STAT3. In this study we compare IL-10 and IL-6 signaling in primary human macrophages derived from blood monocytes. Pretreatment of macrophages with PMA or the proinflammatory mediators LPS and TNF-alpha blocks IL-6-induced STAT3 activation, whereas IL-10-induced activation of STAT3 remains largely unaffected. Although LPS induces the feedback inhibitor suppressor of cytokine signaling 3 (SOCS3) in macrophages, inhibition of IL-6 signal transduction by LPS occurs rapidly and does not depend on gene transcription. We also found that pretreatment of macrophages with IL-10 inhibits subsequent STAT3 activation by IL-6, whereas IL-10-induced STAT3 activation is not affected by preincubation with IL-6. This cross-inhibition is dependent on active transcription and might therefore be explained by different sensitivities of IL-10 and IL-6 signaling toward the feedback inhibitor SOCS3, which is induced by both cytokines. In contrast to the IL-6 signal transducer gp130, which has been previously shown to recruit SOCS3 to one of its phosphotyrosine residues (Y759), peptide precipitation experiments suggest that SOCS3 does not interact with phosphorylated tyrosine motifs of the IL-10R. Taken together, different sensitivities of IL-10 and IL-6 signaling toward mechanisms that inhibit the Janus kinase/STAT pathway define an important mechanism that contributes to the different anti-inflammatory potencies of these two cytokines.

gp130 is the common signal transducing receptor subunit of interleukin (IL)-6-type cytokines. gp130 either homodimerizes in response to IL-6 and IL-11 or forms heterodimers with the leukemia inhibitory factor (LIF) receptor (LIFR) in response to LIF, oncostatin M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1) or cardiotrophin-like cytokine resulting in the onset of cytoplasmic tyrosine phosphorylation cascades. The extracellular parts of both gp130 and LIFR consist of several Ig-like and fibronectin type III-like domains. The role of the membrane-distal domains of gp130 (D1, D2, D3) and LIFR in ligand binding is well established. In this study we investigated the functional significance of the membrane-proximal domains of gp130 (D4, D5, D6) in respect to heterodimerization with LIFR. Deletion of each of the membrane-proximal domains of gp130 (Delta 4, Delta 5 and Delta 6) leads to LIF unresponsiveness. Replacement of the gp130 domains by the corresponding domains of the related GCSF receptor either restores weak LIF responsiveness (D4-GCSFR), leads to constitutive activation of gp130 (D5-GCSFR) or results in an inactive receptor (D6-GCSFR). Mutation of a specific cysteine in D5 of gp130 (C458A) leads to constitutive heterodimerization with the LIFR and increased sensitivity towards LIF stimulation. Based on these findings, a functional model of the gp130-LIFR heterodimer is proposed that includes contacts between D5 of gp130 and the corresponding domain D7 of the LIFR and highlights the requirement for both receptor dimerization and adequate receptor orientation as a prerequisite for signal transduction.

Glycoprotein 130 (gp130) is a type I transmembrane protein and serves as the common signal-transducing receptor subunit of the interleukin-6-type cytokines. Whereas the membrane-distal half of the gp130 extracellular part confers ligand binding and has been subject to intense investigation, the structural and functional features of its membrane-proximal half are poorly understood. On the basis of predictions of tertiary structure, the membrane-proximal part consists of three fibronectin-type-III-like domains D4, D5 and D6. Here we describe the bacterial expression of the polypeptides predicted to comprise each of these three domains. The recombinant proteins were refolded from solubilized inclusion bodies in vitro, purified to homogeneity and characterized by means of size-exclusion chromatography and CD spectroscopy. For the first time the prediction of three individual membrane-proximal protein domains for gp130 has been verified experimentally. The three domains do not show intermediate-affinity or high-affinity interactions between each other. Mapping of a neutralizing gp130 monoclonal antibody against D4 suggested a particular functional role of this domain for gp130 activation, because above that an intrinsic tendency for low-affinity oligomerization was demonstrated for D4.

The interleukin-11 receptor (IL-11R) belongs to the hematopoietic receptor superfamily. The functional receptor complex comprises IL-11, IL-11R and the signal-transducing subunit gp130. The extracellular part of the IL-11R consists of three domains: an N-terminal immunoglobulin-like domain, D1, and two fibronectin-type III-like (FNIII) domains and D2 and D3. The two FNIII domains comprise the cytokine receptor-homology region defined by a set of four conserved cysteine residues in the N-terminal domain (D2) and a WSXWS sequence motif in the C-terminal domain (D3). We investigated the structural and functional role of the third extracellular receptor domain of IL-11R. A molecular model of the human IL-11/IL-11R complex allowed the identification of amino acid residues in IL-11R to be involved in ligand binding. Most of them were located in the third extracellular domain, which therefore should be able to bind with high affinity to IL-11. To prove this prediction, domain D3 of the IL-11R was expressed in Escherichia coli, refolded and purified. For structural characterization, circular dichroism, fluorescence and NMR spectroscopy were used. By plasmon resonance experiments, we show that the ligand-binding capacity of this domain is as high as that one for the whole receptor. These results provide a basis for further structural investigations that could be used for the rational design of potential agonists and antagonists essential in human therapy.

Cytokines are key mediators for the regulation of hemopoiesis and the coordination of immune responses. They exert their various functions through activation of specific cell surface receptors, thereby initiating intracellular signal transduction cascades which lead to defined cellular responses. As the common signal-transducing receptor subunit of at least seven different cytokines, gp130 is an important member of the family of hemopoietic cytokine receptors which are characterized by the presence of at least one cytokine-binding module. Mutants of gp130 that either lack the Ig-like domain D1 (DeltaD1) or contain a distinct mutation (F191E) within the cytokine-binding module have been shown to be severely impaired with respect to IL-6 induced signal transduction. After cotransfection of COS-7 cells with a combination of both inactive gp130 mutants, signal transduction in response to IL-6 is restored. Whereas cells transfected with DeltaD1 do not bind IL-6/sIL-6R complexes, cells transfected with the F191E mutant bind IL-6/sIL-6R with low affinity. Combination of DeltaD1 and F191E, however, leads to high-affinity ligand binding. These data suggest that two different gp130 epitopes, one on each receptor chain, sequentially cooperate in asymmetrical binding of IL-6/IL-6R in a tetrameric signaling complex. On the basis of our data, a model for the mechanism of IL-6-induced gp130 activation is proposed.

A panel of 14 hybridoma cell lines secreting monoclonal antibodies against the human interleukin-11 receptor alpha chain (hIL-11Ralpha) was obtained using two different approaches. Two antibodies were raised against peptides of the N- and C-terminal sequences, respectively, of the extracellular part of the hIL-11Ralpha. Another group of 12 antibodies was generated against a hybrid protein consisting of the extracellular part of the hIL-11Ralpha fused to mature full-length human IL-2. All these antibodies recognized native hIL-11Ralpha and most also recognized the denatured receptor on immunoblots after SDS-PAGE. Four different epitopes were identified on the extracellular part of the hIL-11Ralpha. One epitope, defined by the E27 antibody, is located at the N-terminus and the other three epitopes are clustered in the membrane-proximal, C-terminal region. The antibodies defining epitopes I and II recognized membrane-bound hIL-11Ralpha expressed in gp130/hIL-11Ralpha-co-transfected Ba/F3 cells. The E27 antibody cross-reacted with murine IL-11Ralpha, in agreement with the fact that the N-terminal region is highly conserved between species. The other 13 antibodies all recognized a region between amino acids 319 and 363, which is the membrane-proximal part of the hIL-11Ralpha. This region, which is less conserved between mouse and human, is shown here to be an immunodominant region. Anti-IL-11Ralpha monoclonal antibodies, which have not been described previously enabled us to explore the expression and tissue distribution of IL-11Ralpha on human peripheral blood mononuclear cells and cell lines. The antibodies provide powerful tools for the study of the regulation and function of the receptor.

Gp130 is the common signal transducing receptor subunit of interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor and cardiotrophin-1. IL-6 and IL-11 induce gp130 homodimerization whereas the others lead to the formation of heterodimers with LIFR or OSMR. Binding epitopes for IL-6 and IL-11 are located in the immunoglobulin-like domain and the cytokine binding module (CBM). Here we show that a gp130 mutant lacking domain 1, although unresponsive to IL-6 and IL-11, can still activate signal transducer and activator of transcription (STAT) transcription factors in response to LIF or OSM. Moreover, point mutations in the CBM of gp130 (F191E and V252D) that severely impair signal transduction in response to IL-6 and IL-11 differentially interfere with gp130 activation in response to LIF and OSM. Thus, epitopes involved in gp130 homodimerization are distinct from those leading to the formation of gp130/LIFR or gp130/OSMR heterodimers. These findings may serve as the base for rational design of gp130 antagonists that specifically interfere with bioactivity of distinct IL-6-type cytokines.

The function of the signal-transducing receptor subunit glycoprotein 130 (gp130) in the IL-6-receptor complex has previously been studied using carboxyl-terminal deletion mutants or a truncated molecule of approximately 60 membrane-proximal amino acids (containing box 1 and box 2) linked to the individual gp130 tyrosine motifs. However, the redundancy of the tyrosine motifs within the cytoplasmic part of gp130 has been neglected. Here we describe the analysis of the function of the individual cytoplasmic tyrosine residues of gp130 in the context of the full-length receptor protein in IL-6 signaling as measured by STAT activation, acute phase protein induction, and stimulation of proliferation. Add-back receptor mutants containing only one cytoplasmic tyrosine have been generated and tested for their efficiency in IL-6 signal transduction. Our studies revealed that tyrosine motifs which have been described to recruit STAT proteins are not equivalent with respect to their potential to activate STAT factors and acute phase protein gene promoters: the two distal tyrosines, Tyr905 and Tyr915, of gp130 were more potent than Tyr767 and Tyr814. Surprisingly, Tyr905 and Tyr915 mediate acute phase protein gene promoter activation stronger than the wild-type receptor containing all six cytoplasmic tyrosine residues. In contrast, Ba/F3 cells stably transfected with add-back receptors containing Tyr767 or Tyr905 were more sensitive to IL-6-induced proliferation than cells expressing the other add-back receptor mutants. Thus, the tyrosine residues in the cytoplasmic part of gp130 were found to contribute differentially to IL-6 signal transduction in the full- length gp130 protein.

The transmembrane glycoprotein gp130 is the common signal transducing receptor subunit of the IL-6-type cytokines. The gp130 extracellular part is predicted to consist of six individual domains. Whereas the role of the three membrane-distal domains (D1-D3) in binding of IL-6 and IL-11 is well established, the function of the membrane-proximal domains (D4-D6) is unclear. Mapping of a neutralizing mAb to the membrane-proximal part of gp130 suggests a functional role of D4-D6 in receptor activation. Individual deletion of these three domains differentially interferes with ligand binding of the soluble and membrane-bound receptors. All deletion mutants do not signal in response to IL-6 and IL-11. The deletion mutants Delta4 and, to a lesser extent, Delta6 are still activated by agonistic monoclonal gp130 Abs, whereas the deletion mutant Delta5 does not respond. Because membrane-bound Delta5 binds IL-6/soluble IL-6R as does wild-type gp130, but does not transduce a signal in response to various stimuli, this domain plays a prominent role in coupling of ligand binding and signal transduction. Replacement of the fifth domain of gp130 by the corresponding domain of the homologous G-CSF receptor leads to constitutive activation of the chimera upon overexpression in COS-7 cells. In HepG2 cells this mutant responds to IL-6 comparable to wild-type gp130. Our findings suggest a functional role of the membrane-proximal domains of gp130 in receptor activation. Thus, within the hematopoietic receptor family the mechanism of receptor activation critically depends on the architecture of the receptor ectodomain.

Interleukin-11 (IL-11) belongs to the interleukin-6 (IL-6)-type subfamily of long-chain helical cytokines including IL-6, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M, and cardiotrophin-1, which all share the glycoprotein gp130 as a signal transducing receptor component. IL-11 acts on cells expressing gp130 and the IL-11 receptor (IL-11R) alpha-subunit (IL-11Ralpha). The structural epitopes of IL-11 required for the recruitment of the individual receptor subunits have not yet been defined. Based on the structure of CNTF, a three-dimensional model of human IL-11 was built. Using this model, 10 surface exposed amino acid residues of IL-11 were selected for mutagenesis using analogies to the well-characterized receptor recruitment sites of IL-6, CNTF, and LIF. The respective mutants of human IL-11 were expressed as soluble fusion proteins in bacteria. Their biological activities were determined on HepG2 and Ba/F3-130-11alpha cells. Several mutants with substantially decreased bioactivity and one hyperagonistic mutant were identified and further analyzed with regard to recruitment of IL-11Ralpha and gp130. The low-activity mutant I171D still binds IL-11Ralpha but fails to recruit gp130, whereas the hyperagonistic variant R135E more efficiently engages the IL-11R subunits. The low-activity mutants R190E and L194D failed to bind to IL-11Ralpha. These findings reveal a common mechanism of receptor recruitment in the family of IL-6-type cytokines and offer considerable perspectives for the rational design of IL-11 antagonists and hyperagonists.

Interleukin-11 is a hematopoietic cytokine that signals via the signal transducer gp130. Although gp130 is ubiquitously expressed, interleukine-11 responsiveness is restricted to cells that express the interleukine-11 receptor alpha-subunit. The interleukine-11 receptor alpha-subunit can be functionally replaced by its soluble form indicating that the transmembrane and cytoplasmic parts are not required for signal transduction. Here, we show that a recombinant fusion protein of a fragment of the human interleukine-11 receptor alpha-subunit ectodomain linked to human interleukine-11 acts as a superagonist on cells expressing gp130 but lacking the membrane-bound interleukine-11 receptor alpha-subunit. It induces acute phase protein synthesis in hepatoma cells and efficiently promotes proliferation of Ba/F3 cells stably, transfected with gp130. In these bioassays, the fusion protein of a fragment of the human interleukine-11 receptor alpha-subunit ectodomain linked to human interleukine-11 is 50 times more potent than the combination of interleukine-11 and the soluble interleukine-11 receptor alpha-subunit. Thus, our findings support the concept that covalent fusion of two soluble proteins required for receptor activation dramatically increases their bioactivity.

Leukaemia inhibitory factor (LIF) signals via a heterodimeric receptor complex comprised of the LIF receptor (LIFR) and the interleukin (IL)-6 signal transducer gp130. Upon binding to its cognate receptor LIF is internalized. In this study, we show that the LIFR is endocytosed independently of gp130. By using a heterochimaeric receptor system we identified a dileucine-based internalization motif within the cytoplasmic domain of the LIFR. Our findings suggest that a heterodimeric LIFR/gp130 complex and homodimeric gp130/gp130 complex are endocytosed via distinct internalization signals.

The coordination and regulation of immune responses are primarily mediated by cytokines that bind to specific cell surface receptors. Glycoprotein 130 (gp130) belongs to the family of class I cytokine receptors and is the common signal-transducing receptor subunit shared by the so-called IL-6 type cytokines (IL-6, IL-11, ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M, and cardiotrophin-1). The inflammatory cytokines IL-6 and IL-11 induce gp130 homodimerization after binding to their specific alpha receptors, which leads to the activation of the Janus kinase/STAT signal transduction pathway. A molecular model of IL-6/IL-6R/gp130, which is based on the structure of the growth hormone/growth hormone receptor complex, allowed the selection of several amino acids located in the cytokine-binding module of gp130 for mutagenesis. The mutants were analyzed with regard to IL-6- or IL-11-induced STAT activation and ligand binding. It was found that Y190 and F191 are essential for the interaction of gp130 with IL-6 as well as IL-11, suggesting a common mode of recognition of helical cytokines by class I cytokine receptors. Furthermore, the requirement of the gp130 N-terminal Ig-like domain for ligand binding and signal transduction was demonstrated by the use of deletion mutants. Thus, besides the observed analogy to the growth hormone/growth hormone receptor complex, there is a substantial difference in the mechanism of receptor engagement by cytokines that signal via gp130.

The transmembrane glycoprotein gp130 is the common signal transducing receptor subunit of the interleukin-6-type cytokines. It is a member of the cytokine-receptor superfamily predicted to consist of six domains in its extracellular part. The second and third domain constitute the cytokine-binding module defined by a set of four conserved cysteines and a WSXWS motif, respectively. The three-dimensional structure of the carboxy-terminal domain of this region was determined by multidimensional NMR. The domain consists of seven beta-strands constituting a fibronectin type III-like topology. The structure reveals that the WSDWS motif of gp130 is part of an extended tryptophan/arginine zipper which modulates the conformation of the CD loop.

The transmembrane protein gp130 is the common signalling receptor subunit for the interleukin-6 (IL-6)-type cytokines. It has recently been shown that the cytoplasmic domain of gp130 contains a dileucine internalization motif and that endocytosis of gp130 occurs signal-independent. Here, we have studied whether gp130 itself undergoes constitutive internalization or whether its endocytosis is stimulated by formation of the IL-6/IL-6R/gp130 complex. Using two different assays, we found that gp130 is internalized independent from IL-6/IL-6R stimulation. In addition, we show that gp130 is constitutively associated with the cell surface adaptor complex AP-2. Our findings strongly suggest endocytosis of gp130 to be constitutive.

Soluble receptors for several cytokines have been detected in body fluids and are believed to modulate the cytokine response by binding the ligand and thereby reducing its bioavailability. In the case of IL-6, the situation is more complex. The receptor consists of two components, including a ligand-binding alpha-subunit (IL-6R, gp80, or CD126), which in its soluble (s) form (sIL-6R) acts agonistically by making the ligand accessible to the second subunit, the signal transducer gp130 (CD130). Soluble forms of both receptor subunits are present in human blood. Gel filtration of iodinated IL-6 that had been incubated with human serum revealed that IL-6 is partially trapped in IL-6/sIL-6R/sgp130 ternary complexes. sgp130 from human plasma was enriched by immunoaffinity chromatography and identified as a 100-kDa protein. Functionally equivalent rsgp130 was produced in baculovirus-infected insect cells to study its antagonistic potential on four different cell types. It was found that in situations in which cells lacking membrane-bound IL-6R were stimulated with IL-6/sIL-6R complexes, sgp130 was a much more potent antagonist than it was on IL-6R-positive cells stimulated with IL-6 alone. In the latter case, the neutralizing activity of sgp130 could be markedly enhanced by addition of sIL-6R. As a consequence of these findings, sIL-6R of human plasma must be regarded as an antagonistic molecule that enhances the inhibitory activity of sgp130. Furthermore, in combination with sIL-6R, sgp130 is a promising candidate for the development of IL-6 antagonists.

The family of cytokines signalling through the common receptor subunit gp130 comprises interleukin (IL)-6, IL-11, leukaemia inhibitory factor, oncostatin M, ciliary neurotrophic factor and cardiotrophin-1. These so-called IL-6-type cytokines play an important role in the regulation of complex cellular processes such as gene activation, proliferation and differentiation. The current knowledge on the signal-transduction mechanisms of these cytokines from the plasma membrane to the nucleus is reviewed. In particular, we focus on the assembly of receptor complexes after ligand binding, the activation of receptor-associated kinases of the Janus family, and the recruitment and phosphorylation of transcription factors of the STAT family, which dimerize, translocate to the nucleus, and bind to enhancer elements of respective target genes leading to transcriptional activation. The important players in the signalling pathway, namely the cytokines and the receptor components, the Janus kinases Jak1, Jak2 and Tyk2, the signal transducers and activators of transcription STAT1 and STAT3 and the tyrosine phosphatase SHP2 [SH2 (Src homology 2) domain-containing tyrosine phosphatase] are introduced and their structural/functional properties are discussed. Furthermore, we review various mechanisms involved in the termination of the IL-6-type cytokine signalling, namely the action of tyrosine phosphatases, proteasome, Jak kinase inhibitors SOCS (suppressor of cytokine signalling), protein inhibitors of activated STATs (PIAS), and internalization of the cytokine receptors via gp130. Although all IL-6-type cytokines signal through the gp130/Jak/STAT pathway, the comparison of their physiological properties shows that they elicit not only similar, but also distinct, biological responses. This is reflected in the different phenotypes of IL-6-type-cytokine knock-out animals.

The transmembrane glycoprotein gp130 is involved in many cytokine-mediated cellular responses and acts therein as the signal transducing receptor subunit. Interleukin-6 (IL-6) and interleukin-11 (IL-11), in complex with their specific alpha-receptors, homodimerize gp130 and, as a consequence, activate the Janus kinase (Jak)/signal transducer and activator of transcription (STAT) signalling pathway in their target cells. So far, it is not clear whether gp130 is bound to these cytokines and their specific alpha-receptor subunits through identical or different epitopes. In order to study the interaction of IL-11 and IL-11R with human gp130 the soluble form of the recently cloned human IL-11R was expressed in baculovirus-infected insect cells. By a coprecipitation binding-assay it is demonstrated that IL-11 and IL-6 compete for binding to gp130. Using deletion and point mutants of gp130 it is shown that IL-11-IL-11R and IL-6-IL-6R recognize overlapping binding motifs on gp130. Moreover, using well-established Jak-deficient cell lines we demonstrate that STAT activation by IL-11 requires Jak1. Taken together, our data support the concept that IL-6 and IL-11 activate gp130 by very similar molecular mechanisms.

The acute phase response is the answer of the organism to disturbances of its physiological homeostasis. It consists of a local and a systemic reaction. The latter is characterized by dramatic changes in the concentration of some plasma proteins called acute phase proteins. Interleukin-6 (IL-6) has been identified in vitro and in vivo as the major hepatocyte stimulating factor. Subsequently, additional hepatocyte stimulating factors, such as leukemia inhibitory factor, oncostatin-M, interleukin-11 and ciliary neurotrophic factor have been discovered. IL-t and related cytokines belong to the so-called alpha-helical cytokine family characterized by four antiparallel helices. IL-6 and IL-6-type cytokines exert their action via plasma membrane receptor complexes consisting of specific cytokine binding subunits and a common signal transducing protein gp130. In this presentation we focus on structure/function studies of IL-6, its receptor subunits gp80 and gp130, the internalization of the ligand/receptor complex and a recently elucidated signal transduction pathway. We have shown that protein tyrosine kinases of the JAK family are associated with the cytoplasmic domain of gp130 and are activated in response to IL-6. Subsequently, the transcription factors--named STATs (signal transducers and activators of transcription)--STAT1 alpha and STAT3 are transiently recruited to the cytoplasmic domain of gp130, where they become tyrosine phosphorylated by JAK kinases. In addition to the tyrosine phosphorylation we have observed that IL-6 also induces a serine phosphorylation of STAT3. This modification occurs with a delayed time-course as compared to the tyrosine phosphorylation and is inhibited by the protein kinase inhibitor H7. We propose that the STAT3 serine phosphorylation is required for transactivation of IL-6 target genes which is also inhibited by H7.

Gp130 is the signal transducing receptor subunit of the so-called interleukin-6-type cytokines. This transmembrane protein is a member of the cytokine-receptor superfamily predicted to consist of six fibronectin-type-III-like domains in its extracellular part. The second and the third domain constitute the so-called cytokine-binding module. Domain 2 is characterized by a set of four conserved Cys residues, domain 3 by a conserved WSXWS motif. As a first approach to a more detailed characterization of the cytokine-binding domains of human gp130, we have expressed in Escherichia coli two forms of domain 3 differing in length. Both proteins were purified and refolded in a single step applying size-exclusion chromatography. According to the rotational correlation times deduced from fluorescence anisotropy decay, they do not form aggregates. CD and fluorescence spectroscopy were used to study thermal unfolding and denaturation by guanidinium hydrochloride. It was shown that N- and C-terminal extension by residues of the adjacent hinge regions substantially increase the thermal stability of the domain, which is conceivable from a molecular model. These results are the basis for further structural investigation by NMR spectroscopy.

Long-term stable Ba/F3 transfectants (B13R alpha1 and B13R alpha2) expressing two isoforms of the human IL-IIR alpha receptor (alpha1 full length or alpha2 lacking the cytoplasmic domain) in combination with human gp130 were established. IL-11R alpha1 and IL-11R alpha2 were each expressed and detected as three bands upon Western blot analysis, with apparent molecular masses in agreement with those of the polypeptide backbone (47 and 44 kDa, respectively) with no, one or two N-linked sugars. B13R alpha1 and B13R alpha2 bound IL-11-thioredoxin with similar efficiencies and proliferated with superimposable dose-response curves to IL-11, demonstrating that the intracellular domain of IL-11R alpha has no significant contribution on ligand binding and signaling. Analysis of a set of anti-human gp130 mAbs confirmed the similar responsiveness of B13R alpha1 and B13R alpha2 transfectants.

Soluble cytokine and growth factor receptors are currently believed to play an important role in the regulation of biological activities of their ligands. In this review, the current ideas on the mechanisms and regulation of generation of soluble receptors and their possible physiological functions are presented. Furthermore, implications of soluble cytokine and growth factor receptors as disease markers and therapeutical agents are summarized and discussed.

The soluble human interleukin-6 receptor (shIL6R) was purified from human plasma. In a single immunoaffinity purification step a 140000-fold enrichment with a yield of 95% was achieved. A subsequent IL-6 affinity chromatography resulted in a homogeneous receptor preparation but only in a yield of less than 5%. The biological activity of the soluble receptor was clearly demonstrated by its ability to induce the synthesis of the acute-phase protein 1-antichymotrypsin in HepG2 cells stably transfected with IL-6. Upon gel filtration, the native shIL6R showed an apparent molecular mass of 93 kDa. Analysis by SDS/PAGE revealed an apparent molecular mass of 65 kDa for the soluble receptor. Deglycosylation with peptide N-glycosidase F led to a shift in molecular mass from 65 kDa to 45 kDa. It has previously been shown that the shIL6R can be generated by shedding the membrane-bound form or by expression of an alternatively spliced mRNA. Here we show that the shIL6R isolated from human plasma is recognized by an affinity-purified peptide antibody raised against an amino acid sequence unique for the alternatively spliced isoform. Thus, the shIL6R isoform generated through alternative splicing which has been previously detected in supernatants of cultured cell lines is also an in vivo product circulating in human plasma.

The cytokines interleukin (IL)-6, IL-11, ciliary neurotrophic factor (CNTF), leukemia inhibitor factor (LIF), oncostatin M (OSM) and probably the recently cloned cytokine cardiotrophin-1, signal, in combination with their specific receptors, through the common signal transducer gp130. Here, we report that the signaling activities of IL-6, IL-11, CNTF and OSM/LIF can be specifically blocked by different anti-gp130 monoclonal antibodies (mAb). Furthermore, we found two mAb, B-P8 and B-S12, which directly activate gp130 independently of the presence of cytokines or their receptors. This agonistic activity includes induction of cytokine-dependent cell proliferation and stimulation of acute-phase protein synthesis in liver cells. Compared to B-P8 mAb, the B-S12 mAb exhibited the strongest agonistic activity, while both mAb are synergistic in their action. This activity could not be blocked by inhibiting mAb against IL-6 and the IL-6 receptor. In contrast to F(ab')2 of B-S12 which still could activate gp130, Fab fragments completely lost their agonistic activity. Activation by tyrosine phosphorylation of the transcription factors Stat1 and APRF/Stat3 was also induced by B-S12 and B-P8, suggesting that both mAb induce homodimerization of gp130. Since hematopoietic stem cells express gp130 on their plasma membrane, it was anticipated that the agonistic anti-gp130 mAb could stimulate the proliferation of these stem cells. Indeed, B-S12 and B-P8 were able to stimulate CD34+ cells. In summary, our data show for the first time that mAb against gp130 can specifically block the action of distinct IL-6-type cytokines that signal through gp130. Such mAb might be of great value for therapeutic applications in diseases where a single cytokine action needs to be inhibited. In addition, the agonistic gp130 mAb may be used as growth factors for maintenance and expansion of stem cells prior to grafting.

The extracellular domain of the human interleukin-6 (IL-6) receptor, comprising 339 amino acids following the signal peptide, has been expressed in baculovirus-infected insect cells (Sf158). When the soluble receptor secreted into the culture medium was purified by affinity chromatography, using IL-6 immobilized on Sepharose, 6 mg soluble receptor was isolated from 1 l conditioned medium of Sf158 suspension cultures. A molar absorption coefficient of 9.3 x 10(4) l.mol-1.cm-1 was calculated from the ultraviolet spectrum of the soluble IL-6 receptor. After SDS/PAGE and silver staining, an apparent molecular mass of 48 kDa was estimated for the purified protein. Deglycosylation with peptide N-glycosidase F resulted in an increase in electrophoretic mobility and a decrease in the apparent molecular mass from 48 kDa to about 41-44 kDa. As expected, the soluble human IL-6 receptor bound human 125I-labeled IL-6 with low affinity (Kd = 500 pM). Furthermore, the binding of soluble human IL-6 receptor to immobilized IL-6 was studied using real-time interaction analysis. The recombinant soluble receptor showed biological activity on HepG2 cells stably transfected with a cDNA coding for IL-6 (HepG2-IL-6 cells). Haptoglobin mRNA synthesis was induced by the soluble IL-6 receptor at concentrations as low as 10 ng/ml. Five monoclonal antibodies were generated. Two groups of antibodies were identified mapping to amino acids 1-67 and 68-143 of the soluble IL-6 receptor, respectively. The plasma clearance of soluble 125I-labeled IL-6 receptor in the absence and presence of IL-6 was studied in rats as a model system. The kinetics was biphasic. Soluble IL-6 receptor/IL-6 complexes were cleared more rapidly than the soluble receptor alone. Intravenously injected soluble 125I-labeled IL-6 receptor, as well as complexes with IL-6, rapidly accumulated in liver and to a lesser extent in skeletal muscle, skin and kidneys. Subsequently, the radioactivity appeared in the gut content.

Mitochondrial 2-enoyl-CoA hydratase (mECH) and 3,2-trans-enoyl-CoA isomerase (mECI), two enzymes which catalyze totally different reactions in fatty acid beta-oxidation, belong to the low-similarity hydratase/isomerase enzyme superfamily. Their substrates and reaction mechanisms are similar [Müller-Newen, G. & Stoffel, W. (1993) Biochemistry 32, 11,405-11,412]. Glu164 of mECH is the only amino acid with a protic side chain that is conserved in these monofunctional and polyfunctional enzymes with 2-enoyl-CoA hydratase and 3,2-trans-enoyl-CoA isomerase activities. We tested our hypothesis that Glu164 of mECH is the putative active-site amino acid responsible for the base-catalyzed alpha-deprotonation in the hydratase/dehydrase and isomerase reaction. We functionally expressed rat liver mECH wild-type and [E164Q] mutant enzymes in Escherichia coli. Characterization of the purified wild-type and mutant enzymes revealed that the replacement of Glu164 by Gln lowers the kcat value more than 100,000-fold, whereas the Km value is only moderately affected. We have demonstrated in a previous study that Glu165 is indispensable for the 3,2-trans-enoyl-CoA isomerase activity. Taking these results together, we conclude that the conserved glutamic acid is the essential basic group in the active sites of 2-enoyl-CoA hydratase (Glu164) and 3,2-trans-enoyl-CoA isomerase (Glu165), and that these enzymes are not only evolutionarily but also functionally and mechanistically related.

During beta-oxidation of unsaturated fatty acids, mitochondrial 3,2-trans-enoyl-CoA isomerase (mECI) converts 3-cis- or 3-trans-enoyl-CoA intermediates into their 2-trans isomers. The cDNA-derived amino acid sequence of mECI shows weak but significant homologies to the peroxisomal trifunctional enzyme (pTFE), the alpha-subunit of the fatty acid degradation complex from Escherichia coli (FadB), the mitochondrial 2-enoyl-CoA hydratase (mECH), the naphthoate synthase encoded by the menB gene from Bacillus subtilis (MenB), and the 4-chlorobenzoyl-CoA dehalogenase from Pseudomonas sp. (CBDH). These proteins from the isomerase/hydratase enzyme family. Tyr-150, Arg-151, and Asp-211 of the mECI are the only amino acids with protic side chains conserved within the enzymes with isomerase activity (pTFE and FadB). These amino acids are exchanged in the remaining enzymes of the isomerase/hydratase family. Glu-165 is conserved in all enzymes with isomerase and/or hydratase activity (pTFE, FadB, and mECH). We argue that these amino acids are possibly involved in the proton transfer at the active site of mECI. To test this hypothesis, mECI was functionally expressed in E. coli. The recombinant enzyme (rmECI) exhibits the same specific activity as the enzyme from rat liver. Exchange of the candidate active-site amino acids by site-directed mutagenesis revealed that Tyr-150 is not involved in isomerase catalysis. The exchange of Arg-151 and Asp-211 leads to a reduced expression of the recombinant enzyme accompanied by a reduced specific activity. The replacement of Glu-165 by Gln leads to a strongly reduced enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

3-2trans-Enoyl-CoA isomerase (EC 5.3.3.8) is a key enzyme in mitochondrial beta-oxidation of unsaturated fatty acids in bacteria, plant and animal cells. The enzyme was isolated from rat liver mitochondria and purified to homogeneity by two chromatographic steps. Partial polypeptide sequences of the 29 kDa protein were derived from cyanogen bromide, tryptic, Lys-C, and protease V8 fragments by Edman degradation. Peptide-derived synthetic oligonucleotides were used for the isolation of a 990 bp long isomerase-specific cDNA from rat liver cDNA libraries. 867 bp encode the 289 amino-acid residues of the preisomerase with a molecular mass of 32,254 Da. The 1.3-kb mRNA is most strongly expressed in skeletal muscle followed by liver, heart, kidney, and weakly expressed in spleen and brain. In vitro transcription and translation yielded a 32 kDa polypeptide which was immunoprecipitated by anti rat isomerase antibodies. In the presence of mitochondria the 32 kDa precursor isomerase was processed during mitochondrial import to the 29 kDa mature form of the 3-2trans-enoyl-CoA isomerase with 264 amino-acid residues (Mr 29,706). A N-terminal signal sequence of 25 amino-acid residues directs the import into the mitochondrial matrix and is cleaved in two successive steps passing through an intermediate form of Mr 30,475. The two cysteine residues in positions 142 and 148 of the preisomerase are present as free thiol groups as shown by derivatization of the mature, native protein with the fluorescent label N-(iodoacetaminoethyl)-1-naphthylamine-5-sulfonic acid. The mitochondrial 3-2trans enoyl-CoA isomerase shows significant homology and conserved amino-acid exchanges with the mitochondrial enoyl-CoA hydratase, the N-terminal domain of the bifunctional peroxisomal enoyl-CoA-hydratase:3-hydroxyacyl-CoA dehydrogenase and to extended domains of the alpha-subunit of the procaryotic beta-oxidation complex sharing enoyl-CoA isomerase, D(-)3-hydroxyacyl-CoA epimerase, enoyl-CoA hydratase and L(+)3-hydroxyacyl-CoA dehydrogenase activity, encoded by the fad B operon of E. coli.