Adenosine activates ATP-sensitive potassium channels in arterial myocytes via A2 receptors and cAMP-dependent protein kinase.

The mechanism by which the endogenous vasodilator adenosine causes ATP-sensitive potassium (KATP) channels in arterial smooth muscle to open was investigated by the whole-cell patch-clamp technique. Adenosine induced voltage-independent, potassium-selective currents, which were inhibited by glibenclamide, a blocker of KATP currents. Glibenclamide-sensitive currents were also activated by the selective adenosine A2-receptor agonist 2-p-(2-carboxethyl)-phenethylamino-5'-N- ethylcarboxamidoadenosine hydrochloride (CGS-21680), whereas 2-chloro-N6-cyclopentyladenosine (CCPA), a selective adenosine A1-receptor agonist, failed to induce potassium currents. Glibenclamide-sensitive currents induced by adenosine and CGS-21680 were largely reduced by blockers of the cAMP-dependent protein kinase (Rp-cAMP[S], H-89, protein kinase A inhibitor peptide). Therefore, we conclude that adenosine can activate KATP currents in arterial smooth muscle through the following pathway: (i) Adenosine stimulates A2 receptors, which activates adenylyl cyclase; (ii) the resulting increase intracellular cAMP stimulates protein kinase A, which, probably through a phosphorylation step, opens KATP channels.

A Fas-associated protein factor, FAF1, potentiates Fas-mediated apoptosis.

Fas, a member of the tumor necrosis factor receptor family, can induce apoptosis when activated by Fas ligand binding or anti-Fas antibody crosslinking. Genetic studies have shown that a defect in Fas-mediated apoptosis resulted in abnormal development and function of the immune system in mice. A point mutation in the cytoplasmic domain of Fas (a single base change from T to A at base 786), replacing isoleucine with asparagine, abolishes the signal transducing property of Fas. Mice homozygous for this mutant allele (lprcg/lprcg mice) develop lymphadenopathy and a lupus-like autoimmune disease. Little is known about the mechanism of signal transduction in Fas-mediated apoptosis. In this study, we used the two-hybrid screen in yeast to isolate a Fas-associated protein factor, FAF1, which specifically interacts with the cytoplasmic domain of wild-type Fas but not the lprcg-mutated Fas protein. This interaction occurs not only in yeast but also in mammalian cells. When transiently expressed in L cells, FAF1 potentiated Fas-induced apoptosis. A search of available DNA and protein sequence data banks did not reveal significant homology between FAF1 and known proteins. Therefore, FAF1 is an unusual protein that binds to the wild type but not the inactive point mutant of Fas. FAF1 potentiates Fas-induced cell killing and is a candidate signal transducing molecule in the regulation of apoptosis.

Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids.

The orphan nuclear receptor steroidogenic factor 1 (SF-1) is expressed in the adrenal cortex and gonads and regulates the expression of several P450 steroid hydroxylases in vitro. We examined the role of SF-1 in the adrenal glands and gonads in vivo by a targeted disruption of the mouse SF-1 gene. All SF-1-deficient mice died shortly after delivery. Their adrenal glands and gonads were absent, and persistent Mullerian structures were found in all genotypic males. While serum levels of corticosterone in SF-1-deficient mice were diminished, levels of adrenocorticotropic hormone (ACTH) were elevated, consistent with intact pituitary corticotrophs. Intrauterine survival of SF-1-deficient mice appeared normal, and they had normal serum level of corticosterone and ACTH, probably reflecting transplacental passage of maternal steroids. We tested whether SF-1 is required for P450 side-chain-cleavage enzyme (P450scc) expression in the placenta, which expresses both SF-1 and P450scc, and found that in contrast to its strong activation of the P450scc gene promoter in vitro, the absence of SF-1 had no effect on P450scc mRNA levels in vivo. Although the region targeted by our disruption is shared by SF-1 and by embryonal long terminal repeat-binding protein (ELP), a hypothesized alternatively spliced product, we believe that the observed phenotype reflects absent SF-1 alone, as PCR analysis failed to detect ELP transcripts in any mouse tissue, and sequences corresponding to ELP are not conserved across species. These results confirm that SF-1 is an important regulator of adrenal and gonadal development, but its regulation of steroid hydroxylase expression in vivo remains to be established.

Unimpaired autoreactive T-cell traffic within the central nervous system during tumor necrosis factor receptor-mediated inhibition of experimental autoimmune encephalomyelitis.

The critical role of tumor necrosis factor (TNF) as a mediator in autoimmune inflammatory processes is evident from in vivo studies with TNF-blocking agents. However, the mechanisms by which TNF, and possibly also its homologue lymphotoxin alpha, contributes to development of pathology in rheumatoid arthritis and Crohn disease and in animal models like experimental autoimmune encephalomyelitis is unclear. Possibilities include regulation of vascular adhesion molecules enabling leukocyte movement into tissues or direct cytokine-mediated effector functions such as mediation of tissue damage. Here we show that administration of a TNF receptor (55 kDa)-IgG fusion protein prevented clinical signs of actively induced experimental autoimmune encephalomyelitis. Significantly, the total number of CD4+ T lymphocytes isolated from the central nervous system of clinically healthy treated versus diseased control animals was comparable. By using a CD45 congenic model of passively transferred experimental autoimmune encephalomyelitis to enable tracking of myelin basic protein-specific effector T lymphocytes, prevention of clinical signs of disease was again demonstrated in treated animals but without quantitative or qualitative impediment to the movement of autoreactive T lymphocytes to and within the central nervous system. Thus, despite the uninterrupted movement of specific T lymphocytes into the target tissue, subsequent disease development was blocked. This provides compelling evidence for a direct effector role of TNF/lymphotoxin alpha in autoimmune tissue damage.

A cooperative model for receptor recognition and cell adhesion: evidence from the molecular packing in the 1.6-A crystal structure of the pheromone Er-1 from the ciliated protozoan Euplotes raikovi.

The crystal structure of the pheromone Er-1 from the unicellular eukaryotic organism Euplotes raikovi was determined at 1.6 A resolution and refined to a crystallographic R factor of 19.9%. In the tightly packed crystal, two extensive intermolecular helix-helix interactions arrange the Er-1 molecules into layers. Since the putative receptor of the pheromone is a membrane-bound protein, whose extracellular C-terminal domain is identical in amino acid sequence to the soluble pheromone, the interactions found in the crystal may mimic the pheromone-receptor interactions as they occur on a cell surface. Based on this, we propose a model for the interaction between soluble pheromone molecules and their receptors. In this model, strong pheromone-receptor binding emerges as a consequence of the cooperative utilization of several weak interactions. The model offers an explanation for the results of binding studies and may also explain the adhesion between cells that occurs during mating.

Low density lipoprotein receptor-related protein mediates apolipoprotein E-dependent neurite outgrowth in a central nervous system-derived neuronal cell line.

The epsilon 4 allele of apolipoprotein E (apoE) is a major risk factor for Alzheimer disease, suggesting that apoE may directly influence neurons in the aging brain. Recent data suggest that apoE-containing lipoproteins can influence neurite outgrowth in an isoform-specific fashion. The neuronal mediators of apoE effects have not been clarified. We show here that in a central nervous system-derived neuronal cell line, apoE3 but not apoE4 increases neurite extension. The effect of apoE3 was blocked at low nanomolar concentrations by purified 39-kDa protein that regulates ligand binding to the low density lipoprotein receptor-related protein (LRP). Anti-LRP antibody also completely abolished the neurite-promoting effect of apoE3. Understanding isoform-specific cell biological processes mediated by apoE-LRP interactions in central nervous system neurons may provide insight into Alzheimer disease pathogenesis.

G protein beta gamma subunits stimulate phosphorylation of Shc adapter protein.

The mechanism of mitogen-activated protein (MAP) kinase activation by pertussis toxin-sensitive Gi-coupled receptors is known to involve the beta gamma subunits of heterotrimeric G proteins (G beta gamma), p21ras activation, and an as-yet-unidentified tyrosine kinase. To investigate the mechanism of G beta gamma-stimulated p21ras activation, G beta gamma-mediated tyrosine phosphorylation was examined by overexpressing G beta gamma or alpha 2-C10 adrenergic receptors (ARs) that couple to Gi in COS-7 cells. Immunoprecipitation of phosphotyrosine-containing proteins revealed a 2- to 3-fold increase in the phosphorylation of two proteins of approximately 50 kDa (designated as p52) in G beta gamma-transfected cells or in alpha 2-C10 AR-transfected cells stimulated with the agonist UK-14304. The latter response was pertussis toxin sensitive. These proteins (p52) were also specifically immunoprecipitated with anti-Shc antibodies and comigrated with two Shc proteins, 46 and 52 kDa. The G beta gamma- or alpha 2-C10 AR-stimulated p52 (Shc) phosphorylation was inhibited by coexpression of the carboxyl terminus of beta-adrenergic receptor kinase (a G beta gamma-binding pleckstrin homology domain peptide) or by the tyrosine kinase inhibitors genistein and herbimycin A, but not by a dominant negative mutant of p21ras. Worthmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) inhibited phosphorylation of p52 (Shc), implying involvement of PI3K. These results suggest that G beta gamma-stimulated Shc phosphorylation represents an early step in the pathway leading to p21ras activation, similar to the mechanism utilized by growth factor tyrosine kinase receptors.

12(S)-hydroxyeicosatetraenoic acid and 13(S)-hydroxyoctadecadienoic acid regulation of protein kinase C-alpha in melanoma cells: role of receptor-mediated hydrolysis of inositol phospholipids.

Protein kinase C (PKC) isoenzymes are essential components of cell signaling. In this study, we investigated the regulation of PKC-alpha in murine B16 amelanotic melanoma (B16a) cells by the monohydroxy fatty acids 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] and 13(S)-hydroxyoctadecadienoic acid [13(S)-HODE]. 12(S)-HETE induced a translocation of PKC-alpha to the plasma membrane and focal adhesion plaques, leading to enhanced adhesion of B16a cells to the matrix protein fibronectin. However, 13(S)-HODE inhibited these 12(S)-HETE effects on PKC-alpha. A receptor-mediated mechanism of action for 12(S)-HETE and 13(S)-HODE is supported by the following findings. First, 12(S)-HETE triggered a rapid increase in cellular levels of diacylglycerol and inositol trisphosphate in B16a cells. 13(S)-HODE blocked the 12(S)-HETE-induced bursts of both second messengers. Second, the 12(S)-HETE-increased adhesion of B16a cells to fibronectin was sensitive to inhibition by a phospholipase C inhibitor and pertussis toxin. Finally, a high-affinity binding site (Kd = 1 nM) for 12(S)-HETE was detected in B16a cells, and binding of 12(S)-HETE to B16a cells was effectively inhibited by 13(S)-HODE (IC50 = 4 nM). In summary, our data provide evidence that regulation of PKC-alpha by 12(S)-HETE and 13(S)-HODE may be through a guanine nucleotide-binding protein-linked receptor-mediated hydrolysis of inositol phospholipids.

D1 cap region involved in the receptor recognition and neural cell survival activity of human ciliary neurotrophic factor.

Human ciliary neurotrophic factor (hCNTF), which promotes the cell survival and differentiation of motor and other neurons, is a protein belonging structurally to the alpha-helical cytokine family. hCNTF was subjected to three-dimensional structure modeling and site-directed mutagenesis to analyze its structure-function relationship. The replacement of Lys-155 with any other amino acid residue resulted in abolishment of neural cell survival activity, and some of the Glu-153 mutant proteins had 5- to 10-fold higher biological activity. The D1 cap region (around the boundary between the CD loop and helix D) of hCNTF, including both Glu-153 and Lys-155, was shown to play a key role in the biological activity of hCNTF as one of the putative receptor-recognition sites. In this article, the D1 cap region of the 4-helix-bundle proteins is proposed to be important in receptor recognition and biological activity common to alpha-helical cytokine proteins reactive with gp130, a component protein of the receptors.

A trace component of ginseng that inhibits Ca2+ channels through a pertussis toxin-sensitive G protein.

A crude extract from ginseng root inhibits high-threshold, voltage-dependent Ca2+ channels through an unknown receptor linked to a pertussis toxin-sensitive G protein. We now have found the particular compound that seems responsible for the effect: it is a saponin, called ginsenoside Rf (Rf), that is present in only trace amounts within ginseng. At saturating concentrations, Rf rapidly and reversibly inhibits N-type, and other high-threshold, Ca2+ channels in rat sensory neurons to the same degree as a maximal dose of opioids. The effect is dose-dependent (half-maximal inhibition: 40 microM) and it is virtually eliminated by pretreatment of the neurons with pertussis toxin, an inhibitor of G(o) and Gi GTP-binding proteins. Other ginseng saponins--ginsenosides Rb1, Rc, Re, and Rg1--caused relatively little inhibition of Ca2+ channels, and lipophilic components of ginseng root had no effect. Antagonists of a variety of neurotransmitter receptors that inhibit Ca2+ channels fail to alter the effect of Rf, raising the possibility that Rf acts through another G protein-linked receptor. Rf also inhibits Ca2+ channels in the hybrid F-11 cell line, which might, therefore, be useful for molecular characterization of the putative receptor for Rf. Because it is not a peptide and it shares important cellular and molecular targets with opioids, Rf might be useful in itself or as a template for designing additional modulators of neuronal Ca2+ channels.

Tumor necrosis factor-induced apoptosis is mediated by a CrmA-sensitive cell death pathway.

We report here that the activation of the interleukin 1 beta (IL-1 beta)-converting enzyme (ICE) family is likely to be one of the crucial events of tumor necrosis factor (TNF) cytotoxicity. The cowpox virus CrmA protein, a member of the serpin superfamily, inhibits the enzymatic activity of ICE and ICE-mediated apoptosis. HeLa cells overexpressing crmA are resistant to apoptosis induced by Ice but not by Ich-1, another member of the Ice/ced-3 family of genes. We found that the CrmA-expressing HeLa cells are resistant to TNF-alpha/cycloheximide (CHX)-induced apoptosis. Induction of apoptosis in HeLa cells by TNF-alpha/CHX is associated with secretion of mature IL-1 beta, suggesting that an IL-1 beta-processing enzyme, most likely ICE itself, is activated by TNF-alpha/CHX stimulation. These results suggest that one or more members of the ICE family sensitive to CrmA inhibition are activated and play a critical role in apoptosis induced by TNF.

The G-protein-coupled receptor phosphatase: a protein phosphatase type 2A with a distinct subcellular distribution and substrate specificity.

Phosphorylation of G-protein-coupled receptors plays an important role in regulating their function. In this study the G-protein-coupled receptor phosphatase (GRP) capable of dephosphorylating G-protein-coupled receptor kinase-phosphorylated receptors is described. The GRP activity of bovine brain is a latent oligomeric form of protein phosphatase type 2A (PP-2A) exclusively associated with the particulate fraction. GRP activity is observed only when assayed in the presence of protamine or when phosphatase-containing fractions are subjected to freeze/thaw treatment under reducing conditions. Consistent with its identification as a member of the PP-2A family, the GRP is potently inhibited by okadaic acid but not by I-2, the specific inhibitor of protein phosphatase type 1. Solubilization of the membrane-associated GRP followed by gel filtration in the absence of detergent yields a 150-kDa peak of latent receptor phosphatase activity. Western blot analysis of this phosphatase reveals a likely subunit composition of AB alpha C. PP-2A of this subunit composition has previously been characterized as a soluble enzyme, yet negligible soluble GRP activity was observed. The subcellular distribution and substrate specificity of the GRP suggests significant differences between it and previously characterized forms of PP-2A.

cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells.

Incubating rat aortic smooth muscle cells with either platelet-derived growth factor BB (PDGF) or insulin-like growth factor I (IGF-I) increased the phosphorylation of PHAS-I, an inhibitor of the mRNA cap binding protein, eukaryotic initiation factor (eIF) 4E. Phosphorylation of PHAS-I promoted dissociation of the PHAS-I-eIF-4E complex, an effect that could partly explain the stimulation of protein synthesis by the two growth factors. Increasing cAMP with forskolin decreased PHAS-I phosphorylation and markedly increased the amount of eIF-4E bound to PHAS-I, effects consistent with an action of cAMP to inhibit protein synthesis. Both PDGF and IGF-I activated p70S6K, but only PDGF increased mitogen-activated protein kinase activity. Forskolin decreased by 50% the effect of PDGF on increasing p70S6K, and forskolin abolished the effect of IGF-I on the kinase. The effects of PDGF and IGF-I on increasing PHAS-I phosphorylation, on dissociating the PHAS-I-eIF-4E complex, and on increasing p70S6K were abolished by rapamycin. The results indicate that IGF-I and PDGF increase PHAS-I phosphorylation in smooth muscle cells by the same rapamycin-sensitive pathway that leads to activation of p70S6K.

Activation of mitogen-activated protein kinases by vascular endothelial growth factor and basic fibroblast growth factor in capillary endothelial cells is inhibited by the antiangiogenic factor 16-kDa N-terminal fragment of prolactin.

A number of factors both stimulating and inhibiting angiogenesis have been described. In the current work, we demonstrate that the angiogenic factor vascular endothelial growth factor (VEGF) activates mitogen-activated protein kinase (MAPK) as has been previously shown for basic fibroblast growth factor. The antiagiogenic factor 16-kDa N-terminal fragment of human prolactin inhibits activation of MAPK distal to autophosphorylation of the putative VEGF receptor, Flk-1, and phospholipase C-gamma. These data show that activation and inhibition of MAPK may play a central role in the control of angiogenesis.

A kinase associated with chromatin that can be activated by ligand-p185c-Neu or epidermal growth factor-receptor interactions.

Some growth factors transduce positive growth signals, while others can act as growth inhibitors. Nuclear signaling events of previously quiescent cells stimulated with various growth factors have been studied by isolating the complexed chromatin-associated proteins and chromatin-associated proteins. Signals from the plasma membrane are integrated within the cells and quickly transduced to the nucleus. It is clear that several growth factors, such as epidermal growth factor, transforming growth factor alpha (but not transforming growth factor beta), and platelet-derived growth factor, utilize similar intracellular signaling biochemistries to modulate nucleosomal characteristics. The very rapid and consistent phosphorylation of nuclear p33, p54, and low molecular mass proteins in the range of 15-18 kDa after growth factor stimulation implies that there is a coordination and integration of the cellular signaling processes. Additionally, phosphorylation of p33 and some low molecular mass histones has been found to occur within 5 min of growth factor treatment and to reach a maximum by 30 min. In this study, we report that Neu receptor activating factor also utilizes the same signaling mechanism and causes p33 to become phosphorylated. In addition, both the tumor promoter okadaic acid (which inhibits protein phosphatases 1 and 2A) and phorbol ester (phorbol 12-tetradecanoate 13-acetate) stimulate phosphorylation of p33, p54, and low molecular mass histones. However, transforming growth factor beta, which is a growth inhibitor for fibroblasts, fails to increase p33 phosphorylation. In general, p33 phosphorylation patterns correspond to positive and negative mitogenic signal transduction. p33 isolated from the complexed chromatin-associated protein fraction appears to be a kinase, or tightly associated with a kinase, and shares antigenicity with the cell division cycle-dependent Cdk2 kinase as determined by antibody-dependent analysis. The rapid phosphorylation of nucleosomal proteins may influence sets of early genes needed for the induction and progression of the cell cycle.