Expression analysis of PCSTE3, a putative pheromone receptor from the lung pathogenic fungus Pneumocystis carinii

The fungal pathogen Pneumocystis carinii remains the most prevalent opportunistic infection in patients infected with HIV. Fungal pheromone receptors are seven transmembrane domain G-protein-coupled receptors which are expressed on specific mating types, and have ligand-binding extracellular domains for specific pheromones from cells of the opposite mating type. We have cloned and characterized PCSTE3 from P. carinii, which encodes a seven transmembrane domain protein orthologous to the Saccharomyces cerevisiae pheromone receptor Ste3. We detect PCSTE3 by indirect immunofluorescence using antibodies designed to extracellular domains of the receptor in yeast expressing the protein. Using a downstream Fus1-lacZ reporter gene, we determined that PCSTE3 does not recognize a- or alpha-factor pheromones as ligands for the receptor. We isolated P. carinii life cycle stages and examined PCSTE3 expression by immunofluorescence microscopy and flow cytometry, and found PCSTE3 expression exclusively on a population of trophic forms. PCSTE3 receptor expression was not found on cysts.     

Oncostatin M receptor-mediated signal transduction is negatively regulated by SOCS3 through a receptor tyrosine-independent mechanism

Down-regulation of interleukin (IL)-6-type cytokine signaling has been shown to occur, among other mechanisms, via induction of the feedback inhibitor SOCS3 (suppressor of cytokine signaling 3). Binding of SOCS3 to the phosphorylated Tyr(759) in the cytoplasmic region of gp130, the common signal transducing receptor chain of all IL-6-type cytokines, is necessary for inhibition of Janus kinase-mediated signaling. In the present study, we analyzed the effect of SOCS3 on signal transduction by the proinflammatory cytokine oncostatin M (OSM), which signals through a receptor complex of gp130 and the OSM receptor (OSMR). OSM leads to a much stronger and prolonged induction of SOCS3 in HepG2 hepatoma cells and murine embryonal fibroblasts (MEF) compared with IL-6. A negative effect of SOCS3 on OSM signaling was confirmed using MEF cells lacking SOCS3. We can show that the OSMR-mediated signaling is inhibited by SOCS3 to a similar extent as previously described for gp130. However, the inhibition occurs independent of tyrosine motifs within the OSMR. Instead, SOCS3 interacts directly with JAK1 in a stimulation-dependent manner, a mechanism so far only known for SOCS1.     

Pig whey acidic protein gene is surrounded by two ubiquitously expressed genes

A 140-kb pig DNA fragment containing the whey acidic protein (WAP) gene cloned in a bacterial artificial chromosome (BAC344H5) has been shown to contain all of the cis-elements necessary for position-independent, copy-dependent and tissue-specific expression in transgenic mice. The insert from this BAC was sequenced. This revealed the presence of two other genes with quite different expression patterns in pig tissues and in transfected HC11 mouse mammary cells. The RAMP3 gene is located 15 kb upstream of the WAP gene in reverse orientation. The CPR2 gene is located 5 kb downstream of the WAP gene in the same orientation. The same locus organization was found in the human genome. The region between RAMP3 and CPR2 in the human genome contains a WAP gene-like sequence with several points of mutation which may account for the absence of WAP from human milk.     

The genes of plant signal transduction

Due to the enormous importance of plants as a source of both food and raw materials, an understanding of the control of their growth and development is imperative. Basic to this understanding is the identification of the genes that enable the plant to adapt to its environment while at the same time adhering to its basic developmental plan. The aims of this review are first to briefly summarize the various approaches that have been used to identify these plant signaling genes and second to give an overview of the genes that have been cloned so far and what these genes may tell us about the nature of signal transduction in plants.

The advent of modern molecular biology and molecular genetics has revolutionized our ability to unravel the complexities of plant signal transduction pathways. A whole battery of techniques are now available to identify the genes that control the plants development and ability to adapt to its environment.⁶⁵ Each technique has its own strengths and weaknesses and must be carefully selected by the researcher according to the question that he or she would like to ask.     

The yeast G-protein homolog is involved in the mating pheromone signal transduction system.

I have isolated a new type of sterile mutant of Saccharomyces cerevisiae, carrying a single mutant allele, designated dac1, which was mapped near the centromere on chromosome VIII. The dac1 mutation caused specific defects in the pheromone responsiveness of both a and alpha cells and did not seem to be associated with any pleiotropic phenotypes. Thus, in contrast to the ste4, ste5, ste7, ste11, and ste12 mutations, the dac1 mutation had no significant effect on such constitutive functions of haploid cells as pheromone production and alpha-factor destruction. The characteristics of this phenotype suggest that the DAC1 gene encodes a component of the pheromone response pathway common to both a and alpha cells. Introduction of the GPA1 gene encoding an S. cerevisiae homolog of the alpha subunit of mammalian guanine nucleotide-binding regulatory proteins (G proteins) into sterile dac1 mutants resulted in restoration of pheromone responsiveness and mating competence to both a and alpha cells. These results suggest that the dac1 mutation is an allele of the GPA1 gene and thus provide genetic evidence that the yeast G protein homolog is directly involved in the mating pheromone signal transduction pathway.     

The G protein-coupled receptor kinase-2 is a TGFbeta-inducible antagonist of TGFbeta signal transduction

Signaling from the activin/transforming growth factor beta (TGFbeta) family of cytokines is a tightly regulated process. Disregulation of TGFbeta signaling is often the underlying basis for various cancers, tumor metastasis, inflammatory and autoimmune diseases. In this study, we identify the protein G-coupled receptor kinase 2 (GRK2), a kinase involved in the desensitization of G protein-coupled receptors (GPCR), as a downstream target and regulator of the TGFbeta-signaling cascade. TGFbeta-induced expression of GRK2 acts in a negative feedback loop to control TGFbeta biological responses. Upon TGFbeta stimulation, GRK2 associates with the receptor-regulated Smads (R-Smads) through their MH1 and MH2 domains and phosphorylates their linker region. GRK2 phosphorylation of the R-Smads inhibits their carboxyl-terminal, activating phosphorylation by the type I receptor kinase, thus preventing nuclear translocation of the Smad complex, leading to the inhibition of TGFbeta-mediated target gene expression, cell growth inhibition and apoptosis. Furthermore, we demonstrate that GRK2 antagonizes TGFbeta-induced target gene expression and apoptosis ex vivo in primary hepatocytes, establishing a new role for GRK2 in modulating single-transmembrane serine/threonine kinase receptor-mediated signal transduction.     

The mating-type locus B alpha 1 of Schizophyllum commune contains a pheromone receptor gene and putative pheromone genes.

Analysis of the multispecific B alpha mating-type locus of Schizophyllum commune provided evidence that pheromones and pheromone receptors govern recognition of self versus non-self and sexual development in this homobasidiomycetous fungus. Four subclones of an 8.2 kb genomic fragment carrying B alpha 1 specificity induced B-regulated sexual morphogenesis when introduced into a strain with one of the eight compatible B alpha specificities that are known to exist in nature. One of these clones, which activated all other B alpha specificities, contains a gene termed bar1. The predicted protein product of bar1, as well as that of bar2, a homologous gene isolated from a B alpha 2 strain, has significant homology to known fungal pheromone receptor proteins in the rhodopsin-like superfamily of G protein-linked receptors. The other three active B alpha 1 clones were subcloned further to identify the minimal active element in each clone. Every active subclone contains a putative pheromone gene ending in a signal for possible isoprenylation. A message of approximately 600 bp was observed for one of these genes, bap1(1). This paper presents the first evidence for a system of multiple pheromones and pheromone receptors as a basis for multispecific mating types in a fungus.     

Yeast pheromone receptor endocytosis and hyperphosphorylation are independent of G protein-mediated signal transduction.

When alpha factor binds to the yeast alpha factor receptor a signal is transmitted via a tripartite G protein that prepares the cell for conjugation. As a result of alpha factor binding the receptor also undergoes a regulated internalization and hyperphosphorylation. Using cells that lack activity of this tripartite G protein, we show that G protein-mediated pheromone signal transduction is not necessary for regulation of receptor internalization or hyperphosphorylation. Therefore, the processes of signal transduction and down regulation can be uncoupled. We propose that binding of alpha factor to its receptor results in a receptor conformation change that permits receptor hyperphosphorylation and interaction with the endocytic machinery.     

The TRPC3 channel has a large internal chamber surrounded by signal sensing antennas

Transient receptor potential (TRP) channels are intrinsic sensors adapted for response to all manner of stimuli both from inside and from outside the cell. Within the TRP superfamily, the canonical TRP-3 (TRPC3) has been widely studied and is involved in various biological processes such as neuronal differentiation, blood vessel constriction, and immune cell maturation. Upon stimulation of surface membrane receptors linked to phospholipase C, TRPC3 mediates transmembrane Ca(2+) influx from outside the cell to control Ca(2+) signaling, in concert with the Ca(2+) release from internal stores. The structural basis of TRP superfamily has, however, been poorly understood. Here we present a structure of the TRPC3 at 15 A resolution. This first 3D depiction of TRP superfamily was reconstructed from 135,909 particle images obtained with cryo-electron microscopy. The large intracellular domain represents a "nested-box" structure: a wireframe outer shell is functionable as sensors for activators and modulators, and a globular inner chamber may modulate ion flow, since it is aligned tandem along the central axis with the dense membrane-spanning core. The transmembrane domain demonstrates a pore-forming property. This structure implies that the TRP superfamily has diversely evolved as sensors specialized for various signals, rather than as simple ion-conducting apparatuses.     

The neutrophil chemiluminescence response to Pneumocystis carinii is stimulated by GM-CSF and gamma interferon

An enzyme-linked immunosorbent assay (ELISA) with antibodies specific to beta, epsilon and iota ib toxins of Clostridium perfringens was developed to detect beta, epsilon and iota ib toxins, respectively. The ELISA was sensitive enough to detect as little as 1.0 ng/ml of purified beta and iota ib toxins and 0.1 ng/ml of purified epsilon toxin. By means of the ELISA method, 192 isolates of C. perfringens from food samples in Japan and Thailand, and 58 isolates from patients suffering from gas gangrene or gastroenteritis were examined. One isolate from food samples in Japan, three from food samples in Thailand and five from stools of patients with gastroenteritis were C. perfringens type D. One type B and one type C were detected from the stools of patients with gastroenteritis.     

Fibroblast growth factor receptor 3 induces gene expression primarily through Ras-independent signal transduction pathways

Fibroblast growth factor receptors (FGFR) are widely expressed in many tissues and cell types, and the temporal expression of these receptors and their ligands play important roles in the control of development. There are four FGFR family members, FGFR-1-4, and understanding the ability of these receptors to transduce signals is central to understanding how they function in controlling differentiation and development. We have utilized signal transduction by FGF-1 in PC12 cells to compare the ability of FGFR-1 and FGFR-3 to elicit the neuronal phenotype. In PC12 cells FGFR-1 is much more potent in the induction of neurite outgrowth than FGFR-3. This correlated with the ability of FGFR-1 to induce robust and sustained activation of the Ras-dependent mitogen-activated protein kinase pathways. In contrast, FGFR-3 could not induce strong sustained Ras-dependent signals. In this study, we analyzed the ability of FGFR-3 to induce the expression of sodium channels, peripherin, and Thy-1 in PC12 cells because all three of these proteins are known to be induced via Ras-independent pathways. We determined that FGFR-3 was capable of inducing several Ras-independent gene expression pathways important to the neuronal phenotype to a level equivalent of that induced by FGFR-1. Thus, FGFR-3 elicits phenotypic changes primarily though activation of Ras-independent pathways in the absence of robust Ras-dependent signals.     

The human granulocyte-macrophage colony-stimulating factor receptor is capable of initiating signal transduction in NIH3T3 cells.

The ability of the receptor for the hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) to function in non-hematopoietic cells is unknown. NIH3T3 fibroblasts were transfected with cDNAs encoding the alpha and beta subunit of the human GM-CSF receptor and a series of stable transformants were isolated that bound GM-CSF with either low (KD = 860 - > 1000 pM) or high affinity (KD = 20-80 pM). Low affinity receptors were not functional. However, the reconstituted high affinity receptors were found to be capable of activating a number of signal transduction pathways, including tyrosine kinase activity, phosphorylation of Raf-1, and the transient induction of c-fos and c-myc mRNAs. The activation of protein tyrosine phosphorylation by GM-CSF in NIH3T3 cells was rapid (< 1 min) and transient (peaking at 5-20 min) and resulted in the phosphorylation of proteins of estimated molecular weights of 42, 44, 52/53 and 58-60 kDa. Some of these proteins co-migrated with proteins from myeloid cells that were phosphorylated on tyrosine residues in response to GM-CSF. In particular, p42 and p44 were identified as mitogen-activated protein kinases (MAP kinases), and the phosphorylation on tyrosine residues of p42 and p44 MAP kinases occurred at the same time as the phosphorylation of Raf-1. However, despite evidence for activation of many mitogenic signal transduction molecules, GM-CSF did not induce significant proliferation of transfected NIH3T3 cells. These results suggest that murine fibroblasts contain signal transducing molecules that can effectively interact with the human GM-CSF receptor, and that are sufficient to activate at least some of the same signal transduction pathways this receptor activates in myeloid cells, including activation of one or more tyrosine kinase(s). However, the level of activation of signal transduction is either below a threshold of necessary activity or at least one mitogenic signal necessary for proliferation is missing.     

Enhanced defense against Pneumocystis carinii mediated by a novel dectin-1 receptor Fc fusion protein

Pneumocystis carinii (PC) pneumonia is a leading opportunistic infection found among HIV-infected individuals worldwide. Although CD4(+) T cell deficiency clearly correlates with susceptibility to PC pneumonia, murine models of disease indicate that PC-directed Abs may prevent infection and/or inhibit growth of existing PC within the lungs. Recognition of PC by alveolar macrophages involves the beta-glucan receptor Dectin-1 and macrophage effector function against PC is enhanced by Abs derived from PC-vaccinated hosts. We developed a fusion protein consisting of the extracellular domain of Dectin-1 linked to the Fc portion of murine IgG1, which we hypothesized would enhance host recognition and opsonic phagocytosis of PC. The recombinant protein, Dectin-Fc, is dimeric and the Ag recognition site identifies beta-1,3 glucan linkages specifically and with high affinity (K(D) = 2.03 x 10(-7) M). Dectin-Fc enhances RAW264.7 macrophage recognition of the beta-glucan containing particulate zymosan in an FcgammaRII- and FcgammaRIII-dependent manner and preopsonization of PC organisms with Dectin-Fc increased alveolar and peritoneal macrophage-dependent killing of PC. SCID mice treated with a replication incompetent adenoviral vector expressing Dectin-Fc had attenuated growth of PC within the lungs, overall decreased PC lung burden, and diminished correlates of PC-related lung damage relative to SCID mice receiving a control vector. These findings demonstrate that targeting PC beta-glucan with Dectin-Fc enhances host recognition and clearance of PC in the absence of B and T cells, and suggest that FcgammaR-based targeting of PC, via cell wall carbohydrate recognition, may promote resistance against PC pneumonia in the immunodeficient host.     

The neutrophil chemiluminescence response to Pneumocystis carinii is stimulated by GM-CSF and gamma interferon.

The interaction of Pneumocystis carinii purified from rat lung with human polymorphonuclear leukocytes (PMN) was studied using amplified chemiluminescence. Pre-incubation of PMN with granulocyte-macrophage stimulating factor or gamma interferon resulted in phagocyte stimulation.     

Ganglioside GM3 inhibition of EGF receptor mediated signal transduction

Ganglioside GM3 is a membrane component that has been describedto modulate cell growth through inhibition of EGF receptor associatedtyrosine kinase. In order to determine if the inhibition ofcell growth by this ganglioside is specifically mediated throughEGF receptor signaling, the effects of GM3 on key enzymes implicatedin EGF signaling were determined and compared to another inhibitorof the EGF receptor kinase. Treatment of A1S cells in cultureby GM3 or a tyrosine kinase inhibitor, leflunomide, led to theinhibition of MAP kinase and PI3 kinase activities. There wasno detectable effect on phosphotyrosine phosphatases. In a cellfree system, however, GM3 had no effect on the activity of thesesignaling intermediates. Leflunomide was able to directly inhibitMAP kinase activity. GM3 and leflunomide were also found toact differently on the expression of the early immediate genes.The expression of c-fos and c-jun was inhibited by both GM3and leflunomide. The expression of c-myc, however, was onlyinhibited by leflunomide. These findings suggest that the actionof GM3 on cell growth and signaling is specifically mediatedby EGF receptor and that this ganglioside does not act directlyon the intracellular intermediates of EGF receptor signaling.In addition, soluble small molecule tyrosine kinase inhibitorssuch as leflunomide can directly affect the activity of MAPkinases and possibly other signaling intermediates. The directeffects of leflunomide on signaling intermediates may explainthe differential effects of leflunomide and GM3 on gene expressionand cell growth. cell growth epidermal growth factor gangliosides GM3 signal transduction