Membrane attachment of recombinant G-protein alpha-subunits in excess of beta gamma subunits in a eukaryotic expression system

Recombinant cDNAs encoding the alpha-subunits of Gi1, Gi2, Gi3, Go and Gs were transfected into COS cells with the pCD-PS mammalian expression vector. Expression of each G alpha was verified using subtype-specific peptide antisera on immunoblots. Quantitative immunoblotting of alpha and beta subunits indicated: i) that there was no change in expression of endogenous beta subunits, and ii) overexpression of alpha subunits could achieve a ratio of alpha:beta greater than 25:1. Despite the excess of alpha over beta, the G alpha subunits were found predominantly in the membrane fraction. The results demonstrate that G alpha subunits can attach to the membrane independently of beta gamma subunits.     

Phosphorylation and Palmitoylation of the Human D2L Dopamine Receptor in Sf9 Cells

Abstract: We have expressed and biochemically characterized the human D2_long (D2_L) dopamine receptor isoform using the baculovirus/Sf9 cell system. The expressed receptor bound ligands with a pharmacological profile similar to that reported for neuronal and cloned D2_L receptors expressed in mammalian cell lines. Dopamine binding to D2_L receptor was sensitive to guanine nucleotides, indicating receptor coupling to endogenous G proteins. A D2_L receptor-specific antibody identified two major protein species at ∼44 kDa and at ∼93 kDa in immunoblots, suggesting the presence of D2_L receptor monomers and dimers. Both species were purified by immunoprecipitation from digitonin-solubilized preparation of cells expressing D2_L receptor prelabeled with ³²P_i or [³H]-palmitate. These results constitute the first direct evidence for D2_L receptor phosphorylation and palmitoylation.     

Expression of GTP-binding proteins and prostaglandin E2 receptors during human T cell activation.

GTP-binding proteins (G-proteins) are a family of closely related, yet structurally distinct signal transducing proteins. In this study the presence and relative abundance of several G-proteins and of their corresponding mRNAs were measured in resting and activated human T lymphocytes. We found that T lymphocytes contain RNA coding for Gs, Gi2, and Gi3. No Gi1- and Go-specific RNA could be detected. Membrane fractions of resting and activated lymphocytes were studied in immunoblot experiments. Again, Gs, Gi2, and Gi3, but not Gi1 and Go, were detected. Upon mitogenic activation, a relative increase in mRNA for Gs and Gi3, but not for Gi2 could be demonstrated in Northern blot experiments. Immunoblotting indicated an increase in Gs and Gi3 density in membrane fractions of T cells as well. Paralleling the increase in Gs, we found that activated T cells produce five to seven times more cAMP per cell in response to prostaglandin E2 (PGE2) than resting lymphocytes. Finally, PGE2 binding studies showed that the number of receptors for this hormone increased from 435 +/- 322 to 1035 +/- 357 per cell following in vitro stimulation. We propose that in vitro T cell activation is paralleled by an increase in sensitivity to PGE2-induced cAMP generation. This sensitization is accompanied by both an increase in cell surface PGE2 receptor numbers as well as by increased expression of the signal transducing protein Gs and may physiologically be important for limiting an immune response.     

Inhibition of uterine contractility by progesterone and progesterone metabolites: mediation by progesterone and gamma amino butyric acidA receptor systems.

Progesterone and several progesterone metabolites are capable of inhibiting uterine contractility. Some progesterone metabolites have shown little or no affinity for the progesterone receptor but have been found to be potent modulators of the GABAA receptor system. This study examined whether the inhibition of uterine contraction by progesterone and its metabolites was progesterone receptor-mediated or gamma amino butyric acidA (GABAA) receptor-mediated. Uterine contractions were measured in annular rings of uterine tissue, 5 mm in length, from diestrous II rats, under a fixed tension of 1 gram. The steroids tested were 3 beta-hydroxy-5 beta-pregnan-20-one (6 micrograms/ml), 5 beta-pregnane-3,20-dione (10 micrograms/ml), 3 alpha-hydroxy-5 alpha-pregnan- 20-one (3 alpha,5 alpha-THP, 27.5 micrograms/ml), and progesterone (40 micrograms/ml). All compounds significantly inhibited spontaneous uterine contractions when compared to controls. No effect was seen by either 16 micrograms/ml of the progesterone antagonist, RU486, or 32 micrograms/ml of the GABAA antagonist, pictrotoxin, when administered alone. However, when uterine tissues were exposed to a combination of the steroid and the antagonist, the effect of 3 beta-hydroxy-5 beta-pregnan-20-one and 3 alpha,5 alpha-THP was blocked by picrotoxin but not by RU486, indicating that the action of these steroids was mediated through the GABAA system. The effect of 5 beta-pregnane-3,20-dione and progesterone was effectively blocked by RU486 but not by picrotoxin, suggesting that their actions were mediated through the progesterone receptor system. These results indicate that multiple mechanisms exist in the uterus for inhibiting uterine contractility by progesterone and its metabolites.     

Transforming growth factor-beta: a neuroprotective factor in cerebral ischemia

Transforming growth factor-β (TGF-β) has diverse and multiple roles throughout the body. This review focuses on the evidence supporting its functions in the central nervous system, with a particular emphasis on its purported role in cerebral ischemia. Numerous studies have documented that TGF-β1 levels are enhanced in the brain following cerebral ischemia. As evidence that such an upregulation is beneficial, agonist studies have demonstrated that TGF-β1 reduces neuronal cell death and infarct size following middle cerebral artery occlusion (MCAO), while conversely, antagonist studies have shown increased neuronal cell death and infarct size after MCAO. These studies suggest that TGF-β1 has a neuroprotective role in cerebral ischemia. Recent work with adenoviral-mediated overexpression of TGF-β1 in vivo in mice has further implicated a neuroprotective role for TGF-β1 in cerebral ischemia, as evidenced by a reduction in neuronal cell death, infarct size, and neurological outcome. Additionally, numerous in vitro studies have documented the neuroprotective ability of TGF-β1 in neurons from a variety of species, including rats, mice, chicks, and humans. Of significant interest, TGF-β1 was shown to be protective against a wide variety of death-inducing agents/insults, including hypoxia/ischemia, glutamate excitotoxicity, β-amyloid, oxidative damage, and human immunodeficiency virus. The mechanism of TGF-β1-mediated neuroprotection remains to be resolved, but early evidence suggests that TGF-β1 regulates the expression and ratio of apoptotic (Bad) and antiapoptotic proteins (Bcl-2, Bcl-x₁), creating an environment favorable for cell survival of death-inducing insults. Taken as a whole, these results suggest that TGF-β1 is an important neuroprotective factor that can reduce damage from a wide-array of death-inducing agents/insults in vitro, as well as exert protection of the brain during cerebral ischemia. The authors’ research is supported by research grants (HD-28964 and AG-17186 to DWB) from the National Institutes of Health, NICHD, and NIA.     

Identification of bilirubin reduction products formed by Clostridium perfringens isolated from human neonatal fecal flora

Urobilinoids belong to the heterogenous group of degradation products of bilirubin formed in the gastrointestinal tract by intestinal microflora. Among them urobilinogen and stercobilinogen with their respective oxidation products, urobilin and stercobilin, are the most important compounds. The aim of present study was to analyze the products of bacterial reduction of bilirubin in more detail. The strain of Clostridium perfringens isolated from neonatal stools, capable of reducing bilirubin, was used in the study. Bacteria were incubated under anaerobic conditions with various native as well as synthetic bile pigments, including radiolabeled unconjugated bilirubin (UCB). Their reduction products were extracted from media and separated following thin layer chromatography. Pigments isolated were analyzed by spectrophotometry, spectrofluorometry and mass spectrometry. In a special set of experiments, bilirubin diglucuronide was incubated with either bacterial lysate or partially purified bilirubin reductase and beta-glucuronidase to reveal whether bilirubin glucuronides may be directly reduced onto conjugated urobilinoids. A broad substrate activity was detected in the investigated strain of C. perfringens and a series of bilirubin reduction products was identified. These products were separated in the form of their respective chromogens and further oxidized. Based on their physical-chemical properties, as well as mass spectra, end-catabolic bilirubin products were identified to belong to urobilinogen species. The reduction process, catalyzed enzymatically by the studied bacterial strain, does not proceed to stercobilinogen. Bilirubin diglucuronide is not reduced onto urobilinoid conjugates, glucuronide hydrolysis must precede double bond reduction and thus UCB is reduced much faster.     

Nerve growth factor-induced p75-mediated death of cultured hippocampal neurons is age-dependent and transduced through ceramide generated by neutral sphingomyelinase.

Binding of nerve growth factor (NGF) to the p75 neurotrophin receptor (p75) in cultured hippocampal neurons has been reported to cause seemingly contrasting effects, namely ceramide-dependent axonal outgrowth of freshly plated neurons, versus Jun kinase (Jnk)-dependent cell death in older neurons. We now show that the apoptotic effects of NGF in hippocampal neurons are observed only from the 2nd day of culture onward. This switch in the effect of NGF is correlated with an increase in p75 expression levels and increasing levels of ceramide generation as the cultures mature. NGF application to neuronal cultures from p75(exonIII-/-) mice had no effect on ceramide levels and did not affect neuronal viability. The neutral sphingomyelinase inhibitor, scyphostatin, inhibited NGF-induced ceramide generation and neuronal death, whereas hippocampal neurons cultured from acid sphingomyelinase(-/-) mice were as susceptible to NGF-induced death as wild type neurons. The acid ceramidase inhibitor, (1S,2R)-d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol, enhanced cell death, supporting a role for ceramide itself and not a downstream lipid metabolite. Finally, scyphostatin inhibited NGF-induced Jnk phosphorylation in hippocampal neurons. These data indicate an initiating role of ceramide generated by neutral sphingomyelinase in the diverse neuronal responses induced by binding of neurotrophins to p75.     

Medial and Lateral Entorhinal Cortex Differentially Excite Deep versus Superficial CA1 Pyramidal Neurons

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