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The ␣-subunit of eukaryotic initiation factor 2B (eIF-2B), a guanine nucleotide exchange protein that functions in regulation of translation, was observed to associate with the carboxyl-terminal cytoplasmic domains of the ␣ 2A -and ␣ 2B -adrenergic receptors in a yeast twohybrid screen of a cDNA library prepared from 293 cells. This protein association was confirmed in vitro by affinity chromatography and was shown to be specific for a subset of G protein-coupled receptors, including the ␣ 2A -, ␣ 2B -, ␣ 2C -, and ␤ 2 -adrenergic receptors, but not the vasopressin (V 2 ) receptor. Association of these proteins in vivo was confirmed by specific co-immunoprecipitation of eIF-2B␣ with full-length ␤ 2 -adrenergic receptors expressed in transfected 293 cells and by fluorescence microscopy showing co-localization of these proteins in intact cells. Remarkably, eIF-2B␣ co-localized with receptors exclusively in regions of the plasma membrane that are in contact with the extracellular medium, but failed to associate with membranes making cell-cell contacts. Overexpression of eIF-2B␣ in 293 cells caused a small (ϳ15%) but significant enhancement of ␤ 2 -adrenergic receptor-mediated activation of adenylyl cyclase, without affecting forskolin or V 2 receptor-mediated activation. These observations suggest a new role for a previously identified guanine nucleotide exchange protein in membrane biology and cell signaling.G protein-coupled receptors interact with several classes of cytoplasmic proteins, including heterotrimeric G proteins, kinases, phosphatases, and arrestins (1-4). Specific roles of these protein associations in receptor signaling and regulation are now well established. These protein interactions were first inferred from their functional effects on receptor signaling and desensitization before direct physical associations of these proteins with receptors were observed biochemically (5-8). This observation raises the possibility that receptors may interact with additional cellular proteins that could play unanticipated roles in determining the efficacy or specificity of receptor-G protein coupling.We have investigated this possibility by searching for novel protein interactions with adrenergic receptors, focusing on the carboxyl-terminal cytoplasmic domain because mutations within this domain have pleiotropic effects on receptor physiology (9 -12). 1 Using interaction cloning and biochemical techniques, we have observed that several subtypes of adrenergic receptors associate specifically with eIF-2B␣, 2 the smallest subunit of a cytoplasmic guanine nucleotide exchange factor. While this protein has a well defined role in regulation of translation, it has never been shown previously to interact with any membrane receptor. Immunocytochemical studies suggest that receptors associate with eIF-2B␣ only in restricted regions of the plasma membrane, and functional studies suggest that this protein interaction may play a role in the regulation of receptor-mediated signaling. EXPERIMENTAL PROCEDURESYeast Two-hybrid Clo...

We have characterized the interaction of endothelin (ET) with cultured neonatal rat ventricular myocytes. Binding studies indicate a single population of ETA receptors [53,000 sites/cell, apparent dissociation constant (Kd) for ET-1 approximately 0.07 nM]. Analysis of mRNA levels for ET receptors using 35 cycles of reverse transcriptase-polymerase chain reaction demonstrates the presence of only ETA-receptor message. Studies with ET-1 and a variety of congeners and antagonists indicate that ETA receptors couple to both the stimulation of phosphoinositide turnover and the inhibition of adenylyl cyclase. In myocytes transfected with an atrial natriuretic factor (ANF) promoter linked to a luciferase reporter gene, ET-1 stimulates luciferase expression through an ETA receptor. These data indicate that the ETA receptor is the exclusive receptor on neonatal ventricular myocytes and that this receptor couples to both phosphoinositide hydrolysis and adenylyl cyclase. ET-1 also induces a threefold increase in mitogen-activated protein kinase (MAPK) activity, an effect that is not sensitive to pertussis toxin (PTx). By contrast, ET-stimulated ANF-luciferase expression is partially inhibited by treatment of cells with PTx, suggesting that both PTx-sensitive (Gi) and PTx-insensitive (Gq) pathways mediate the effects of ET-1 on ANF gene expression in neonatal myocytes and that hormonal regulation of ANF expression may utilize pathways in addition to the activation of MAPK.

The superantigen toxic shock syndrome toxin (TSST)-1 can induce tumor necrosis factor (TNF)-alpha expression in T cells and monocytes, through different signaling pathways. We have stimulated peripheral blood mononuclear cells with TSST-1 and found that the major cell producers of TNF-alpha as detected by cytofluorimetry and immunocytochemistry were CD4(+) T lymphocytes. The expression of TNF-alpha by CD4(+) T cells can be inhibited by either, wortmannin (WN) or LY 294002, two phosphatidylinositol 3-kinase (PI 3-K) inhibitors. The inhibitory effect is not transcriptional as WN does not change the mRNA steady state of TNF-alpha at any of the concentrations tested and LY 294002 when preincubated with mononuclear cells at its median inhibitory concentration (IC(50) = 1. 4 microM) significantly inhibited the expression of TNF-alpha but not its mRNA. Immunoprecipitation of pulse-labeled intracellular TNF-alpha showed a specific decrease in the synthesis of this cytokine on cells treated with PI 3-K inhibitors. The p38 mitogen-activated protein kinase (MAPK) is involved in control of TNF-alpha translation in human macrophages. In T cells, we have found that the p38 MAPK inhibitor SB 203580 significantly decreased the secretion of TNF-alpha but not its mRNA. In addition, the combined use of WN and SB 203580 had an additive inhibitory effect on secretion of TNF-alpha. Therefore, both PI 3-K and p38 MAPK signaling pathways control TNF-alpha production in T cells.