Adenosine receptor A2B mediates alcoholic hepatitis by regulating cAMP levels and the NF-KB pathway.

Alcoholic hepatitis is a serious form of liver damage. Inflammation is a key factor in alcoholic hepatitis and plays a key role in the progression of alcoholic liver disease. Adenosine receptor A2B (A2BAR) is a member of the adenosine receptor family and generally considered to be a negative regulator of the inflammatory response. We found that A2BAR was the most highly expressed adenosine receptor in ETOH-fed mouse liver tissue and was also highly expressed in primary Kupffer cells and ETOH-induced RAW264.7 cells. In addition, injection of BAY 60-6583 stimulated A2BAR, induced upregulation of the expression levels of cAMP, and reduced ETOH-induced steatosis and inflammation in mice. At the same time, knockdown of A2BAR in vitro increased the inflammatory response in RAW264.7 cells triggered by ETOH. After knockdown of A2BAR in vitro, the release of the inflammatory cytokines IL-6, IL-1ß and TNF-α was increased. After overexpression of A2BAR in vitro, the cAMP level was significantly increased, PKA expression was increased, the expression of phosphorylated proteins in the NF-kB signal transduction pathway was significantly affected, and the expression of the key phosphorylated protein p-P65 was decreased. However, after the simultaneous overexpression of A2BAR and inhibition of PKA, the expression of the key phosphorylated protein p-P65 was still significantly decreased. In addition, after the expression of A2BAR increased or decreased in RAW264.7 cells, AML-12 cells were cultured in the supernatant of RAW264.7 cells stimulated by ETOH, and the apoptosis rate was significantly changed by flow cytometry. These results suggest that A2BAR can reduce alcoholic steatohepatitis by upregulating cAMP levels and negatively regulating the NF-kB pathway. Overall, these findings suggest the significance of A2BAR-mediated inflammation in alcoholic liver disease.

Rice TUTOU1 Encodes a Suppressor of cAMP Receptor-Like Protein That Is Important for Actin Organization and Panicle Development.

Panicle development, a key event in rice (Oryza sativa) reproduction and a critical determinant of grain yield, forms a branched structure containing multiple spikelets. Genetic and environmental factors can perturb panicle development, causing panicles to degenerate and producing characteristic whitish, small spikelets with severely reduced fertility and yield; however, little is known about the molecular basis of the formation of degenerating panicles in rice. Here, we report the identification and characterization of the rice panicle degenerative mutant tutou1 (tut1), which shows severe defects in panicle development. The tut1 also shows a pleiotropic phenotype, characterized by short roots, reduced plant height, and abnormal development of anthers and pollen grains. Molecular genetic studies revealed that TUT1 encodes a suppressor of cAMP receptor/Wiskott-Aldrich syndrome protein family verprolin-homologous (SCAR/WAVE)-like protein. We found that TUT1 contains conserved functional domains found in eukaryotic SCAR/WAVE proteins, and was able to activate Actin-related protein2/3 to promote actin nucleation and polymerization in vitro. Consistently, tut1 mutants show defects in the arrangement of actin filaments in trichome. These results indicate that TUT1 is a functional SCAR/WAVE protein and plays an important role in panicle development.

The novel tyrosine kinase ZAK1 activates GSK3 to direct cell fate specification.

Inhibition of GSK3 by 7-TM Wnt/wg receptor signaling is critical for specifying embryonic cell fate patterns. In Dictyostelium, the 7-TM cAMP receptors regulate GSK3 by parallel, antagonistic pathways to establish a developmental body plan. We describe here a novel tyrosine kinase, ZAK1, downstream of 7-TM cAMP receptor signaling that is required for GSK3 activation during development. zak1-nulls have reduced GSK3 activity and are defective in GSK3-regulated developmental pathways. Moreover, recombinant ZAK1 phosphorylates and activates GSK3 in vitro. We propose that ZAK1 is a positive regulator of GSK3 activity required for cell pattern formation in Dictyostelium and speculate that similar mechanisms exist to antagonize Wnt/wg signaling for metazoan cell fate specification.

The cAMP-binding ectoprotein from Saccharomyces cerevisiae is membrane-anchored by glycosyl-phosphatidylinositol.

Saccharomyces cerevisiae contains an amphiphilic cAMP-binding glycoprotein at the outer face of the plasma membrane (M(r) = 54,000). It is converted to a hydrophilic form by treatment with glycosyl-phosphatidylinositol-specific phospholipases C and D (GPI-PLC/D), suggesting membrane anchorage by a covalently bound glycolipid. Determination of the constituents of the purified anchor by gas-liquid chromatography and amino acid analysis reveals the presence of glycerol, myo-inositol, glucosamine, galactose, mannose, ethanolamine, and asparagine (as the carboxyl-terminal amino acid of the Pronase-digested protein to which the anchor is attached). Complementary results are obtained by metabolic labeling, indicating that fatty acids and phosphorus are additional anchor constituents. The phosphorus is resistant to alkaline phosphatase, whereas approximately half is lost from the protein after treatment with GPI-PLD or nitrous acid, and all is removed by aqueous HF indicating the presence of two phosphodiester bonds. Inhibition of N-glycosylation by tunicamycin or removal of protein-bound glycan chains by N-glycanase or Pronase does not abolish radiolabeling of the anchor structure by any of the above compounds. Analysis of the products obtained after sequential enzymic and chemical degradation of the anchor agrees with the arrangement of constituents in GPIs from higher eucaryotes. Evidence for anchorage of the yeast cAMP-binding protein by a GPI anchor is strengthened additionally by the reactivity of the GPI-PLC-cleaved anchor with antibodies directed against the cross-reacting determinant of trypanosomal variant surface glycoproteins.

19F-n.m.r. studies of ligand binding to 5-fluorotryptophan- and 3-fluorotyrosine-containing cyclic AMP receptor protein from Escherichia coli.

Two fluorine-containing analogues of the cyclic AMP receptor protein (CRP) from Escherichia coli were prepared by biosynthetic incorporation of 5-fluorotryptophan (5-F-Trp) and 3-fluorotyrosine (3-F-Tyr). The 19F-n.m.r. spectrum of the [5-F-Trp]CRP showed two signals corresponding to the two tryptophan residues, and that of the [3-F-Tyr]CRP showed six signals (two overlapping) corresponding to the six tyrosine residues: these results are as expected for a symmetrical dimer. A comparison of the 19F-n.m.r. spectra of the CRP analogues in the presence and in the absence of cyclic AMP reveals that the chemical shifts of both tryptophan residues and of two of the six tyrosine residues show differences. Since none of these residues is in direct contact with the bound nucleotide (although Trp-85 is fairly close), these shift changes must arise from induced conformational effects. The 19F-n.m.r. spectra of complexes with cyclic GMP showed chemical-shift perturbations different from those caused by cyclic AMP, indicating that different conformational changes are induced by the binding of cyclic GMP. The 19F-n.m.r. spectrum of the complex of [3-F-Tyr]CRP with tubercidin 3',5'-(cyclic)monophosphate (which can activate transcription) showed essentially the same chemical-shift changes as seen for the cyclic AMP complex, indicating that similar conformational changes have been induced by the nucleotide binding. [3-F-Tyr]CRP in the presence of an equimolar amount of the 20 bp self-complementary DNA oligomer 5'-AATGTGAGTTAACTCACATT-3' and excess cyclic AMP gave an 19F-n.m.r. spectrum that was almost identical with that for the [3-F-Tyr]CRP-cyclic AMP complex, indicating that the binding of DNA does not induce significant conformational changes involving the tyrosine residues. Proteolysis of [3-F-Tyr]CRP with chymotrypsin produced a 31 kDa fragment that is a dimer containing the cyclic AMP-binding domain. This fragment contains five of the six tyrosine residues, and its 19F-n.m.r. chemical shifts were essentially the same as those of the intact protein except for one missing signal (signal F): this signal could be assigned to Tyr-206 and shown to be unperturbed by the binding of cyclic nucleotide to the intact [3-F-Tyr]CRP. The similarity of the 19F-n.m.r. chemical shifts in the alpha-fragment and the intact CRP indicates that the alpha-fragment retains the same structure as found in the intact protein.(ABSTRACT TRUNCATED AT 400 WORDS)