Phosphorylation-independent beta-arrestin translocation and internalization of leukotriene B4 receptors

Leukotriene B4 (LTB4) activates the G-protein-coupled receptor leukotriene B4 receptor 1 (BLT1) to mediate a diverse array of cellular responses in leukocytes including chemotaxis, calcium mobilization, degranulation, and gene expression. To determine the role of phosphorylation in BLT1 regulation, we generated mutants of BLT1 in which all of the serine/threonine residues in the C-tail are converted to alanine or to aspartate/glutamate. These mutants expressed in rat basophilic leukemia RBL-2H3 cells bound LTB4 with similar affinity and activated all of the known functional activities of BLT1, albeit at different levels. The conversion of phosphorylation sites to alanine resulted in enhanced G-protein-mediated activities, whereas conversion to aspartate/glutamate resulted in reduced responses and a right shift in dose response, indicating that receptor phosphorylation is a critical regulator of G-protein-mediated pathways. Surprisingly, translocation of beta-arrestin and receptor internalization was completely independent of BLT1 phosphorylation. Real-time analysis of beta-arrestin translocation and receptor internalization using digital fluorescence video microscopy in cells expressing a red fluorescent protein labeled BLT1 and a green fluorescent protein-tagged beta-arrestin confirmed phosphorylation-independent beta-arrestin translocation and internalization of BLT1. In beta-arrestin-deficient mouse embryo fibroblasts, the BLT1 receptors failed to display endosomal localization upon stimulation. In these cells, co-expression of beta-arrestin-green fluorescent protein with BLT1-red fluorescent protein resulted in co-localization of BLT1 and beta-arrestin upon activation. Thus, receptor phosphorylation-dependent mechanisms regulate G-protein-mediated pathways; however, phosphorylation-independent mechanisms regulate beta-arrestin association and internalization of BLT1.     

Effects of HU binding on the equilibrium cyclization of mismatched, curved, and normal DNA

The effects of HU, the histone-like protein from Escherichia coli, on the equilibrium cyclization of duplex DNAs have been observed as a function of protein concentration and DNA sequence. The results indicate that the presence of HU significantly enhances the extent of cyclization and increases the melting temperature, T(m), of the cyclized form of the DNA by >10 K. The stabilization of equilibrium cyclization by HU binding is at least -1.2 kcal/mol. The results are consistent with two HU homotypic dimers binding to each of the three 29-mer duplexes studied. One of the 29-mer duplexes contains a central dA tract, one contains mismatched sites, and one a conventional sequence. Stepwise or microscopic association constants, determined from the fluorescence data, range from 1.5 to 0.6 micro M(-1). The binding affinity of the HU dimer is strongest for the mismatched duplex and lowest for the dA tract, consistent with HU dimers having a preference for flexible DNA substrates. These results demonstrate the utility of the equilibrium cyclization approach to monitor DNA-protein interactions. These results have been considered along with those previously obtained to refine a model for the interaction of HU with duplex DNA.     

Mechanical stress and nitric oxide influence leukotriene production in cartilage.

Nitric oxide (NO) and leukotrienes regulate a variety of processes in joint tissues and are frequently elevated in arthritis. Mechanical stress can induce biochemical and functional changes in cartilage that may influence mediator production. To investigate the relationship between mechanical stress and the production of leukotriene B(4) (LTB(4)) and NO, explants of porcine articular cartilage were subjected to mechanical compression for 1 h followed by 23 h recovery in the presence or absence of the NOS2 inhibitor 1400W. Dynamic compression significantly increased LTB(4) and LOX protein production in the presence of 1400W. The induced LTB(4) was functional as evidenced by its ability to promote chemotaxis of RBL-2H3 cells expressing the LTB(4) receptor. Increased LOX protein but not LTB(4) occurred in response to compression alone. These findings provide a direct link between mechanical stress and inflammation in cartilage and may have implications in the pathogenesis and treatment of arthritis.