Receptor-regulated dynamic interaction between endothelial nitric oxide synthase and calmodulin revealed by fluorescence resonance energy transfer in living cells.

The endothelial isoform of nitric oxide synthase (eNOS), a key regulator of vascular tone, is activated in endothelial cells by diverse Ca(2+)-mobilizing agonists, including vascular endothelial growth factor (VEGF). Although the activation state of eNOS and the subcellular localization of the enzyme are both highly regulated, the relationship between enzyme activity and subcellular targeting remains obscure. We aim here to elucidate this relationship by direct dynamic imaging analysis of Ca(2+)/CaM-dependent eNOS activation in living endothelial cells, using high-resolution confocal microscopy and donor dequenching fluorescence resonance energy transfer (FRET) techniques. Confocal images show a complex pattern of eNOS subcellular distribution; the enzyme is concentrated in both the plasma membrane and internal membranes, with robust expression in the perinuclear region. We construct a fusion protein between eNOS and the FRET-based calcium sensor cameleon, and analyze the temporal and spatial pattern of VEGF-mediated calcium mobilization using donor dequenching FRET methods. We find that VEGF promotes rapid mobilization of intracellular calcium throughout the regions of the cell in which eNOS is distributed. We further create a series of fusion proteins and use FRET imaging methods to study the interactions between eNOS and its obligate allosteric activator protein calmodulin. We clone the FRET acceptor EYFP (enhanced yellow fluorescent protein) at the C-terminus of calmodulin, and the FRET donor ECFP (enhanced cyan fluorescent protein) into eNOS at a site adjacent to its calmodulin-binding domain. FRET imaging analysis of individual endothelial cells cotransfected with eNOS-ECFP and calmodulin-EYFP shows that VEGF induces interactions between eNOS and calmodulin wherever both are present in the cell. Our studies provide evidence that the pool of rapidly responsive receptor-activated eNOS is distributed throughout endothelial cells in both plasma membrane and internal membrane structures, and that this distribution parallels the localization of agonist-induced intracellular Ca(2+) changes in the vicinity of eNOS.

CFTR is a pattern recognition molecule that extracts Pseudomonas aeruginosa LPS from the outer membrane into epithelial cells and activates NF-kappa B translocation.

Immune cells are activated during cellular responses to antigen by two described mechanisms: (i) direct uptake of antigen and (ii) extraction and internalization of membrane components from antigen-presenting cells. Although endocytosis of microbial antigens by pattern recognition molecules (PRM) also activates innate immunity, it is not known whether this involves extraction and internalization of microbial surface components. Epithelial cells on mucosal surfaces use a variety of receptors that are distinct from the classical endocytic PRM to bind and internalize intact microorganisms. Nonclassical receptor molecules theoretically could act as a type of endocytic PRM if these molecules could recognize, bind, extract, and internalize a pathogen-associated molecule and initiate cell signaling. We report here that the interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) and the outer core oligosaccharide of the lipopolysaccharide (LPS) in the outer membrane of Pseudomonas aeruginosa satisfies all of these conditions. P. aeruginosa LPS was specifically recognized and bound by CFTR, extracted from the organism's surface, and endocytosed by epithelial cells, leading to a rapid (5- to 15-min) and dynamic translocation of nuclear transcription factor NF-kappa B. Inhibition of epithelial cell internalization of P. aeruginosa LPS prevented NF-kappa B activation. Cellular activation depended on expression of wild-type CFTR, because both cultured Delta F508 CFTR human airway epithelial cells and lung epithelial cells of transgenic-CF mice failed to endocytose LPS and translocate NF-kappa B. CFTR serves as a critical endocytic PRM in the lung epithelium, coordinating the effective innate immune response to P. aeruginosa infection.