Using a bioinformatics approach, we have isolated a novel Gprotein-coupled receptor (GPCR), R527, and have demonstrated that this receptor shows no significant homology to previously deorphanized GPCRs. Quantitative reverse transcription-polymerase chain reaction analysis of the expression of GPCR R527 indicated a very high level of mRNA expression in eosinophils, with high expression also detected in neutrophils and lung macrophages. Stable cell lines were generated expressing this receptor together with the G-protein ␣-subunit G␣ 16 . These cells were used to screen an agonist collection in a calcium mobilization assay and 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) was identified as a putative ligand. 5(S)-Hydroxyperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid was also shown to activate the receptor, whereas the leukotrienes LTB 4 , LTC 4 , LTD 4 , and LTE 4 failed to elicit a response. In cAMP assays, pertussis toxin reversed the inhibitory effects of 5-oxo-ETE on cAMP production, indicating that the receptor is G␣ i -coupled. The GPCR R527 shows pharmacological properties similar to those of the previously described 5-oxo-ETE receptor expressed on eosinophils, neutrophils, and monocytes. These cell types show chemotactic responses to 5-oxo-ETE, and this eicosanoid has been proposed to play a key role in the inflammatory response. The molecular identification of a receptor binding 5-oxo-ETE will expand our understanding of the physiological role of this mediator and may provide new therapeutic opportunities.
Type 1 serine/threonine protein phosphatases (PP1) are important regulators of many cellular and developmental processes, including glycogen metabolism, muscle contraction, and the cell cycle [1] [2] [3] [4] [5]. Drosophila and humans both have multiple genes encoding PP1 isoforms [3] [6] [7]; each has one beta and several alpha isoform genes (alpha(1), alpha(2), alpha(3) in flies, alpha and gamma in humans; mammalian PP1beta is also known as PP1delta). The alpha/beta subtype differences are highly conserved between flies and mammals [6]. Though all these proteins are >85% identical to each other and have indistinguishable activities in vitro, we show here that the Drosophila beta isoform has a distinct biological role. We show that PP1beta9C corresponds to flapwing (flw), previously identified mutants of which are viable but flightless because of defects in indirect flight muscles (IFMs) [8]. We have isolated a new, semi-lethal flw allele that shows a range of defects, especially in muscles, which break away from their attachment sites and degenerate.
The global incidence of the sexually transmitted disease gonorrhea is expected to rise due to the spread of Neisseria gonorrhoeae strains with decreased susceptibility to extended-spectrum cephalosporins (ESCs). ESC resistance is conferred by mosaic variants of penicillin-binding protein 2 (PBP2) that have diminished capacity to form acylated adducts with cephalosporins. To elucidate the molecular mechanisms of ESC resistance, we conducted a biochemical and high-resolution structural analysis of PBP2 variants derived from the decreased-susceptibility N. gonorrhoeae strain 35/02 and ESC-resistant strain H041. Our data reveal that mutations both lower affinity of PBP2 for ceftriaxone and restrict conformational changes that normally accompany acylation. Specifically, we observe that a G545S substitution hinders rotation of the β3 strand necessary to form the oxyanion hole for acylation and also traps ceftriaxone in a noncanonical configuration. In addition, F504L and N512Y substitutions appear to prevent bending of the β3–β4 loop that is required to contact the R1 group of ceftriaxone in the active site. Other mutations also appear to act by reducing flexibility in the protein. Overall, our findings reveal that restriction of protein dynamics in PBP2 underpins the ESC resistance of N. gonorrhoeae.
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