GPR61 anchoring of PKA consolidates GPCR and cAMP signaling

Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein–protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein–protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions

Integrated Detection of Extended-Spectrum-Beta-Lactam Resistance by DNA Microarray-Based Genotyping of TEM, SHV, and CTX-M Genes▿ †

Extended-spectrum beta-lactamases (ESBL) of the TEM, SHV, or CTX-M type confer resistance to beta-lactam antibiotics in Gram-negative bacteria. The activity of these enzymes against beta-lactam antibiotics and their resistance against inhibitors can be influenced by genetic variation at the single-nucleotide level. Here, we describe the development and validation of an oligonucleotide microarray for the rapid identification of ESBLs in Gram-negative bacteria by simultaneously genotyping blaTEM, blaSHV, and blaCTX-M. The array consists of 618 probes that cover mutations responsible for 156 amino acid substitutions. As this comprises unprecedented genotyping coverage, the ESBL array has a high potential for epidemiological studies and infection control. With an assay time of 5 h, the ESBL microarray also could be an attractive option for the development of rapid antimicrobial resistance tests in the future. The validity of the DNA microarray was demonstrated with 60 blinded clinical isolates, which were collected during clinical routines. Fifty-eight of them were characterized phenotypically as ESBL producers. The chip was characterized with regard to its resolution, phenotype-genotype correlation, and ability to resolve mixed genotypes. ESBL phenotypes could be correctly ascribed to ESBL variants of blaCTX-M (76%), blaSHV (22%), or both (2%), whereas no ESBL variant of blaTEM was found. The most prevalent ESBLs identified were CTX-M-15 (57%) and SHV-12 (18%)

A systems biology approach to analyse amplification in the JAK2-STAT5 signalling pathway-5

For transient stimulation with an average duration of ∈ [0.1, 10] minutes, and an average concentration of ∈ [10,500] units/ml.<p><b>Copyright information:</b></p><p>Taken from "A systems biology approach to analyse amplification in the JAK2-STAT5 signalling pathway"</p><p>http://www.biomedcentral.com/1752-0509/2/38</p><p>BMC Systems Biology 2008;2():38-38.</p><p>Published online 25 Apr 2008</p><p>PMCID:PMC2386439.</p><p></p

A systems biology approach to analyse amplification in the JAK2-STAT5 signalling pathway-3

On of ∈ [0.1, 10] minutes, and an average concentration of ∈ [10,500] units/ml.<p><b>Copyright information:</b></p><p>Taken from "A systems biology approach to analyse amplification in the JAK2-STAT5 signalling pathway"</p><p>http://www.biomedcentral.com/1752-0509/2/38</p><p>BMC Systems Biology 2008;2():38-38.</p><p>Published online 25 Apr 2008</p><p>PMCID:PMC2386439.</p><p></p

A systems biology approach to analyse amplification in the JAK2-STAT5 signalling pathway-6

M was analysed for oscillatory stimuli with an average period duration T ∈ [0.5, 1440] minutes, and an average concentration of ∈ [10, 10] units/ml. In all the simulations, the signal was averaged for 12 periods of the oscillatory stimulus.<p><b>Copyright information:</b></p><p>Taken from "A systems biology approach to analyse amplification in the JAK2-STAT5 signalling pathway"</p><p>http://www.biomedcentral.com/1752-0509/2/38</p><p>BMC Systems Biology 2008;2():38-38.</p><p>Published online 25 Apr 2008</p><p>PMCID:PMC2386439.</p><p></p

A systems biology approach to analyse amplification in the JAK2-STAT5 signalling pathway-4

Its/ml. The solid black line is used to highlight the different values of obtained for a total amount of Epo of one hundred units. In this case the average of the system response ranges between 0.05 and 0.17 normalised units. The dashed black line is used to highlight how in the interval of intense stimulation input signals with totally different properties (∈ [10,10] units/ml) produce output signal with identical average intensity .<p><b>Copyright information:</b></p><p>Taken from "A systems biology approach to analyse amplification in the JAK2-STAT5 signalling pathway"</p><p>http://www.biomedcentral.com/1752-0509/2/38</p><p>BMC Systems Biology 2008;2():38-38.</p><p>Published online 25 Apr 2008</p><p>PMCID:PMC2386439.</p><p></p

A systems biology approach to analyse amplification in the JAK2-STAT5 signalling pathway-2

Ological value for serum concentration of Epo (aprox. 7.9·10units/ml), while the finely dashed line indicates the concentration of Epo used in the experiments performed (5 units/ml).<p><b>Copyright information:</b></p><p>Taken from "A systems biology approach to analyse amplification in the JAK2-STAT5 signalling pathway"</p><p>http://www.biomedcentral.com/1752-0509/2/38</p><p>BMC Systems Biology 2008;2():38-38.</p><p>Published online 25 Apr 2008</p><p>PMCID:PMC2386439.</p><p></p

A systems biology approach to analyse amplification in the JAK2-STAT5 signalling pathway-1

Prox. 7.9·10units/ml), while the finely dashed line indicates the concentration of Epo used in the experiments performed (5 units/ml).<p><b>Copyright information:</b></p><p>Taken from "A systems biology approach to analyse amplification in the JAK2-STAT5 signalling pathway"</p><p>http://www.biomedcentral.com/1752-0509/2/38</p><p>BMC Systems Biology 2008;2():38-38.</p><p>Published online 25 Apr 2008</p><p>PMCID:PMC2386439.</p><p></p