Dynamics of the G protein-coupled vasopressin V2 receptor signaling network revealed by quantitative phosphoproteomics.
ABSTRACT G protein-coupled receptors (GPCRs) regulate diverse physiological processes, and many human diseases are due to defects in GPCR signaling. To identify the dynamic response of a signaling network downstream from a prototypical G(s)-coupled GPCR, the vasopressin V2 receptor, we have carried out multireplicate, quantitative phosphoproteomics with iTRAQ labeling at four time points following vasopressin exposure at a physiological concentration in cells isolated from rat kidney. A total of 12,167 phosphopeptides were identified from 2,783 proteins, with 273 changing significantly in abundance with vasopressin. Two-dimensional clustering of phosphopeptide time courses and Gene Ontology terms revealed that ligand binding to the V2 receptor affects more than simply the canonical cyclic adenosine monophosphate-protein kinase A and arrestin pathways under physiological conditions. The regulated proteins included key components of actin cytoskeleton remodeling, cell-cell adhesion, mitogen-activated protein kinase signaling, Wnt/β-catenin signaling, and apoptosis pathways. These data suggest that vasopressin can regulate an array of cellular functions well beyond its classical role in regulating water and solute transport. These results greatly expand the current view of GPCR signaling in a physiological context and shed new light on potential roles for this signaling network in disorders such as polycystic kidney disease. Finally, we provide an online resource of physiologically regulated phosphorylation sites with dynamic quantitative data (http://helixweb.nih.gov/ESBL/Database/TiPD/index.html).
SourceAvailable from: Tiina Öhman[Show abstract] [Hide abstract]
ABSTRACT: BackgroundIt is possible to identify thousands of phosphopeptides and –proteins in a single experiment with mass spectrometry-based phosphoproteomics. However, a current bottleneck is the downstream data analysis which is often laborious and requires a number of manual steps.ResultsToward automating the analysis steps, we have developed and implemented a software, PhosFox, which enables peptide-level processing of phosphoproteomic data generated by multiple protein identification search algorithms, including Mascot, Sequest, and Paragon, as well as cross-comparison of their identification results. The software supports both qualitative and quantitative phosphoproteomics studies, as well as multiple between-group comparisons. Importantly, PhosFox detects uniquely phosphorylated peptides and proteins in one sample compared to another. It also distinguishes differences in phosphorylation sites between phosphorylated proteins in different samples. Using two case study examples, a qualitative phosphoproteome dataset from human keratinocytes and a quantitative phosphoproteome dataset from rat kidney inner medulla, we demonstrate here how PhosFox facilitates an efficient and in-depth phosphoproteome data analysis. PhosFox was implemented in the Perl programming language and it can be run on most common operating systems. Due to its flexible interface and open source distribution, the users can easily incorporate the program into their MS data analysis workflows and extend the program with new features. PhosFox source code, implementation and user instructions are freely available from https://bitbucket.org/phintsan/phosfox.ConclusionsPhosFox facilitates efficient and more in-depth comparisons between phosphoproteins in case–control settings. The open source implementation is easily extendable to accommodate additional features for widespread application use cases.Proteome Science 06/2014; 12:36. DOI:10.1186/1477-5956-12-36 · 1.88 Impact Factor
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ABSTRACT: Glomerular biology is dependent on tightly controlled signal transduction networks that control phosphorylation of signaling proteins such as cytoskeletal regulators or slit diaphragm proteins of kidney podocytes. Cross-species comparison of phosphorylation events is a powerful mean to functionally prioritize and identify physiologically meaningful phosphorylation sites. Here, we present the result of phosphoproteomic analyses of cow and rat glomeruli to allow cross-species comparisons. We discovered several phosphorylation sites with potentially high biological relevance, e.g. tyrosine phosphorylation of the cytoskeletal regulator synaptopodin and the slit diaphragm protein neph-1 (Kirrel). Moreover, cross-species comparisons revealed conserved phosphorylation of the slit diaphragm protein nephrin on an acidic cluster at the intracellular terminus and conserved podocin phosphorylation on the very carboxyl terminus of the protein. We studied a highly conserved podocin phosphorylation site in greater detail and show that phosphorylation regulates affinity of the interaction with nephrin and CD2AP. Taken together, these results suggest that species comparisons of phosphoproteomic data may reveal regulatory principles in glomerular biology.This article is protected by copyright. All rights reservedProteomics 11/2014; 15(7). DOI:10.1002/pmic.201400235 · 3.97 Impact Factor
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ABSTRACT: Biological information is growing at a rapid pace, making it difficult for individual investigators to be familiar with all information that is relevant to their own research. Computers are beginning to be used to extract and curate biological information; however, the complexity of human language used in research papers continues to be a critical barrier to full automation of knowledge extraction. Here, we report a manually curated knowledge base of vasopressin actions in renal epithelial cells that is designed to be readable either by humans or by computer programs using natural language processing algorithms. The knowledge base consists of three related databases accessible at https://helixweb.nih.gov/ESBL/TinyUrls/Vaso_portal.html. One of the component databases reports vasopressin actions on individual proteins expressed in renal epithelia including effects on phosphorylation, protein abundances, protein translocation from one sub-cellular compartment to another, protein-protein binding interactions, etc. The second database reports vasopressin actions on physiological measures in renal epithelia, and the third reports specific mRNA species whose abundances change in response to vasopressin. We illustrate the application of the knowledge base by using it to generate a protein kinase network that connects vasopressin binding in collecting duct cells to physiological effects to regulate the water channel protein, aquaporin-2.American journal of physiology. Renal physiology 07/2014; 307(6). DOI:10.1152/ajprenal.00012.2014 · 3.30 Impact Factor