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Research ArticleSpecial Section on Phosphoproteomic Analysis of G Protein-Coupled Pathways - Axelrod Symposium—Minireview

Phosphoproteomic Identification of Vasopressin/cAMP/Protein Kinase A–Dependent Signaling in Kidney

Karim Salhadar, Allanah Matthews, Viswanathan Raghuram, Kavee Limbutara, Chin-Rang Yang, Arnab Datta, Chung-Lin Chou and Mark A. Knepper
Molecular Pharmacology May 2021, 99 (5) 358-369; DOI: https://doi.org/10.1124/mol.120.119602
Karim Salhadar
Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland
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Allanah Matthews
Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland
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Viswanathan Raghuram
Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland
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Kavee Limbutara
Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland
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Chin-Rang Yang
Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland
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Arnab Datta
Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland
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Chung-Lin Chou
Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland
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Mark A. Knepper
Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland
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    Fig. 1.

    Vasopressin signaling in collecting duct cells of the kidney. The physiologic responses at a cellular level are listed in Table 1. AC VI, adenylyl cyclase 6.

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    Fig. 2.

    Simplified basic protocol for phosphoproteomic analysis. A protein sample is subjected to proteolysis with a purified, recombinant protease (typically trypsin). Phosphopeptides are enriched through use of ion chromatography, e.g., with TiO2 or Fe-IMAC columns, which select peptides with negative charges. The column eluate is subjected to LC-MS/MS analysis to identify and quantify phosphopeptides, often after additional fractionation (not shown). IMAC, immobilized metal affinity chromatography. Circled P indicates phosphorylation.

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    Fig. 3.

    Mapping of vasopressin-responsive phosphoproteins to major physiologic effects of vasopressin at a cellular level. Proteins are indicated by their official gene symbols. Those showing increases in phosphorylation in response to vasopressin are indicated in green, whereas those showing decreases are indicated in red. Known PKA targets are underlined.

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    Fig. 4.

    PKA signaling network. PKA target phosphoproteins were identified by deletion of PKA by genome editing techniques followed by SILAC-based phosphoproteomics (Isobe et al., 2017). Functional groups around the periphery correspond to vasopressin-regulated processes in Table 1. Yellow nodes indicate other protein kinases that undergo PKA-mediated phosphorylation. Gray nodes indicate non-PKA targets included to consolidate phosphoprotein clustering. Analysis used a computer application called STRING (https://string-db.org/) for initial mapping followed by additional classification by Gene Ontology biologic process terms. Final visualization used Cytoscape (https://cytoscape.org/).

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    Fig. 5.

    Lithium signaling network. Phosphoproteins altered in rat inner medullary collecting ducts by in vivo lithium administration were clustered according to protein function. Red nodes indicate proteins with phosphosites downregulated by lithium; green nodes indicate proteins with phosphosites that were upregulated by lithium. Nodes with thick borders are protein kinases. Original data were from Trepiccione et al. (2014).

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    Fig. 6.

    Comparison of phophoproteomic responses to a V2 vasopressin receptor antagonist satavaptan and to a V2 vasopressin receptor agonist dDAVP. Proteins are indicated by official gene symbols, with regulated phosphorylation sites indicated. Dotted line indicates best fit, linear regression.

Tables

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    TABLE 1

    Cellular processes regulated by vasopressin in collecting duct principal cells

    Vasopressin ActionsReferencesFig. 2 Cluster
    Accelerates Aqp2 gene transcriptionHasler et al., 2009; Sandoval et al., 20161
    Mobilizes intracellular calciumStar et al., 1988; Champigneulle et al., 1993; Chou et al., 20002
    Reorganizes filamentous actinSimon et al., 1993; Tamma et al., 2001; Chou et al., 2004; Loo et al., 20133
    Depolymerizes filamentous actinSimon et al., 1993; Tamma et al., 20014
    Causes microtubule-dependent AQP2 redistributionSabolić et al., 19955
    Accelerates exocytosis of AQP2-containing vesiclesNielsen and Knepper, 1993; Nielsen et al., 1995; Nunes et al., 20086
    Decreases AQP2 endocytosisNielsen and Knepper, 1993; Brown, 20037
    Increases tight junction permeabilityMishler et al., 19908
    Accelerates AQP2 translationKhositseth et al., 2011; Sandoval et al., 20139
    Increases AQP2 protein half-lifeNedvetsky et al., 2010; Sandoval et al., 201311
    Slows rate of apoptosisMiller et al., 201310
    Slows rate of proliferationYamaguchi et al., 2003–
    Increases principal cell sizeGanote et al., 1968; Kirk et al., 1984; Nielsen et al., 1993; Chou et al., 2008–
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    TABLE 2

    List of phosphoproteomic data sets; links to browsing and download sites

    Data Set NamePublication YearCell TypeExperimentLink
    Phosphorylation sites in IMCD proteins – response to vasopressin2019Native rat IMCD cellsResponse to V2 agonist dDAVPhttps://hpcwebapps.cit.nih.gov/ESBL/Database/IMCD-Phos/
    Mouse mpkCCD phosphoprotein database2010Cultured mpkCCD cellsResponse to V2 agonist dDAVPhttp://helixweb.nih.gov/ESBL/Database/mpkCCDphos/
    Quantitative phosphoproteomics of vasopressin-sensitive renal cells: regulation of Aquaporin-2 phosphorylation at two sites2006Native rat IMCD cellsResponse to V2 agonist dDAVPhttps://big.nhlbi.nih.gov/index.jsp
    TiPD2011Native rat IMCD cellsTime course of response to V2 agonist dDAVPhttp://helixweb.nih.gov/ESBL/Database/TiPD/
    Phosphopeptides altered by PKA deletion in mouse mpkCCD cells2017Cultured mpkCCD cellsEffect of CRISPR-mediated deletion of both PKA catalytic geneshttps://hpcwebapps.cit.nih.gov/ESBL/Database/PKAKO/
    IMCD phosphoproteome with acute lithium treatment2014Native rat IMCD cellsResponse to short-term treatment of rats with lithiumhttp://helixweb.nih.gov/ESBL/Database/iPALT/
    Effect of satavaptan on phosphoproteome in rat inner medullary collecting duct2014Native rat IMCD cellsEffect of V2 antagonist satavaptanhttps://hpcwebapps.cit.nih.gov/ESBL/Database/Satavaptan/
    • iTRAQ, isobaric tags for relative and absolute quantitation; TiPD, temporal iTRAQ phosphoproteomic database. IMCD, inner medullary collecting duct; mpkCCD, mouse epithelial cell culture line resembling cortical collecting duct.

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    TABLE 3

    Studies in which phosphoprotemic response to vasopressin was measured. Progress has been marked by a progressive improvement in sensitivity of phosphoproteomic methods used.

    YearReferenceTissueNumber of Phosphopeptides QuantifiedQuantification MethodNumber of Phosphorylation Sites ChangedNumber of Phosphorylation Sites Increased
    2006Hoffert et al., 2006Rat IMCD suspensions17Label free94
    2010Rinschen et al., 2010Mouse mpkCCD cells338SILAC4518
    2012Hoffert et al., 2012Rat IMCD suspensions1427Isobaric tags (iTRAQ)4430
    2019Deshpande et al., 2019Rat IMCD suspensions10,738Isobaric tags (TMT)219156
    2020Datta et al., 2020Mouse mpkCCD cells19,221SILAC452205
    • IMCD, inner medullary collecting duct; iTRAQ, isobaric tags for relative and absolute quantitation; mpkCCD, mouse epithelial cell culture line resembling cortical collecting duct; SILAC, stable isotope labeling in cell culture; TMT, tandem mass tag.

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Molecular Pharmacology: 99 (5)
Molecular Pharmacology
Vol. 99, Issue 5
1 May 2021
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Research ArticleSpecial Section on Phosphoproteomic Analysis of G Protein-Coupled Pathways - Axelrod Symposium—Minireview

Phosphoproteomic Identification of Signaling Pathways

Karim Salhadar, Allanah Matthews, Viswanathan Raghuram, Kavee Limbutara, Chin-Rang Yang, Arnab Datta, Chung-Lin Chou and Mark A. Knepper
Molecular Pharmacology May 1, 2021, 99 (5) 358-369; DOI: https://doi.org/10.1124/mol.120.119602

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Research ArticleSpecial Section on Phosphoproteomic Analysis of G Protein-Coupled Pathways - Axelrod Symposium—Minireview

Phosphoproteomic Identification of Signaling Pathways

Karim Salhadar, Allanah Matthews, Viswanathan Raghuram, Kavee Limbutara, Chin-Rang Yang, Arnab Datta, Chung-Lin Chou and Mark A. Knepper
Molecular Pharmacology May 1, 2021, 99 (5) 358-369; DOI: https://doi.org/10.1124/mol.120.119602
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  • Article
    • Abstract
    • Introduction
    • Cellular Physiology of AQP2-Expressing Renal Collecting Duct Cells
    • Phosphoproteomic Methodology and Bioinformatics
    • Effect of Vasopressin on the Phosphoproteome of the Renal Collecting Duct
    • Effect of PKA Deletion on the Phosphoproteome of the Renal Collecting Duct
    • Identification of Potential PKA Targets by In Vitro Phosphorylation and Mass Spectrometry
    • Effect of Kinase Inhibitor H89 on Protein Phosphorylation in PKA-Null Collecting Duct Cells
    • Polyuric Disorders: Phosphoproteomics of Lithium-Induced Nephrogenic Diabetes Insipidus
    • V2 Receptor Antagonists and Hyponatremic Disorders: How Vaptans Affect the Collecting Duct Phosphoproteome
    • Integration of Phosphoproteomic Results
    • Authorship Contributions
    • Footnotes
    • Abbreviations
    • References
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