Abstract:Vasopressin regulates renal water excretion by binding to a Gαs‐coupled receptor (V2R) in collecting duct cells, resulting in increased water permeability through regulation of the aquaporin‐2 (AQP2) water channel. This action is widely accepted to be associated with cAMP‐mediated activation of protein kinase A (PKA). Here, we use phosphoproteomics in collecting duct cells in which PKA has been deleted (CRISPR‐Cas9) to identify PKA‐independent responses to vasopressin. The results show that V2R‐mediated vasopr… Show more
“…The simplest experiment to identify vasopressin signaling pathways in the renal collecting duct is to expose collecting duct cells to vasopressin or its vehicle and carry out mass spectrometry-based quantitative phosphoproteomics. Experiments following this strategy have been done in both cultured collecting duct cells (mouse mpkCCD) (Rinschen et al, 2010;Datta et al, 2020) and in suspensions of native inner medullary collecting duct cells from rats (Hoffert et al, 2006(Hoffert et al, , 2012Deshpande et al, 2019). The studies have utilized a V2 receptor-selective vasopressin analog dDAVP (desmopressin, D-amino D-arginine vasopressin), which is used clinically for treatment of central diabetes insipidus (Christensen and Rittig, 2006;Oiso et al, 2013).…”
Section: Effect Of Vasopressin On the Phosphoproteome Of The Renamentioning
confidence: 99%
“…Many of those that had basophilic phosphorylation sites that increased with vasopressin are known targets of PKA (underlined, Figure 3), cementing the conclusion that vasopressin signaling is largely mediated by PKA activation. Nevertheless, some elements of vasopressin signaling appear to be PKA-independent (Datta et al, 2020). This includes PKA-independent, vasopressin induced phosphorylation changes associated with activation of one or more kinases of the AMPK-related kinase familiy (also known as SNF1family kinases).…”
Section: Mol#119602mentioning
confidence: 99%
“…The two known effectors of cAMP in collecting duct cells are protein kinase A (PKA) and 'exchange protein directly activated by cAMP', EPAC1 and EPAC2 (coded by Rapgef3 and Rapgef4). Deletion of PKA in collecting duct cells abolishes nearly all of the protein phosphorylation changes associated with vasopressin stimulation, in addition to eliminating Aqp2 gene transcription (Isobe et al, 2017;Datta et al 2020). On the other hand, EPAC gene deletions did not have a pronounced effect on AQP2 regulation when knocked out in This article has not been copyedited and formatted.…”
Water excretion by the kidney is regulated by the neurohypophyseal peptide hormone vasopressin through actions in renal collecting duct cells to regulate the water channel protein, aquaporin-2. Vasopressin signalling is initiated by binding to a G-protein coupled receptor V2R, which signals through Gsα, adenylyl cyclase 6, and activation of the cAMP-regulated protein kinase (PKA). Signaling events coupling PKA activation and aquaporin-2 regulation were largely unknown until the advent of modern protein mass spectrometry techniques that allow proteomewide quantification of protein phosphorylation changes (phosphoproteomics). This short review documents phosphoproteomic findings in collecting duct cells describing the response to V2selective vasopressin agonists and antagonists, the response to CRISPR-mediated deletion of PKA, results from in vitro phosphorylation studies using recombinant PKA, the response to the broad spectrum kinase inhibitor H89, and the responses underlying lithium-induced nephrogenic diabetes insipidus. These phosphoproteomic datasets have been made available online for modelling vasopressin signalling and signalling downstream from other Gsα-coupled receptors.
“…The simplest experiment to identify vasopressin signaling pathways in the renal collecting duct is to expose collecting duct cells to vasopressin or its vehicle and carry out mass spectrometry-based quantitative phosphoproteomics. Experiments following this strategy have been done in both cultured collecting duct cells (mouse mpkCCD) (Rinschen et al, 2010;Datta et al, 2020) and in suspensions of native inner medullary collecting duct cells from rats (Hoffert et al, 2006(Hoffert et al, , 2012Deshpande et al, 2019). The studies have utilized a V2 receptor-selective vasopressin analog dDAVP (desmopressin, D-amino D-arginine vasopressin), which is used clinically for treatment of central diabetes insipidus (Christensen and Rittig, 2006;Oiso et al, 2013).…”
Section: Effect Of Vasopressin On the Phosphoproteome Of The Renamentioning
confidence: 99%
“…Many of those that had basophilic phosphorylation sites that increased with vasopressin are known targets of PKA (underlined, Figure 3), cementing the conclusion that vasopressin signaling is largely mediated by PKA activation. Nevertheless, some elements of vasopressin signaling appear to be PKA-independent (Datta et al, 2020). This includes PKA-independent, vasopressin induced phosphorylation changes associated with activation of one or more kinases of the AMPK-related kinase familiy (also known as SNF1family kinases).…”
Section: Mol#119602mentioning
confidence: 99%
“…The two known effectors of cAMP in collecting duct cells are protein kinase A (PKA) and 'exchange protein directly activated by cAMP', EPAC1 and EPAC2 (coded by Rapgef3 and Rapgef4). Deletion of PKA in collecting duct cells abolishes nearly all of the protein phosphorylation changes associated with vasopressin stimulation, in addition to eliminating Aqp2 gene transcription (Isobe et al, 2017;Datta et al 2020). On the other hand, EPAC gene deletions did not have a pronounced effect on AQP2 regulation when knocked out in This article has not been copyedited and formatted.…”
Water excretion by the kidney is regulated by the neurohypophyseal peptide hormone vasopressin through actions in renal collecting duct cells to regulate the water channel protein, aquaporin-2. Vasopressin signalling is initiated by binding to a G-protein coupled receptor V2R, which signals through Gsα, adenylyl cyclase 6, and activation of the cAMP-regulated protein kinase (PKA). Signaling events coupling PKA activation and aquaporin-2 regulation were largely unknown until the advent of modern protein mass spectrometry techniques that allow proteomewide quantification of protein phosphorylation changes (phosphoproteomics). This short review documents phosphoproteomic findings in collecting duct cells describing the response to V2selective vasopressin agonists and antagonists, the response to CRISPR-mediated deletion of PKA, results from in vitro phosphorylation studies using recombinant PKA, the response to the broad spectrum kinase inhibitor H89, and the responses underlying lithium-induced nephrogenic diabetes insipidus. These phosphoproteomic datasets have been made available online for modelling vasopressin signalling and signalling downstream from other Gsα-coupled receptors.
“…The raw data used for this paper were reported as control experiments in our prior publication about PKA-independent signaling in collecting duct cells (35), but were not analyzed bioinformatically with respect to physiological mechanisms. For the reader's convenience, we provide the methods used in a supplemental file (Supplemental Information -Methods).…”
Section: Methodsmentioning
confidence: 99%
“…Briefly, mpkCCD, a collecting duct cell line, was grown on permeable supports using the Stable Isotope Labeling with Amino acids in Cell culture (SILAC) protocol for labeling (35). The cells were exposed to the vasopressin analog dDAVP (0.1 nM for 30 min) versus vehicle in three pairs of SILAC experiments.…”
Background and PurposeThe peptide hormone vasopressin regulates water transport in the renal collecting duct largely via the V2 receptor, which triggers a cAMP-mediated activation of a protein kinase A (PKA)-dependent signaling network. The protein kinases downstream from PKA have not been fully identified or mapped to regulated phosphoproteins.Experimental ApproachWe carried out systems-level analysis of large-scale phosphoproteomic data quantifying vasopressin-induced changes in phosphorylation in aquaporin-2-expressing cultured collecting duct cells (mpkCCD). Quantification was done using stable isotope labeling (SILAC method).Key Results9640 phosphopeptides were quantified. Stringent statistical analysis identified significant changes in response to vasopressin in 429 of these phosphopeptides. The corresponding phosphoproteins were mapped to known vasopressin-regulated cellular processes. The vasopressin-regulated sites were classified according to the sequences surrounding the phosphorylated amino acids giving 11 groups distinguished predominantly by the amino acids at positions +1, −3, −2 and −5 relative to the phosphorylated amino acid. Among the vasopressin-regulated phosphoproteins were 25 distinct protein kinases. Among these, six of them plus PKA appeared to account for phosphorylation of more than 80% of the 313 vasopressin-regulated phosphorylation sites. The six downstream kinases were salt-inducible kinase 2 (Sik2), cyclin-dependent kinase 18 (PCTAIRE-3, Cdk18), calmodulin-dependent kinase kinase 2 (Camkk2), protein kinase D2 (Prkd2), mitogen-activated kinase 3 (ERK1; Mapk3), and myosin light chain kinase (Mylk).Conclusion and ImplicationsIn V2 receptor-mediated signaling, PKA is at the head of a complex network that includes at least 6 downstream vasopressin-regulated protein kinases that are prime targets for future study. The extensive phosphoproteomic data generated in this study is provided as a web-based data resource for future studies of G-protein coupled receptors.
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