Dataset Information

PKA-independent vasopressin signaling in renal collecting duct.

ABSTRACT: Vasopressin regulates renal water excretion by binding to a G_α s-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 vasopressin signaling is predominantly, but not entirely, PKA-dependent. Upregulated sites in PKA-null cells include Ser256 of AQP2, which is critical to regulation of AQP2 trafficking. In addition, phosphorylation changes in the protein kinases Stk39 (SPAK) and Prkci (an atypical PKC) are consistent with PKA-independent regulation of these protein kinases. Target motif analysis of the phosphopeptides increased in PKA-null cells indicates that vasopressin activates one or more members of the AMPK/SNF1-subfamily of basophilic protein kinases. In vitro phosphorylation assays using recombinant, purified SNF1-subfamily kinases confirmed postulated target specificities. Of interest, measured IBMX-dependent cAMP levels were an order of magnitude higher in PKA-null than in PKA-intact cells, indicative of a PKA-dependent feedback mechanism. Overall, the findings support the conclusion that V2-receptor mediated signaling in collecting duct cells is in part PKA-independent.

SUBMITTER: Datta A

PROVIDER: S-EPMC9200475 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

PKA-independent vasopressin signaling in renal collecting duct.

Datta Arnab A Yang Chin-Rang CR Limbutara Kavee K Chou Chung-Lin CL Rinschen Markus M MM Raghuram Viswanathan V Knepper Mark A MA

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20200326 5

Vasopressin regulates renal water excretion by binding to a G<sub>α</sub> s-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 sh ...[more]

PMID: 32219907

Similar Datasets

Project description:Vasopressin controls water balance largely through PKA-dependent effects to regulate the collecting duct water channel aquaporin-2 (AQP2). Although considerable information has accrued regarding the regulation of water and solute transport in collecting duct cells, information is sparse regarding the signaling connections between PKA and transport responses. Here, we exploited recent advancements in protein mass spectrometry to perform a comprehensive, multiple-replicate analysis of changes in the phosphoproteome of native rat inner medullary collecting duct cells in response to the vasopressin V2 receptor-selective agonist 1-desamino-8D-arginine vasopressin. Of the 10,738 phosphopeptides quantified, only 156 phosphopeptides were significantly increased in abundance, and only 63 phosphopeptides were decreased, indicative of a highly selective response to vasopressin. The list of upregulated phosphosites showed several general characteristics: 1) a preponderance of sites with basic (positively charged) amino acids arginine (R) and lysine (K) in position -2 and -3 relative to the phosphorylated amino acid, consistent with phosphorylation by PKA and/or other basophilic kinases; 2) a greater-than-random likelihood of sites previously demonstrated to be phosphorylated by PKA; 3) a preponderance of sites in membrane proteins, consistent with regulation by membrane association; and 4) a greater-than-random likelihood of sites in proteins with class I COOH-terminal PDZ ligand motifs. The list of downregulated phosphosites showed a preponderance of those with proline in position +1 relative to the phosphorylated amino acid, consistent with either downregulation of proline-directed kinases (e.g., MAPKs or cyclin-dependent kinases) or upregulation of one or more protein phosphatases that selectively dephosphorylate such sites (e.g., protein phosphatase 2A). The phosphoproteomic data were used to create a web resource for the investigation of G protein-coupled receptor signaling and regulation of AQP2-mediated water transport.

Project description:Transient receptor potential channels TRPC3 and TRPC6 are expressed in principal cells of the collecting duct (CD) along with the water channel aquaporin-2 (AQP2) both in vivo and in the cultured mouse CD cell line IMCD-3. The channels are primarily localized to intracellular vesicles, but upon stimulation with the antidiuretic hormone arginine vasopressin (AVP), TRPC3 and AQP2 translocate to the apical membrane. In the present study, the effect of various activators and inhibitors of the adenylyl cyclase (AC)/cAMP/PKA signaling cascade on channel trafficking was examined using immunohistochemical techniques and by biotinylation of surface membrane proteins. Both in vivo in rat kidney and in IMCD-3 cells, translocation of AQP2 and TRPC3 (but not TRPC6) was stimulated by [deamino-Cys(1), d-Arg(8)]-vasopressin (dDAVP), a specific V2-receptor agonist, and blocked by [adamantaneacetyl(1), O-Et-d-Tyr(2), Val(4), aminobutyryl(6), Arg(8,9)]-vasopressin (AEAVP), a specific V2-receptor antagonist. In IMCD-3 cells, translocation of TRPC3 and AQP2 was activated by forskolin, a direct activator of AC, or by dibutyryl-cAMP, a membrane-permeable cAMP analog. AVP-, dDAVP-, and forskolin-induced translocation in IMCD-3 cells was blocked by SQ22536 and H89, specific inhibitors of AC and PKA, respectively. Translocation stimulated by dibutyryl-cAMP was unaffected by AEAVP but could be blocked by H89. AVP- and forskolin-induced translocation of TRPC3 in IMCD-3 cells was also blocked by two additional inhibitors of PKA, specifically Rp-cAMPS and the myristoylated inhibitor of PKA (m-PKI). Quantification of TRPC3 membrane insertion in IMCD-3 cells under each assay condition using a surface membrane biotinylation assay, confirmed the translocation results observed by immunofluorescence. Importantly, AVP-induced translocation of TRPC3 as estimated by biotinylation was blocked on average 95.2 +/- 1.0% by H89, Rp-cAMPS, or m-PKI. Taken together, these results demonstrate that AVP stimulation of V2 receptors in principal cells of the CD causes translocation of TRPC3 to the apical membrane via stimulation of the AC/cAMP/PKA signaling cascade.

Project description:Tolvaptan, a vasopressin antagonist selective for the V2-subtype vasopressin receptor (V2R), is widely used in the treatment of hyponatremia and autosomal-dominant polycystic kidney disease (ADPKD). Its effects on signaling in collecting duct cells have not been fully characterized. Here, we perform RNA-seq in a collecting duct cell line (mpkCCD). The data show that tolvaptan inhibits the expression of mRNAs that were previously shown to be increased in response to vasopressin including aquaporin-2, but also reveals mRNA changes that were not readily predictable and suggest off-target actions of tolvaptan. One such action is activation of the MAPK kinase (ERK1/ERK2) pathway. Prior studies have shown that ERK1/ERK2 activation is essential in the regulation of a variety of cellular and physiological processes and can be associated with cell proliferation. In immunoblotting experiments, we demonstrated that ERK1/ERK2 phosphorylation in mpkCCD cells was significantly reduced by vasopressin, in contrast to the increases seen in non-collecting-duct cells overexpressing V2R in prior studies. We also found that tolvaptan has a strong effect to increase ERK1/ERK2 phosphorylation in the presence of vasopressin and that tolvaptan's effect to increase ERK1/ERK2 phosphorylation is absent in mpkCCD cells in which both protein kinase A (PKA)-catalytic subunits have been deleted. Thus, it appears that the tolvaptan effect to increase ERK activation is PKA-dependent and is not due to an off-target effect of tolvaptan. We conclude that in cells expressing V2R at endogenous levels: 1) vasopressin decreases ERK1/ERK2 activation; 2) in the presence of vasopressin, tolvaptan increases ERK1/ERK2 activation; and 3) these effects are PKA-dependent.NEW & NOTEWORTHY Vasopressin is a key hormone that regulates the function of the collecting duct of the kidney. ERK1 and ERK2 are enzymes that play key roles in physiological regulation in all cells. The authors used collecting duct cell cultures to investigate the effects of vasopressin and the vasopressin receptor antagonist tolvaptan on ERK1 and ERK2 phosphorylation and activation.

Dataset Information

PKA-independent vasopressin signaling in renal collecting duct.

Publications

PKA-independent vasopressin signaling in renal collecting duct.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets