Dataset Information


PKA-Independent Vasopressin Signaling in Renal Collecting Duct

ABSTRACT: Vasopressin regulates renal water excretion by binding to the Gs-coupled vasopressin receptor (V2R) in collecting duct cells, resulting in cyclic AMP-dependent increases in epithelial water permeability through regulation of the aquaporin-2 (AQP2) water channel. Our prior studies showed that CRISPR-mediated deletion of protein kinase A (PKA) in cultured mpkCCD cells largely eliminates these regulatory events. These PKA-null cells provide a means of identifying PKA-independent signaling downstream from the V2 receptor. We carried out large-scale quantitative protein mass spectrometry (SILAC) to identify PKA-independent phosphorylation changes in response to V2R-selective vasopressin analog, dDAVP. The results show that V2R-mediated vasopressin signaling is predominantly, but not entirely, PKA-dependent. Target motif analysis of the phosphopeptides increased in response to dDAVP in PKA-null cells indicates that the vasopressin activates of one or more members of the AMPK/SNF1 subfamily of basophilic protein kinases. Among the upregulated phosphorylation sites were three known targets of SNF1-subfamily kinases, namely Lipe (S559), Crtc1 (S151) and Arhgef2 (S151). One of the phosphorylation sites that increased in occupancy in PKA-null cells was Ser256 of AQP2, a site critical for vasopressin-mediated trafficking of AQP2 to the cell surface. Beyond this, PKA-independent active site phosphorylation changes were also seen for protein kinases Stk39 (SPAK) and Prkci (Protein kinase C iota). Cyclic AMP levels were ~10-fold higher in PKA-null than in PKA-intact cells in the presence of phosphodiesterase inhibitor IBMX, consistent with a marked acceleration of cAMP production in PKA-null cells. The findings are indicative of substantial PKA-independent signaling downstream from the Gs-coupled V2 receptor.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Epithelial Cell, Kidney


LAB HEAD: Mark A. Knepper

PROVIDER: PXD015719 | Pride | 2020-03-05


altmetric image


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]

Similar Datasets

2017-11-23 | PXD005938 | Pride
| E-GEOD-13667 | biostudies-arrayexpress
| E-GEOD-19938 | biostudies-arrayexpress
2018-03-26 | GSE98076 | GEO
2023-11-17 | GSE243773 | GEO
2020-03-04 | PXD014985 | Pride
2018-03-26 | GSE108786 | GEO
2018-01-02 | GSE95007 | GEO
2022-05-16 | GSE194044 | GEO
2022-07-23 | PXD033859 | Pride