PKA‐independent vasopressin signaling in renal collecting duct

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 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.

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

“…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).…”

“…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).…”

“…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.…”

Phosphoproteomic Identification of Vasopressin-Regulated Protein Kinases in Collecting Duct Cells

“…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).…”

“…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.…”