Phosphoproteomic identification of vasopressin V2 receptor-dependent signaling in the renal collecting duct

Abstract

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.

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).…”

“…These results suggest therefore that, with regard to vasopressin signaling in the collecting duct, cAMP-mediated signaling dominates over -arrestin signaling. There is also evidence that vasopressin increases the activity of a proline-directed phosphatase, PP2A, through PKA-mediated phosphorylation of a phosphatase inhibitor, cAMPregulated phosphoprotein 19 (Arpp19) (Deshpande et al, 2019), which could explain some of the downregulated -X-p(S/T)-P sites seen in response to vasopressin.…”

“…Futhermore, parathyroid hormone binding to the PTH1R, a Gsα coupled receptor, in MC3T3-E1 preosteoblast cells produced the same general pattern of phosphorylation changes (Williams et al, 2016), demonstrating this response pattern is not unique to the V2 receptor or to the kidney. Figure 3 shows some of the results from the most comprehensive vasopressin-response phosphoproteomic study in native collecting duct cells (Deshpande et al, 2019). Several of the phosphoprotein targets of vasopressin signaling mapped to cellular physiological functions corresponding to those in Table 1.…”

“…Specifically, protein kinase D1 (Prkd1), PCTAIRE kinase 1 (Cdk16), PCTAIRE kinase 3 (Cdk18) and Src undergo phosphorylation changes consistent with increases in activity in response to vasopressin, while A-Raf (Araf) and Raf1 undergo apparent decreases in activity in response to vasopressin. Prkd1, Cdk16, and Cdk18 are involved in regulation of membrane trafficking, while Src, Araf and Raf1 regulate MAP kinase activities (Deshpande et al, 2019). The full data set describing the This article has not been copyedited and formatted.…”

“…Because protein kinases must come into physical contact with their targets to phosphorylate them, phosphoproteomic analysis can be construed as a form of proximity analysis, potentially identifying subcompartments in the cell that contain the regulated forms of the relevant protein kinases. For example, in the Deshpande et al analysis (Deshpande et al, 2019), it was concluded that PKA phosphorylates its targets predominantly at membrane surfaces, particularly targeting plasma membrane proteins.…”