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

Abstract

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.

Phosphoproteomic identification of vasopressin‐regulated protein kinases in collecting duct cells

“…The physiological responses at the cellular level elicited by vasopressin incollecting duct epithelial cells as documented in prior literature (Salhadar et al, 2020)…”

“…The physiological responses at the cellular level elicited by vasopressin incollecting duct epithelial cells as documented in prior literature (Salhadar et al, 2020) 2.3.2 | Network construction STRING (https://string-db.org/, RRID: SCR_005223) was used to create a relational network for proteins with increased and decreased phosphorylation sites using default settings to construct a preliminary signalling network. This network was the starting point for manual curation based on knowledge gleaned from prior literature to classify vasopressin signalling into various cellular processes, functions or components.…”

“…The functional effects of vasopressin at a cellular level in collecting duct principal cells are shown in Figure 1, which summarizes an extensive literature on the topic (Salhadar et al, 2020). Most of these downstream effects are believed to be mediated by PKA through cAMP (Isobe et al, 2017) (Figure 1).…”

Protein kinase A catalytic-α and catalytic-β proteins have non-redundant functions

“…Vasopressin's actions in collecting duct principal cells (23) are mediated by a G-protein coupled receptor (GPCR) V2R (Gene symbol: Avpr2), which couples to the heterotrimeric G-protein stimulatory a subunit (G s a) with activation of adenylyl cyclase 6 (22). Increased intracellular cyclic AMP levels result in physiological effects in collecting duct principal cells largely through activation of PKA (4,5,8,11,12,17,(19)(20)(21) .…”

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

“…The physiologic responses at the cellular level elicited by vasopressin in the collecting duct epithelial cells as documented in prior literature(81).…”