Dynamics of the G Protein-coupled Vasopressin V2 Receptor Signaling Network Revealed by Quantitative Phosphoproteomics

Abstract

G protein-coupled receptors (GPCRs) regulate diverse physiological processes, and many human diseases are due to defects in GPCR signaling. To identify the dynamic response of a signaling network downstream from a prototypical G s -coupled GPCR, the vasopressin V2 receptor, we have carried out multireplicate, quantitative phosphoproteomics with iTRAQ labeling at four time points following vasopressin exposure at a physiological concentration in cells isolated from rat kidney. A total of 12,167 phosphopeptides were identified from 2,783 proteins, with 273 changing significantly in abundance with vasopressin. Two-dimensional clustering of phosphopeptide time courses and Gene Ontology terms revealed that ligand binding to the V2 receptor affects more than simply the canonical cyclic adenosine monophosphateprotein kinase A and arrestin pathways under physiological conditions. The regulated proteins included key components of actin cytoskeleton remodeling, cell-cell adhesion, mitogen-activated protein kinase signaling, Wnt/␤-catenin signaling, and apoptosis pathways. These data suggest that vasopressin can regulate an array of cellular functions well beyond its classical role in regulating water and solute transport. These results greatly expand the current view of GPCR signaling in a physiological context and shed new light on

Vasopressin inhibits apoptosis in renal collecting duct cells

“…This site lies within a strong PKA consensus motif (...VLRRSS*VF...). In addition, PKA is capable of phosphorylating this residue as determined by in vitro kinase assays (17). The current study reveals that Bok-pS8 is sensitive to both H89 and is significantly elevated by cAMP.…”

“…Multiple Bcl-2 family member proteins are regulated in response to short-term AVP treatment. Previous studies have shown that the proapoptotic proteins Bad and Bok, both members of the Bcl-2 family, become phosphorylated with short-term dDAVP treatment (17,30). Phosphorylation at these sites by various basophilic kinases (predominantly PKA and Akt) has either been predicted or directly demonstrated to inhibit apoptosis in other cell types (25, 39, 46).…”

“…Previously, we demonstrated that AVP reduced the levels of cleaved caspases in isolated native rat IMCD cells treated with dDAVP (17). Caspase cleavage is considered one of the hallmarks of apoptosis (8,10,27,35).…”

“…In these cells, AVP signals through the heterotrimeric G protein G s and subsequent activation of adenylyl cyclases, which mediate a rise in intracellular cAMP, activation of protein kinase A (PKA), and phosphorylation of the water channel aquaporin-2 (11,18) as well as the urea channel UT-A1 (2). A recent large-scale phosphoproteomics study by our group revealed that AVP may regulate additional signaling pathways in the kidney collecting duct, including various MAP kinase pathways, Wnt/␤-catenin signaling, and apoptosis (17). In that study, we provided preliminary evidence that AVP may attenuate apoptosis in isolated rat inner medullary collecting duct (IMCD) cells, a process that may influence cell survival in a variety of physiological [e.g., adaptive osmotic tolerance (4)] and pathophysiological [e.g., autosomal dominant polycystic kidney disease; ADPKD (37)] conditions.…”

Vasopressin-2 Receptor Signaling and Autosomal Dominant Polycystic Kidney Disease

“…Quantitative global phosphoproteomic analyses using both native tissue, as well as mpkCCD cells, revealed that treatment with a V2R agonist caused a significant decrease in phosphorylation of proline-directed phosphorylation motifs, thereby suggesting inhibited activity of MAPKs. 43,[52][53][54] This effect is a quantitatively important effect. In primary cultured IMCD cells, p38 activity is decreased in response to cAMP and dDAVP.…”