Parathyroid Hormone Activates TRPV5 via PKA-Dependent Phosphorylation

Abstract

Low extracellular calcium (Ca 2ϩ ) promotes release of parathyroid hormone (PTH), which acts on multiple organs to maintain overall Ca 2ϩ balance. In the distal part of the nephron, PTH stimulates active Ca 2ϩ reabsorption via the adenylyl cyclase-cAMP-protein kinase A (PKA) pathway, but the molecular target of this pathway is unknown. The transient receptor potential vanilloid 5 (TRPV5) channel constitutes the luminal gate for Ca 2ϩ entry in the distal convoluted tubule and has several putative PKA phosphorylation sites. Here, we investigated the effect of PTH-induced cAMP signaling on TRPV5 activity. Using fluorescence resonance energy transfer, we studied cAMP and Ca 2ϩ dynamics during PTH stimulation of HEK293 cells that coexpressed the PTH receptor and TRPV5. PTH increased cAMP levels, followed by a rise in TRPV5-mediated Ca 2ϩ influx. PTH (1 to 31) and forskolin, which activate the cAMP pathway, mimicked the stimulation of TRPV5 activity. Remarkably, TRPV5 activation was limited to conditions of strong intracellular Ca 2ϩ buffering. Cell surface biotinylation studies demonstrated that forskolin did not affect TRPV5 expression on the cell surface, suggesting that it alters the single-channel activity of a fixed number of TRPV5 channels. Application of the PKA catalytic subunit, which phosphorylated TRPV5, directly increased TRPV5 channel open probability. Alanine substitution of threonine-709 abolished both in vitro phosphorylation and PTH-mediated stimulation of TRPV5. In summary, PTH activates the cAMP-PKA signaling cascade, which rapidly phosphorylates threonine-709 of TRPV5, increasing the channel's open probability and promoting Ca 2ϩ reabsorption in the distal nephron.

Parathyroid hormone (PTH) rapidly enhances CFTR-mediated HCO3− secretion in intestinal epithelium-like Caco-2 monolayer: a novel ion regulatory action of PTH

“…The downstream signaling pathways of PTH were herein demonstrated to be cAMP/PKA-and PI3K-dependent. In general, the cAMP/PKA pathway is of utmost importance for the classical PTH actions, including the production of osteoblastderived osteoclastogenic factors (35), activation of transient receptor potential vanilloid 5 Ca 2ϩ channel for distal tubular Ca 2ϩ reabsorption (14), and upregulation of 1␣-hydroxylase for renal 1,25(OH) 2 D 3 synthesis (8). Since CFTR, NBCe1, NHE1, NHE3, carbonic anhydrase, and Na ϩ -K ϩ -ATPase are known to be under cAMP/PKA regulation (3, 4, 9, 57, 62), PKA and/or its downstream molecules might direct PTH signals to activate all of these transporters and related proteins, thereby leading to the enhanced HCO 3 Ϫ secretion.…”

β1-Adrenergic Receptor Signaling Activates the Epithelial Calcium Channel, Transient Receptor Potential Vanilloid Type 5 (TRPV5), via the Protein Kinase A Pathway

“…Finally, biotinylated proteins were precipitated using NeutrAvidin beads (Pierce). TRPV5 expression was analyzed by immunoblotting for the precipitates (plasma membrane fraction) and for the total cell lysates using the anti-HA antibody (6). [Ca 2ϩ ] i and cAMP measurements, HEK293 cells were seeded onto coverslips (Ø 25 mm) and cotransfected with the cAMP sensor EPAC-EGFP-mCherry (26), kindly provided by Dr. K. Jalink) for cAMP measurements, and the appropriate TRPV5 pCINeo/IRES-EGFP construct from which the sequence encoding EGFP was deleted.…”

“…TRPV5 activity and plasma membrane abundance are regulated by various factors, including GPCRs. For example, activation of bradykinin receptor type 2 and PTH receptor type 1 initiate phosphorylation of TRPV5 through PLC/DAG/PKC and adenylyl cyclase/cAMP/PKA signaling cascades, respectively (6,12).…”

“…Dobutamine Stimulates TRPV5-mediated Ca 2ϩ Uptake in HEK293 Cells via PKA Phosphorylation of Thr-709 Residue-TRPV5 channel activity is known to be stimulated by cAMP/ PKA-mediated pathways (6). HEK293 cells were transfected with TRPV5 and loaded with the Fura-2 Ca 2ϩ sensor to measure Ca 2ϩ uptake.…”

Reduced Renal Calcium Excretion in the Absence of Sclerostin Expression

“…PTH increases the activity of TRPV5 channels in the kidney by activating cAMP-protein kinase A signaling, and phosphorylating a threonine residue within the channel, resulting in an increase in the open probability of the channel. 86,87 PTH also activates the protein kinase C pathway and increases the numbers of TRPV5 channels on the surface of tubular cells by inhibiting endocytosis of caveolae in which the channels are located. 88 1a,25(OH) 2 D 3 enhances the expression of TRPV5 and TRPV6 channels present in the distal and connecting tubule and cortical collecting duct by increasing respective mRNA concentrations through increased binding of the vitamin D receptor to response elements in the gene promoters.…”