1999 European Regional Conference Proceeding

May 12 - 16, 1999

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Conference: 1999 European Regional Conference
Title: Identification of proteins involved in the vasopressin-induced shuttle of aquaporin-2
Authors: Rosenthal, Walter; Klussmann, Enno; Maric, Kenan; Wiesner, Burkhard; Krause, Eberhard; Beyermann, Michael; Tamma, Grazia; Valenti, Giovanna
Institutions: Charite - Universitatsmedizin Berlin, Max Delbrueck Center for Molecular Medicine Berlin (MDC), German Federal Ministry of Health and Social Security, Forschunginstitut fur Molekulare Pharmakologie (FMP), Forschungsinstitut fur Molekulare Pharmakologie, Universita di Bari
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Walter Rosenthal

In man and other mammals, vasopressin plays a key role in maintaining water homeostasis. Acting via V2 receptors, it induces a rapid increase in the osmotic water permeability of principal cells contributing to the epithelial monolayer of renal collecting ducts, thereby permitting reabsorption of water from the lumen. As a consequence, urine osmolality increases and urinary output decreases. The increase in osmotic water permeability is achieved by a cAMP/protein kinase A (PKA) triggered exocytotic insertion of the water channel aquaporin 2 (AQP2) into the plasma membrane of principal cells. The proteins involved in the translocation of AQP2 and the signal cascade downstream of the cAMP/PKA signal remain largely elusive. Mutations of the V2 receptor or AQP2 are the cause of congenital nephrogenic diabetes insipidus (1).

In a fraction enriched for AQP2-bearing vesicles we have identified more than 30 proteins, among them several GTP-binding proteins (Gi2, Gi3, G13, Rab3a, Rab5, Rab7), the v-SNARE protein synaptobrevin (VAMP2) and several protein kinase A anchoring proteins (AKAPs) including the AKAP moesin. Using rabbit cortical collecting duct cells stably transfected with the rat AQP2 cDNA (CD8 cells), we identified G-proteins of the Gi family as a crucial component of the signal cascade (2). In contrast to the G protein Gs which is involved in transmembrane signaling via the vasopressin V2 receptor, Gi proteins are involved in the pathway downstream of the cAMP/PKA signal. Using primary cultured rat kidney inner medulla collecting duct (IMCD) cells (3), we identified AKAPs as another component required for the translocation of AQP2 in IMCD cells (4). Experiments with clostridial toxins revealed that the AQP2 shuttle is controlled by proteins of the Rho family.

Elucidation of the signal cascade underlying the AQP2 shuttle will greatly facilitate the development of pharmacological strategies for the treatment of congenital and acquired diuretic states, in particular of those strategies circumventing activation of the V2 receptor.

When the antidiuretic hormone, vasopressin (VP), binds with its vasopressin-2 receptor, it begins a molecular sequence which moves tiny sacs called vesicles with aquaporin-2 (AQP2) proteins from inside the principal cells of the kidney collecting duct to the apical membranes of those cells. Once the AQP2-bearing vesicles arrive at the apical membranes, they fuse with them and the AQP2s are inserted into those membranes. This enables the kidneys to absorb much of the water flowing through its collecting ducts. In nephrogenic diabetes insipidus (NDI), this molecular sequence is interrupted and the kidneys cannot absorb the body water flowing through their collecting ducts. Thus the NDI patient cannot easily maintain proper body water balance.

Researchers have a clear picture of a portion of this molecular sequence: VP binds to its vasopressin-2 receptor, which is coupled to a stimulatory G protein (Gs). This activates the enzyme complex, adenylyl cyclase (AC), and this elevates the level of cAMP, an important intracellular messenger, cAMP activates protein kinase A (PKA), which helps induce AQP2-bearing vesicles to travel to the apical membrane. But there are many proteins involved in this process that have not yet been identified. In addition, the elements -- and the dynamics of these elements -- involved in the sequence immediately after PKA remained largely unknown.

Rosenthal, et al, examined the AQP2-bearing vesicles and identified more than 30 proteins associated with them, including guanosine triphosphate (GTP)-binding proteins, the v-SNARE protein synaptobrevin 2 (VAMP2) and several PKA anchoring proteins (AKAPs). The researchers proved that AKAPs are required for the translocation of AQP2s to the apical membrane and that guanosine (G)-proteins of the inhibitory G family were crucial to the molecular sequence. Whereas Gs proteins are involved in the molecular sequence before cAMP and PKA have been activated, Gi proteins are involved in the sequence after the cAMP/PKA signal. The researchers also showed that the movement of AQP2-vesicles to the apical membrane is controlled by proteins of the Rho family.

This research will help develop pharmacological strategies for NDI, particularly those that aim to operate independently of the activation of the V2 receptor.