A Heterotrimeric G Protein of the Gi Family is Required for cAMP-triggered Trafficking of Aquaporin 2 in Kidney Epithelial Cells

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Title: A Heterotrimeric G Protein of the Gi Family is Required for cAMP-triggered Trafficking of Aquaporin 2 in Kidney Epithelial Cells
Authors: Liebenhoff, Ursula; Frigeri, Antonio; Rosenthal, Walter; Valenti, Giovanna; Procino, Giuseppe; Benedetti, Pio Alberto; Ahnert-Hilger, Gudrun; Nurnberg, Bernd; Svelto, Maria
Publisher: Journal of Biological Chemistry
Date Published: August 28, 1998
Reference Number: 233
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Vasopressin is the key regulator of water homeostasis in vertebrates. Central to its antidiuretic action in mammals is the redistribution of the water channel aquaporin 2 (AQP2) from intracellular vesicles to the apical membrane of kidney epithelial cells, an event initiated by an increase in cAMP and activation of protein kinase A. The subsequent steps of the signaling cascade are not known. To identify proteins involved in the AQP2 shuttle we exploited a recently developed cell line (CD8) derived from the rabbit cortical collecting duct and stably transfected with rat AQP2 cDNA. Treatment of CD8 cells with pertussis toxin (PTX) inhibited both the vasopressin-induced increase in water permeability and the redistribution of AQP2 from an intracellular compartment to the apical membrane. ADP-ribosylation studies revealed the presence of at least two major PTX substrates. Correspondingly, two alpha subunits of PTX-sensitive G proteins, Galphai2 and Galphai3, were identified by Western blotting. Introduction of a synthetic peptide corresponding to the C terminus of the Gi3 alpha subunit into permeabilized CD8 cells efficiently inhibited the cAMP-induced AQP2 translocation; a peptide corresponding to the alpha subunits of Gi1/2 was much less potent. Thus a member of the Gi family, most likely Gi3, is involved in the cAMP-triggered targeting of AQP2-bearing vesicles to the apical membrane of kidney epithelial cells.
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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
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Aquaporin-2 is a water-transporting protein located in the principal cells of the kidney collecting duct. Its function is to insert itself into the apex of the collecting duct cell membrane in order to make it more water permeable. AQP2, sitting inside sacs called vesicles, waits within holding places called the endoplasmic reticulum (ER). When the antidiuretic hormone, arginine vasopressin (AVP) binds with the vasopressin-2 receptor (V2R) it sends a signal to AQP2 vesicles waiting in the ER to shuttle to the cell membrane so the AQP2 can be inserted in it.

Researchers know this much of the molecular sequence that gets AQP2s to the cell membrane: V2R is coupled to a stimulatory G-protein. When AVP binds with V2R, via the G-protein, it stimulates the enzyme, adenylyl cyclase (AdC). AdC, in turn, elevates levels of the metabolic regulator, cyclic adenosine monophosphate (cAMP). cAMP activates protein kinase A (PKA). The steps between this and the insertion of AQP2 are not known. Valenti, et al. report on their research, which takes steps toward clarifying what happens between the step where PKA gets stimulated and the AQP2 gets inserted.

The authors created an experimental design to identify some of the proteins involved in shuttling the AQP2-bearing vesicles from the ER to the cell membrane surface. They used a specific cell line called CD8 which, under laboratory conditions, responds to AVP just as the collecting duct principal cells would. When the authors fused the CD8 cells with AVP, AQP2-bearing vesicles shuttled from the ER to the cell surface and the cells expressed a 4 - 6 fold increase in their ability to let water pass through them.

When the cells were treated with pertussis toxin (PTX) and again infused with AVP, the cells showed no redistribution of AQP2s and no increase in their ability to let water pass through. This indicated that PTX was able to inhibit AQP2 transport to the cell surface. Previous research with other vesicle transport systems proved that PTX uncouples G proteins of the Gi and Go types, thereby inhibiting vesicle transport. That PTX could inhibit AQP2 translocation in this experiment indicated to the authors that Gi and Go proteins are involved in transporting AQP2-bearing vesicles to the cell surface. Further experiments identified the proteins involved in the transport as Gai2 and Gai3.

Wanting to further clarify which of the G proteins were involved in AQP2 vesicle transport, the researchers introduced different synthetic peptides at different times into the CD8 cells, then analyzed their affects on AQP2 movement from ER to cell surface. The peptides corresponded to a part of the G proteins called the C-terminus (the tail end of the protein). Each G protein had a different peptide associated with it. Since each peptide would inhibit the functioning of the G-protein with which it was associated, the researchers could determine which of the G proteins was involved in AQP2 transport by noting which peptide induced a failure in AQP2 transport. They concluded the protein Gi3 is most likely involved in shuttling AQP2-bearing vesicles to the cell membrane surface.