A Heterotrimeric G Protein of the Gi Family Is Required for cAMP-triggered Trafficking of Aquaporin 2 in Kidney Epithelial Cells*
- Giovanna Valenti‡,
- Giuseppe Procino‡,
- Ursula Liebenhoff§,
- Antonio Frigeri‡,
- Pio Alberto Benedetti¶,
- Gudrun Ahnert-Hilger‖,
- Bernd Nürnberg**,
- Maria Svelto‡ and
- Walter Rosenthalत
- From the ‡Dipartimento di Fisiologia Generale e Ambientale, Universitá degli Studi, 70126 Bari, Italy,§Rudolf-Buchheim-Institut für Pharmakologie, Justus-Liebig-Universität Gießen, 35392 Gießen, Germany,‡Forschungsinstitut für Molekulare Pharmakologie, 10315 Berlin, Germany, ¶Istituto di Biofisica-CNR, 56127 Pisa, Italy, ‖Institut for Anatomie, Humboldt-Universität zu Berlin, 10115 Berlin, Germany, and**Institut für Pharmakologie, Freie Universität Berlin, 14195 Berlin, Germany
Abstract
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 α subunits of PTX-sensitive G proteins, Gαi2 and Gαi3, were identified by Western blotting. Introduction of a synthetic peptide corresponding to the C terminus of the Gi3 α subunit into permeabilized CD8 cells efficiently inhibited the cAMP-induced AQP2 translocation; a peptide corresponding to the α 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.
Footnotes
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↵* This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Ro 597/6) and a grant from the Italian “Ministero Ricerca Scientifica e Tecnologica” (MURST, ex 40%).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§§ To whom correspondence should be addressed. Tel.: 49-30-51551 218; Fax: 49-30-51551 291; E-mail: rosenthal{at}fmp-berlin.de.
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↵2 U. Liebenhoff and W. Rosenthal, unpublished results.
- Abbreviations:
- AQP2
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aquaporin 2
- PTX
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pertussis toxin
- TIR
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total internal reflection
- Pf
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osmotic water permeability coefficient
- AS
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antiserum
- IC
-
intracellular
- HSP
-
high speed pellet.
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- Received April 17, 1998.
- Revision received June 9, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
