A HETEROTRIMERIC G-PROTEIN OF THE G(I) FAMILY IS REQUIRED FOR CAMP-TRIGGERED TRAFFICKING OF AQUAPORIN-2 IN KIDNEY EPITHELIAL-CELLS

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 t he apical membrane of kidney epithelial cells, an event initiated by a n increase in cAMP and activation of protein kinase A. The subsequent steps of the signaling cascade are not known. To identify proteins inv olved in the AQP2 shuttle we exploited a recently developed cell line (CD8) derived from the rabbit cortical collecting duct and stably tran sfected with rat AQP2 cDNA, Treatment of CD8 cells with pertussis toxi n (PTX) inhibited both the vasopressin-induced increase in water perme ability and the redistribution of AQP2 from an intracellular compartme nt to the apical membrane. ADP-ribosylation studies revealed the prese nce of at least two major PTX substrates, Correspondingly, two a: subu nits of PTX-sensitive G proteins, G alpha(i2), and G alpha(i3), were i dentified by Western blotting. Introduction of a synthetic peptide cor responding to the C terminus of the G(i3) alpha subunit into permeabil ized CD8 cells efficiently inhibited the cAMP-induced AQP2 translocati on; a peptide corresponding to the a subunits of G(i1/2) was much less potent. Thus a member of the G(i) family, most likely G(i3), is invol ved in the cAMP-triggered targeting of AQP2-bearing vesicles to the ap ical membrane of kidney epithelial cells.