The long-term objective of this proposal is to provide a fundamental understanding of the cell biology of the neurohypophyseal hormone, vasopressin, which plays a major role in the regulation of salt and water balance through its action on the kidney. Its dysfunction is a primary cause of congenital nephrogenic diabetes insipidus (CNDI) and a secondary cause of fluid and electrolyte abnormalities in such diseases as congestive heart failure, cirrhosis of the liver, and nephrotic syndrome. The initial aims of this proposal are to provide an analysis of the structural motifs of the vasopressin V2 receptor (V2R) responsible for 1) proper folding and processing in the ER and Golgi and appropriate targeting to basolateral (or apical) membranes of kidney cells; and 2) regulation of desensitization, downregulation or resensitization of the V2R that we propose is mediated by G protein receptor kinase phosphorylation, arrestin binding, and endocytosis and exocytosis initiated by clathrin-coated pits or caveolae. We also propose that one or more of these events is coordinated by the heterotrimeric G protein subunit, G/alpha/i3. These studies will make use of epitope-tagged or green fluorescent protein-tagged V2Rs or its mutations, CNDI mutants, or """"""""split"""""""" V2Rs to monitor these processes by immunocytochemistry, confocal and electron microscopy or cell surface biotinylation in fixed and living cells. Association of the V2R with similarly tagged auxiliary proteins (e.g. G/alpha/i3, clathrin and adaptor proteins, dynamin or caveolin) will be determined by immunocytochemistry and immunoprecipation or by a new method of surface plasmon resonance. Transduction of the vasopressin receptor signaling pathway to the distal elements in its physiologic action also requires G/alpha/i3 regulation of vasopressin-sensitive Na+ channels, referred to as the 5 pS (rENaC) or the 9 pS channel.
The final aim of our proposal is to determine the ability of G/alpha/i3 to regulate the 5 pS rENaC, evaluated by electrophysiological techniques in oocytes or MDCK cells co-expressing rENaC and a constitutively active, pertussis-toxin insensitive G/alpha/i3 (G/alpha/i3*PTneg). Studies will be conducted to determine if G/alpha/i3 action depends on its membrane targeting sequences previously determined in our laboratory. Direct demonstration of the action of G/alpha/i3*PTneg on its mutations on the 5 pS and 9 pS Na+ channels will be determined by electrophysiological techniques after the addition of the purified protein to the cytosolic surface of membranes of either A6 cells or MDCK cells expressing rENaC, or following addition to purified bovine renal 9 pS Na+ channels reconstituted into lipid bilayers. Finally, utilizing G/alpha/i3*PTneg coupled via a hexahistidine to nickel-agarose beads, effector proteins for G/alpha/i3 action on Na+ channels will be expression cloned.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Method to Extend Research in Time (MERIT) Award (R37)
Project #
Application #
Study Section
General Medicine B Study Section (GMB)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Massachusetts General Hospital
United States
Zip Code
Yi, Xianhua; Bouley, Richard; Lin, Herbert Y et al. (2007) Alix (AIP1) is a vasopressin receptor (V2R)-interacting protein that increases lysosomal degradation of the V2R. Am J Physiol Renal Physiol 292:F1303-13
Bouley, Richard; Hawthorn, Gayle; Russo, Leileata M et al. (2006) Aquaporin 2 (AQP2) and vasopressin type 2 receptor (V2R) endocytosis in kidney epithelial cells: AQP2 is located in 'endocytosis-resistant' membrane domains after vasopressin treatment. Biol Cell 98:215-32
Bouley, Richard; Lin, Herbert Y; Raychowdhury, Malay K et al. (2005) Downregulation of the vasopressin type 2 receptor after vasopressin-induced internalization: involvement of a lysosomal degradation pathway. Am J Physiol Cell Physiol 288:C1390-401
de Sousa Lopes, Susana M Chuva; Roelen, Bernard A J; Monteiro, Rui M et al. (2004) BMP signaling mediated by ALK2 in the visceral endoderm is necessary for the generation of primordial germ cells in the mouse embryo. Genes Dev 18:1838-49
Roelen, Bernard A J; Cohen, Ori S; Raychowdhury, Malay K et al. (2003) Phosphorylation of threonine 276 in Smad4 is involved in transforming growth factor-beta-induced nuclear accumulation. Am J Physiol Cell Physiol 285:C823-30
Bouley, Richard; Sun, Tian-Xiao; Chenard, Melissa et al. (2003) Functional role of the NPxxY motif in internalization of the type 2 vasopressin receptor in LLC-PK1 cells. Am J Physiol Cell Physiol 285:C750-62
Bouley, R; Breton, S; Sun, T et al. (2000) Nitric oxide and atrial natriuretic factor stimulate cGMP-dependent membrane insertion of aquaporin 2 in renal epithelial cells. J Clin Invest 106:1115-26
Berdiev, B K; Prat, A G; Cantiello, H F et al. (1996) Regulation of epithelial sodium channels by short actin filaments. J Biol Chem 271:17704-10
Brand, S H; Holtzman, E J; Scher, D A et al. (1996) Role of myristoylation in membrane attachment and function of G alpha i-3 on Golgi membranes. Am J Physiol 270:C1362-9
Hozawa, S; Holtzman, E J; Ausiello, D A (1996) cAMP motifs regulating transcription in the aquaporin 2 gene. Am J Physiol 270:C1695-702

Showing the most recent 10 out of 27 publications