Abstract
We have generated a specific antibody against phosphorylated aquaporin-h2 (pAQP-h2) protein to investigate the role of phosphorylation in the translocation of AQP-h2 protein within the granule cells of the urinary bladder of the frog (Hyla japonica). The antibody was generated against a synthetic peptide (ST-160) corresponding to amino acids 255–268, with a phosphorylated Ser-262, a residue that is putatively phosphorylated by protein A kinase. Using this antibody, we found, by Western blot analysis, that phosphorylation of the AQP-h2 protein rapidly increased within 2 min after vasotocin (AVT) stimulation and remained at a higher than normal level for 15 min. Moreover, quantitative immunoelectron microscopy indicated that the location of the AQP-h2 protein dramatically changed after AVT stimulation. Before stimulation, pAQP-h2 protein was localized in only a small number of intracellular vesicles near the nucleus of the granular cells, whereas the labeling density of the intracellular vesicles and the apical membrane rapidly increased after stimulation. This finding was also confirmed by the results of an immunofluorescence study. Thus, phosphorylation of AQP-h2 protein seems to be essential for translocation of the protein from the cytoplasmic pool to the apical plasma membrane of the granular cells in frog urinary bladder.
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References
Bentley PJ (2002) The Amphibia. In: Bentley PJ (ed) Endocrines and osmoregulation. A comparative account in vertebrates, vol 39. Springer, Berlin Heidelberg New York, pp 155–186
Brown D (1989) Membrane recycling and epithelial cell function. Am J Physiol 256:F1–F12
Brown D, Grosso A, DeSousa RC (1983) Correlation between water flow and intramembrane particle aggregates in toad epidermis. Am J Physiol 245:C334–C342
Brown D, Katsura T, Gustafson CE (1998) Cellular mechanisms of aquaporin trafficking. Am J Physiol 275:F328–F331
Chevalier J, Bourguet J, Hugon JS (1974) Membrane associated particles: distribution in frog urinary bladder epithelium at rest and after oxytocin treatment. Cell Tissue Res 152:129–140
Christensen BM, Zelenina M, Aperia A, Nielsen S (2000) Localization and regulation of PKA-phosphorylated AQP2 in response to V(2)-receptor agonist/antagonist treatment. Am J Physiol Renal Physiol 278:F29–F42
Fushimi K, Uchida S, Hara Y, Hirata Y, Marumo F, Sasaki S (1993) Cloning and expression of apical membrane water channel of rat kidney collecting tubule. Nature 361:549–552
Fushimi K, Sasaki S, Marumo F (1997) Phosphorylation of serine 256 is required for cAMP-dependent regulatory exocytosis of the aquaporin-2 water channel. J Biol Chem 272:14800–14804
Hasegawa T, Tanii H, Suzuki M, Tanaka S (2003) Regulation of water absorption in the frog skins by 2 vasotocin-dependent water-channel aquaporins, AQP-h2 and AQP-h3. Endocrinology 144:4087–4096
Ishibashi K, Kuwahara M, Sasaki S (2000) Molecular biology of aquaporins. Rev Physiol Biochem Pharmacol 141:1–32
Kachadorian WA, Wade JB, DiScala VA (1975) Vasopressin: induced structural change in toad bladder luminal membrane. Science 190:67–69
Kachadorian WA, Muller J, Ellis SJ (1986) Time-dependent attenuation of water flow in antidiuretic hormone-treated toad bladder. Am J Physiol 250:F845–F849
Kachadorian WA, Spring KR, Shinowara NL, Muller J, Palaia TA, DiScala VA (1990) Effects of serosal hypertonicity on water permeability in toad urinary bladder. Am J Physiol 258:C871–C878
Katsura T, Gustafson CE, Ausiello DA, Brown D (1997) Protein kinase A phosphorylation is involved in regulated exocytosis of aquaporin-2 in transfected LLC-PK1 cells. Am J Physiol 272:F817–F822
Keller GA, Tokuyasu KT, Dutton AH, Singer SJ (1984) An improved procedure for immunoelectron microscopy: ultrathin plastic embedding of immunolabeled ultrathin frozen sections. Proc Natl Acad Sci U S A 81:5744–5747
Kuwahara M, Fushimi K, Terada Y, Bai L, Marumo F, Sasaki S (1995) cAMP-dependent phosphorylation stimulates water permeability of aquaporin-collecting duct water channel protein expressed in Xenopus oocytes. J Biol Chem 270:10384–10387
Lorenz D, Krylov A, Hahm D, Hagen V, Rosenthal W, Pohl P, Maric K (2003) Cyclic AMP is sufficient for triggering the exocytic recruitment of aquaporin-2 in renal epithelial cells. EMBO Rep 4:88–93
Martin S, Slot JW, James DE (1999) GLUT4 trafficking in insulin-sensitive cells. A morphological review. Cell Biochem Biophys 30:89–113
Nielsen S, DiGiovanni SR, Christensen EI, Knepper MA, Harris HW (1993) Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney. Proc Natl Acad Sci U S A 90:11663–11667
Nielsen S, Chou CL, Marples D, Christensen EI, Kishore BK, Knepper MA (1995) Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to plasma membrane. Proc Natl Acad Sci U S A 92:1013–1017
Nishimoto G, Zelenina M, Li D, Yasui M, Aperia A, Nielsen S, Nairn AC (1999) Arginine vasopressin stimulates phosphorylation of aquaporin-2 in rat renal tissue. Am J Physiol 276:F254–F259
Orci L, Montesano R, Brown D (1980) Heterogeneity of toad bladder granular cell luminal membranes. Distribution of filipin-sterol complexes in freeze-fracture. Biochim Biophys Acta 601:443–452
Park JH, Saier MH Jr (1996) Phylogenetic characterization of the MIP family of transmembrane channel proteins. J Membr Biol 153:171–180
Preston GM, Agre P (1991) Isolation of the cDNA for erythrocyte integral membrane protein of 28 kilodaltons: member of an ancient channel family. Proc Natl Acad Sci U S A 88:11110–11114
Rodnick KJ, Slot JW, Studelska DR, Hanpeter DE, Robinson LJ, Geuze HJ, James DE (1992) Immunocytochemical and biochemical studies of GLUT4 in rat skeletal muscle. J Biol Chem 267:6278–6285
Slot JW, Geuze HJ, Gigengack S, Lienhard GE, James DE (1991) Immuno-localization of the regulatable glucose transporter in brown adipose tissue of the rat. J Cell Biol 113:123–135
Tajika Y, Matsuzaki T, Suzuki T, Aoki T, Hagiwara H, Tanaka S, Kominami E, Takata K (2002) Immunohistochemical characterization of the intracellular pool of water channel aquaporin-2 in the kidney. Anat Sci Inter 77:189–195
Takata K, Matsuzaki T, Tajika Y (2004) Aquaporins: water channel proteins of the cell membrane. Prog Histochem Cytochem 39:1–83
Tanaka S, Kurosumi K (1992) A certain step of proteolytic processing of proopiomelanocortin occurs during the transition between two distinct stages of secretory granules maturation in rat anterior pituitary corticotrophs. Endocrinology 131:779–786
Tanaka S, Nomizu M, Kurosumi K (1991) Intracellular sites of proteolytic processing of pro-opiomelanocortin in melanotrophs and corticotrophs in the rat pituitary. J Histochem Cytochem 39:809–821
Tanaka S, Yora T, Nakayama K, Inoue K, Kurosumi K (1997) Proteolytic processing of pro-opiomelanocortin occurs in acidifying secretory granules of AtT-20 cells. J Histochem Cytochem 45:425–436
Tanii H, Hasegawa T, Hirakawa N, Suzuki M, Tanaka S (2002) Molecular and cellular characterization of a water channel protein, AQP-h3, specifically expressed in the frog ventral skin. J Membr Biol 188:43–53
Tokuyasu K (1986) Application of cryomicrotomy to immunocytochemistry. J Microsc 143:139–149
Wade JB, Stetson DL, Lewis SA (1981) ADH action: evidence for a membrane shuttle mechanism. Ann N Y Acad Sci 372:106–117
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This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan to S.T.
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Hasegawa, T., Suzuki, M. & Tanaka, S. Immunocytochemical studies on translocation of phosphorylated aquaporin-h2 protein in granular cells of the frog urinary bladder before and after stimulation with vasotocin. Cell Tissue Res 322, 407–415 (2005). https://doi.org/10.1007/s00441-005-0037-8
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DOI: https://doi.org/10.1007/s00441-005-0037-8