Immunocytochemical studies on translocation of phosphorylated aquaporin-h2 protein in granular cells of the frog urinary bladder before and after stimulation with vasotocin.
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.
Hasegawa
Takahiro
T
Department of Biology, Faculty of Science, Shizuoka University, Ohya 836, Shizuoka, 422-8529, Japan.
Suzuki
Masakazu
M
Tanaka
Shigeyasu
S
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D000918:Antibody Specificity; D001001:Anura / Q000378:metabolism*; D020346:Aquaporins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D010766:Phosphorylation; D001743:Urinary Bladder / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D014668:Vasotocin / Q000494:pharmacology
D000906:Antibodies; D020346:Aquaporins; D010446:Peptide Fragments; D014668:Vasotocin