Molecular and cellular characterization of a water-channel protein, AQP-h3, specifically expressed in the frog ventral skin.
Two cDNAs encoding frog aquaporin (AQP) were cloned from a cDNA library constructed for the ventral skin of the tree frog, Hyla japonica and sequenced. One AQP (Hyla AQP-h1) consisted of 271 amino-acid residues with high homology to toad AQP-t1, Rana CHIP28 (AQP1), and rat AQP1. The other AQP (AQP-h3) consisted of 271 amino-acid residues with higher homology to mammalian AQP2 than to mammalian AQP3. The predicted amino-acid sequence contained the conserved two NPA motifs found in all MIP family members and the putative six transmembrane domains. The sequence also confers mercurial sensitivity, which is common to all the AQPs except AQP0, AQP4 and AQP7. Potential N-glycosylation sites were present at Asn-44 in AQP-h1, and at Asn-124 and Asn-125 in AQP-h3. In addition, AQP-h3 had a putative phosphorylation site by protein kinase A at Ser-255, which is identical to mammalian AQP2. In swelling assays using Xenopus oocytes, AQP-h1 facilitates water permeability, whereas AQP-h3 displayed weak water permeability. Searching for the expression of these two AQP mRNAs revealed that AQP-h1 was expressed in most tissues, whereas AQP-h3 was observed only in the ventral skin. An antibody (ST-141) against the C-terminal peptide of the AQP-h3 protein recognized a 29.0 kDa-protein with a molecular mass close to that of the Hyla AQP-h3 protein and immunostained predominantly in the abdominal pelvic skin. In pelvic skin, the label for AQP-h3 was more intense in the upper layer of the stratum granulosum and was localized to both the apical and basolateral plasma membranes of the principal cells. These findings suggest that Hyla AQP-h3 plays a pivotal role in constitutively absorbing water from ventral pelvic skin.
Tanii
H
H
Department of Biology, Faculty of Science, Shizuoka University, Ohya 836, Shizuoka 422-8529, Japan.
Hasegawa
T
T
Hirakawa
N
N
Suzuki
M
M
Tanaka
S
S
D000818:Animals; D001001:Anura; D020346:Aquaporins / Q000235:genetics*; D001483:Base Sequence; D018076:DNA, Complementary / Q000032:analysis*; D015870:Gene Expression; D005786:Gene Expression Regulation; D015723:Gene Library; D008969:Molecular Sequence Data; D009865:Oocytes; D010388:Pelvis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012867:Skin / Q000166:cytology*; D014981:Xenopus
D020346:Aquaporins; D018076:DNA, Complementary