Journal of Biological Chemistry
Volume 285, Issue 4, 22 January 2010, Pages 2653-2664
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Membrane Transport, Structure, Function, and Biogenesis
Rhesus Glycoprotein P2 (Rhp2) Is a Novel Member of the Rh Family of Ammonia Transporters Highly Expressed in Shark Kidney*

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Rhesus (Rh) glycoproteins are a family of membrane proteins capable of transporting ammonia. We isolated the full-length cDNA of a novel Rh glycoprotein, Rhp2, from a kidney cDNA library from the banded hound shark, Triakis scyllium. Molecular cloning and characterization indicated that Rhp2 consists of 476 amino acid residues and has 12 putative transmembrane spans, consistent with the structure of other family members. The shark Rhp2 gene was found to consist of only one coding exon. Northern blotting and in situ hybridization revealed that Rhp2 mRNA is exclusively expressed in the renal tubules of the sinus zone but not in the bundle zone and renal corpuscles. Immunohistochemical staining with a specific antiserum showed that Rhp2 is localized in the basolateral membranes of renal tubule cells. Double fluorescence labeling with phalloidin or labeling of the Na+/K+-ATPase further narrowed the location to the second and fourth loops in the sinus zone. Vacuolar type H+-ATPase was localized in apical membranes of the Rhp2-expressing tubule cells. Quantitative real-time PCR analysis and Western blotting showed that expression of Rhp2 was increased in response to elevation of environmental salinity. Functional analysis using the Xenopus oocyte expression system showed that Rhp2 has transport activity for methylammonium, an analog of ammonia. This transport activity was inhibited by NH4Cl but not trimethylamine-N-oxide and urea. These results suggested that Rhp2 is involved in ammonia reabsorption in the kidney of the elasmobranch group of cartilaginous fish comprising the sharks and rays.

Glycoproteins
Membrane/Channels
Metabolism/Nitrogen
Tissue/Organ Systems/Kidney
Transport
Rh Glycoprotein
Ammonia
Ammonium

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The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB287007.

*

This work was supported by Grants-in-aid for Scientific Research 14104002 and 18059010 (to S. H.) and 18-5667 (to T. N.) from the Ministry of Education, Culture, Sport, Science and Technology of Japan (MEXT), the Japan Society for the Promotion of Science Research Fellowships for Young Scientists, and the 21st Century and Global Centers of Excellence Program of MEXT.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and Fig. 1.

1

Present address: T. Nakada, Dept. of Molecular Pharmacology, Shinshu University School of Medicine, Nagano 390-8621, Japan.