Ligand-dependent complex formation between the Angiotensin II receptor subtype AT2 and Na+/H+ exchanger NHE6 in mammalian cells.
Involvement of Angiotensin II (Ang II) in the regulation of sodium levels by modulating the Na+/H+ exchangers is demonstrated in many tissues. Screening of a mouse 17-day fetus cDNA library with the Angiotensin II receptor AT2 as the bait in yeast two-hybrid assay led us to identify an AT2-interacting mouse fetus peptide that shared 98% amino acid identity with the corresponding region of the human NHE6. NCBI Blast search showed that the clone 6430520C02 (GenBank Accession # AK032326) of the mouse genome project carried the complete sequence of this new mouse NHE6 isoform. The human and mouse NHE6 peptides share 97% overall homology. Further analysis showed that the region spanning the third intracellular loop and C-terminal cytoplasmic tail of the AT2 directly interacted with a 182 amino acid region that spans the predicted 5th intracellular loop and the initial part of the C-terminus of the mouse NHE6 in yeast two-hybrid assay. This 182-amino acid region that interacted with the AT2 also shares 98% homology with the corresponding region of rat NHE6 and therefore is highly conserved across species. We detected widespread expression of this NHE6 isoform in several rat tissues including 10-day fetus, 17-day fetus, and 30-day post-natal tissues of heart, brain, kidney and muscle. Moreover, the AT2 co-immunoiprecipitated with a hemagglutinin tagged NHE6 when expressed in human cell line MCF-7, and activated by AngII. This ligand-dependent complex formation between the AT2 and NHE6 suggests that the hormone Ang II may act as a regulator of NHE6, and Ang II-mediated direct protein-protein interaction between AT2 and NHE6 could be a mechanism for modulating the functions of the ubiquitously expressed NHE6 in different tissues.
Pulakat
Lakshmi
L
Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403, USA. pulakat@bgnet.bgsu.edu
Cooper
Shannon
S
Knowle
Dieter
D
Mandavia
Chirag
C
Bruhl
Steven
S
Hetrick
Mary
M
Gavini
Nara
N
D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017124:Conserved Sequence; D015870:Gene Expression; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D044139:Receptor, Angiotensin, Type 2 / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D008024:Ligands; D008565:Membrane Proteins; C503961:NHE6 protein, mouse; D010455:Peptides; D044139:Receptor, Angiotensin, Type 2; C445316:SLC9A6 protein, human; D017923:Sodium-Hydrogen Exchangers; D000804:Angiotensin II