The COOH termini of NBC3 and the 56-kDa H+-ATPase subunit are PDZ motifs involved in their interaction.
The electroneutral sodium bicarbonate cotransporter 3 (NBC3) coimmunoprecipitates from renal lysates with the vacuolar H(+)-ATPase. In renal type A and B intercalated cells, NBC3 colocalizes with the vacuolar H(+)-ATPase. The involvement of the COOH termini of NBC3 and the 56-kDa subunit of the proton pump in the interaction of these proteins was investigated. The intact and modified COOH termini of NBC3 and the 56-kDa subunit of the proton pump were synthesized, coupled to Sepharose beads, and used to pull down kidney membrane proteins. Both the 56- and the 70-kDa subunits of the proton pump, as well as a PDZ domain containing protein Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1), were bound to the intact 18 amino acid NBC3 COOH terminus. A peptide truncated by five COOH-terminal amino acids did not bind these proteins. Replacement of the COOH-terminal leucine with glycine blocked binding of both the proton pump subunits but did not affect binding of NHERF-1. The 18 amino acid COOH terminus of the 56-kDa subunit of the proton pump bound NHERF-1 and NBC3, but the truncated and modified peptide did not. A complex of NBC3, the 56-kDa subunit of the proton pump, and NHERF-1 was identified in rat kidney. The data indicate that the COOH termini of NBC3 and the 56-kDa subunit of the vacuolar proton pump are PDZ-interacting motifs that are necessary for the interaction of these proteins. NHERF-1 is involved in the interaction of NBC3 and the vacuolar proton pump.
Pushkin
Alexander
A
Division of Nephrology, David Geffen School of Medicine, University of California, Los Angeles 90095-1689, USA.
Abuladze
Natalia
N
Newman
Debra
D
Muronets
Vladimir
V
Sassani
Pejvak
P
Tatishchev
Sergei
S
Kurtz
Ira
I
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D004847:Epithelial Cells / Q000201:enzymology*; D007684:Kidney Tubules / Q000201:enzymology*; D015394:Molecular Structure; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D051381:Rats; D027982:Sodium-Bicarbonate Symporters / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers; D013347:Subcellular Fractions
D010750:Phosphoproteins; D021122:Protein Subunits; C498902:Slc4a7 protein, rat; D027982:Sodium-Bicarbonate Symporters; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D006180:Proton-Translocating ATPases