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Glyceraldehyde 3-phosphate dehydrogenase is required for band 3 (anion exchanger 1) membrane residency in the mammalian kidney.

Authors: Su, Y  Blake-Palmer, KG  Fry, AC  Best, A  Brown, AC  Hiemstra, TF  Horita, S  Zhou, A  Toye, AM  Karet, FE 
Citation: Su Y, etal., Am J Physiol Renal Physiol. 2011 Jan;300(1):F157-66. doi: 10.1152/ajprenal.00228.2010. Epub 2010 Oct 27.
Pubmed: (View Article at PubMed) PMID:20980406
DOI: Full-text: DOI:10.1152/ajprenal.00228.2010

The mammalian kidney isoform of the essential chloride-bicarbonate exchanger AE1 differs from its erythrocyte counterpart, being shorter at its N terminus. It has previously been reported that the glycolytic enzyme GAPDH interacts only with erythrocyte AE1, by binding to the portion not found in the kidney isoform. (Chu H, Low PS. Biochem J 400:143-151, 2006). We have identified GAPDH as a candidate binding partner for the C terminus of both AE1 and AE2. We show that full-length AE1 and GAPDH coimmunoprecipitated from both human and rat kidney as well as from Madin-Darby canine kidney (MDCK) cells stably expressing kidney AE1, while in human liver, AE2 coprecipitated with GAPDH. ELISA and glutathione S-transferase (GST) pull-down assays using GST-tagged C-terminal AE1 fusion protein confirmed that the interaction is direct; fluorescence titration revealed saturable binding kinetics with Kd 2.3+/-0.2 muM. Further GST precipitation assays demonstrated that the D902EY residues in the D902EYDE motif located within the C terminus of AE1 are important for GAPDH binding. In vitro GAPDH activity was unaffected by C-terminal AE1 binding, unlike in erythrocytes. Also, differently from red cell N-terminal binding, GAPDH-AE1 C-terminal binding was not disrupted by phosphorylation of AE1 in kidney AE1-expressing MDCK cells. Importantly, small interfering RNA knockdown of GAPDH in these cells resulted in significant intracellular retention of AE1, with a concomitant reduction in AE1 at the cell membrane. These results indicate differences between kidney and erythrocyte AE1/GAPDH behavior and show that in the kidney, GAPDH is required for kidney AE1 to achieve stable basolateral residency.

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CRRD Object Information
CRRD ID: 10450535
Created: 2016-01-15
Species: All species
Last Modified: 2016-01-15
Status: ACTIVE



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