During chronic metabolic acidosis, increased renal ammoniagenesis and gluconeogenesis from glutamine are sustained, in part,, by a cell specific increase in expression of the mitochondrial glutaminase (GA) that results from the selective stabilization of the GA mRNA. Previous experiments identified an 8-base AU- sequence within the 3'- non-translated region of the GA mRNA that functions as a pH-response element (pHRE) when introduced into a chimeric reporter mRNA. This sequence was used as an affinity ligand to purify and identify zeta-crystallin/NADPH; quinone reductase as the pHRE-binding protein. The functional characterization of the pHRE was carried out in LLC-PK1-FBPase+ cells, a porcine proximal tubule-like cell line that expresses multiple forms of GA mRNAs. The corresponding porcine GA cDNAs were cloned and sequenced. Only the 4.5-kb GA mRNA contains pHREs that are identical to the sequence identified in the rat GA mRNA and only this form of GA mRNA is stabilized and increased by incubating the cells in acidic medium (Ph=6.9, 10 mM HCO3). Thus this cell lines provides a system to further characterized the molecular mechanism of GA mRNA stabilization and the associated signal transduction pathway. Finally, the 4.50kb GA mRNA is the ortholog of a newly identified isoform of the human kidney-type GA that is generated by alternative splicing of exons within the GA gene. This isoform contains a unique C-terminal domain of unknown function.
The specific aims of the proposed research are: to express and characterize the isoforms of the kidney-type GA; to characterize the mechanism of GA mRNA turnover; to characterize the role of zeta-crystallin/NADPH; quinone reductase in the stabilization of the GA mRNA; and to identify the signal transduction pathway that leads to enhanced binding of zeta-crystallin/NADPH; quinone reductase to the pHRE. The results of the proposed experiments should significantly increase the understanding of the molecular mechanism that regulates this essential adaptive response and may provide insight to improve the clinical treatment of chronic acidosis.
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