Stephen L. Gluck
Vacuolar Proton ATPaseOur laboratory is investigating the control of gene expression and biochemical regulation of the mammalian vacuolar H+-ATPase. Vacuolar H+-ATPases are complex enzymes with multiple subunits found in all eukaryotic cells throughout nature. In most cells, vacuolar H+-ATPases reside at low densities primarily in intracellular vacuoles, acidifying endosomes, Iysosomes, and other compartments. In certain specialized cells, such as the kidney intercalated cells, activated macrophages, and osteoclasts in bone, vacuolar H+-ATPases reside at high densities on the plasma membrane, and are polarized to one pole of the cell to serve in transcellular proton transport. Work in our laboratory has focussed on the mechanisms for cell-specific amplified H+-ATPase expression in these cells, and on the regulation of the vacuolar H+-ATPase in proton-transporting cells.
Our laboratory has also obtained evidence that a family of H+-ATPases exists with different enzymatic and structural properties. In order to examine the structural basis for such diverse roles of the enzyme, we isolated cDNA clones for several subunits of the enzyme that demonstrate structural heterogeneity. One subunit, the 56 kD subunit, has two isoforms encoded by different genes. One isoform exhibits enhanced transcriptional regulation in differentiating macrophages, and we are studying the regulation of its expression. We found that the other isoform is expressed at high levels specifically in kidney intercalated cells. We are currently using transgenic mice to identify the controlling elements in the 5' flanking region of this gene that are responsible for the cell-specific amplification exhibited by intercalated cells. We are examining the expression of H+-ATPase genes during renal development in kidney explants taken from 13 day-old rat embryos that undergo differentiation in vitro.
We have discovered H+-ATPase regulatory proteins in kidney cytosol. We have purified an H+-ATPase inhibitor protein, and we are currently attempting to obtain some protein sequence from it with the goals of preparing antibodies to the protein, isolating a cDNA clone for it, and investigating its role in the regulation of kidney, macrophage, and osteoclast H+ secretion.