Identification of specific apical membrane polypeptides associated with the antidiuretic hormone-elicited water...

Antidiuretic hormone (ADH) increases the water permeability of the toad urinary bladder. The increase occurs in the apical plasma membrane of granular cells that line the urinary surface of the bladder and is produced by the insertion of water permeability units that have been identified by freeze-fracture electron microscopy as intramembrane particle aggregates. Under water-impermeable conditions, particle aggregates reside in intracellular vesicles called "aggrephores." In response to ADH, the aggrephores fuse with the apical plasma membrane and render it water permeable. When ADH is removed, intramembrane particle aggregates and aggrephores are retrieved from the apical membrane, and it returns to a water-impermeable state. To identify proteins involved in the water permeability response, we used lactoperoxidase/glucose oxidase to 125I-label external apical membrane proteins to compare control and ADH-treated bladders. Several polypeptides were consistently labeled in ADH-treated bladders and not in paired controls. After demonstrating that lactoperoxidase behaves as a fluid-phase marker and is sequestered in aggrephore-like vesicles when ADH is withdrawn, we used the technique of Mellman et al. [Mellman, I.S., Steinman, R. M., Unkeless, J. C. & Cohn, Z. A. (1980) J. Cell Biol. 86, 712-722] to label proteins endocytosed when water permeability declines after ADH is withdrawn to test whether the membrane proteins labeled in ADH-treated bladders behaved like particle aggregates. The internalized membranes contained polypeptides of the same molecular weights (55,000, 17,000-14,000, and 7,000) as those labeled on the apical surface of ADH-treated but not control bladders. These polypeptides are evidently involved in the ADH-stimulated water permeability response and may be components of particle aggregates.