Urothelium covers most of the urinary tract including the bladder, and performs two functions. Its apical surface area expands during bladder distention, but decreases upon bladder contraction. At all times during this micturition cycle, the apical surface also functions as an effective permeability barrier. Exactly how bladder epithelium accomplishes these is unclear. However, since the apical surface of mammalian urothelium is covered with numerous rigid-looking plaques that are highly urothelium- specific, it is generally assumed that these plaques must be important for these urothelial functions. When the plaques are viewed in cross sections, their outer leaflets are almost twice as thick as the inner ones, hence the term asymmetric unit membrane (AUM). The thickened outer leaflet actually contains crystalline hexagonal arrays of 12-nm protein particles, each of which can be further resolved by image enhancement into 6 inner and 6 outer domains. It has been suggested that these AUM plaques, which cover over 70% of urothelial apical surface, contribute to the urothelial permeability barrier; that they stabilize urothelial surface through their anchorage into an underlying cytoskeleton thus preventing the apical surface from rupturing during bladder distention; and that some of the apical plaques can reform vesicles to be retrieved into the cytoplasm for later use thus allowing a reversible adjustment of the apical surface area. Little was known, however, about the biochemical nature, let alone the real function, of the AUM plaques. The PI has recently succeeded in purifying milligram quantities of bovine AUMs, which contain 4 major integral membrane proteins (the """"""""uroplakins"""""""" or UP) - the 27-kD UPIa, the 28-kD UPIb, the 15-kD UPII and the 47-kD UPIII. cDNA cloning data showed that UPIa and Ib are closely related isoforms with 4 transmembrane domains (TMD), while UPII and UPIII have only one TMD. The bulk of the hydrophilic domains of all UP's are extracellular, and thus are probably involved ill forming the luminal 12-nm protein particles. To better understand the structure and function of uroplakins -which are synthesized as major differentiation products by terminally differentiated urothelial umbrella cells -we will perform three series of experiments. First, we will determine how the UP molecules interact with one another to form the highly ordered 12-nm protein particles. We will determine whether there exist 2 distinct populations of 12-nm protein particles, one consisting of UPIa/II while another of UPIb/III, and whether the pro sequence of UPII regulates UPIl assembly. Second, we will study the regulation of AUM assembly, which is retarded in many hyperplastic urothelial conditions. Finally, we will study AUM/cytoskeletal interaction 'which is believed to play a central role in stabilizing the urothelial apical surface. Results from these studies should enhance our understanding on the structure and function of AUM - a fascinating biomembrane, and on normal and eventually abnormal bladder epithelial function.
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